DC-CD4 bispecific tolerogenic nanovesicles induce antigen-specific regulatory T cells and ameliorate collagen-induced arthritis in mice

Domain: biology
Published: 2026-03-19
Editorial outcome: major_revision

Abstract
Inducing antigen-specific regulatory T (Treg) cells is a promising strategy for treating autoimmune diseases (AID). Here, we present dendritic cells-CD4⁺ T cells (DC-CD4) bispecific tolerogenic nanovesicles (NV) co-loaded with an antigenic peptide and rapamycin. We show that these NVs bring DCs and CD4⁺ T cells into close spatial proximity through CTLA4-CD80/86 and anti-CD4 (aCD4)-CD4 interactions, thereby promoting antigen-specific Treg cell generation. In a collagen-induced arthritis mouse model, the bispecific NVs display enhanced lymph node (LN) tropism and increase antigen-specific Treg cells in LNs and spleen, when administered in both therapeutic and prophylactic settings, resulting in robust efficacy against CIA. Furthermore, arthritogenic cell transfer and adoptive transfer of Treg cells into Treg-ablated mice confirm the pivotal role of bispecific NV-induced antigen-specific Treg cells in mediating the anti-inflammatory efficacy. Collectively, our data support DC-CD4 bispecific tolerogenic NVs as a platform to induce antigen-specific Treg cells for precise immune tolerance in AIDs.

Introduction
Immune tolerance refers to the immune system’s ability to distinguish self from non-self, actively preventing immune responses against the body’s own tissues1-3. This mechanism is essential for maintaining immune homeostasis and preventing immune disorders1, 3. In recent years, the concept of therapeutically inducing immune tolerance has garnered increasing attention as a promising strategy for treating autoimmune disorders4-7, allergies8, 9, and transplant rejection10, 11. Being distinct from conventional immunosuppression that broadly dampens immunity, restoring immune tolerance offers significant advantages in terms of antigen-specificity (targeting only pathogenic immune responses), long-term efficacy (reprogramming immune memory), and minimal systemic toxicity (preserving host defense against infections)1, 12. Therefore, immune tolerance is an attractive therapeutic strategy for a wide range of immune-mediated conditions.

Regulatory T (Treg) cells, a specialized subset of CD4+ T lymphocytes characterized by Foxp3 expression, play a pivotal role in maintaining immune tolerance and preventing autoimmune disorders13-17. Current clinical strategies in Treg-mediated immunotherapy primarily focus on two major approaches: direct adoptive transfer of ex vivo-expanded Treg cells 18, 19 and in vivo induction of endogenous Treg cells using tolerogenic dendritic cells (tDC)20, 21 or Treg-biased immunostimulants such as IL-2 variants17. However, cell-based approaches face significant challenges, including the patient-specific production in a clinical setup and labor-intensive process of in vitro cell expansion, induction and reinfusion22-24. To overcome these limitations, nanotechnology-based strategies have been explored to deliver antigens and immunosuppressive agents specifically to antigen presenting cells (APC), particularly dendritic cells (DC), thereby boosting the generation of antigen-specific Treg cells in situ4, 6, 12, 25, 26. Among these approaches, Sohn et al. reported lipid nanoparticles (LNP) co-loaded with type II collagen peptide and rapamycin (RM), which reduced synovial inflammation and ameliorated osteoarthritis in mice, providing a compelling proof-of-concept for NP-mediated antigen-specific tolerance in joint disease6. However, this strategy relies on passive uptake by DCs followed by intradermal (i.d.) injection, without actively targeting or programming DC fate to coordinate crosstalk with other immune cells. Therefore, there is an urgent need to develop a targeted tolerogenic strategy that actively reprograms DCs and amplifies their interactions with immune partners, driving more efficient and durable induction of antigen-specific Treg cells.

Bispecific systems, such as bispecific NPs and antibodies, have emerged as innovative tools capable of bridging distinct immune cell types to enhance intercellular communication27-31. By simultaneously engaging specific receptors on both types of cells, these systems can precisely orchestrate cellular crosstalk27, 28. To date, bispecific strategies have been extensively explored in cancer therapies, where they are primarily used to link immune effector cells with tumor cells, thereby promoting immune-mediated tumor eradication27-31. Nevertheless, their application in non-cancer settings to induce immune tolerance, particularly in inflammatory diseases such as autoimmune diseases (AID), remains limited. Considering the critical role of cellular crosstalk in shaping inflammatory responses, there is significant potential for harnessing bispecific nanosystem to modulate pathological immune interactions in these contexts. Therefore, developing bispecific nanoplatforms tailored for inflammatory disease intervention via generating antigen-specific Treg cells represents a promising yet underexplored frontier.

Here we develop a DC-CD4 bispecific tolerogenic nanovesicle (NV) platform that actively couples DCs with CD4⁺ T cells to enhance tolerogenic crosstalk and drive antigen-specific Treg cell induction in vivo. The platform enhances lymph node (LN) accumulation after i.d. administration, strengthens DC-CD4 engagement, and amplifies antigen-specific Treg responses in lymphoid organs. These effects support a coordinated tolerogenic program that promotes tDC differentiation, immunosuppressive CD4+ T cell (iCD4) induction, and subsequent antigen-specific Treg cell generation. Collectively, these activities establish durable, antigen-specific immune tolerance in a collagen-induced arthritis model. This work provides a generalizable strategy for programming immune cell interactions to achieve precise immune tolerance, and offers a modular framework for antigen-specific immunotherapy across autoimmune and other immune-mediated inflammatory diseases.

Results
1 Preparation and characterization of CII-RM@aCD4Tn
The fabrication process for CII-RM@aCD4Tn encapsulating bovine type II collagen 259 to 273 peptide (CII) is shown in Fig. 1a. iCD4 with stabled high CTLA4 express were successfully induced from CD4+ T cells isolated from spleen and LNs, showing approximately threefold expansion at 10 ng mL-1 TGF-β (Supplementary Fig. 1a-e). Additionally, the key immunomodulatory markers PD-1 (P<0.0001) and LAG-3 (P=0.0012) were significantly upregulated following stimulation (Supplementary Fig. 1f). For CII-RM@aCD4Tn preparation, NVs derived from iCD4 (referred to as Tn) were prepared through sequential extrusion and used for LN-targeted delivery (Supplementary Fig. 2). Protein analysis confirmed that Tn shared a protein expression profile closely mirroring that of parent iCD4 (Supplementary Fig. 3).

To optimize the binding affinity of CII-RM@aCD4Tn to CD4+ T cells, DP-aCD4 were synthesized with varying reaction molar ratios of DSPE-PEG2000-NHS (DP) to anti-CD4 Fab fragment (aCD4). Membrane insertion capacity of DP-aCD4 on CD4+ T cells reached a plateau of approximately 100% when the molar ratio up to 8 (Supplementary Fig. 4a). Nevertheless, when the reaction molar ratio exceeded 8, the affinity of CII-RM@aCD4Tn for CD4+ T cells gradually declined with increased DP modification on aCD4 (Supplementary Fig. 4b), primarily due to partial functional inactivation of aCD4. Consequently, a DP:aCD4 ratio of 8:1 was selected as optimal, yielding CII-RM@aCD4Tn with efficient attachment to CD4+ T cell membrane (Supplementary Fig. 4c).

Under these conditions, CII-RM@aCD4Tn loaded with CII and rapamycin (RM), and functionalized with aCD4 was successfully fabricated (Fig. 1b), with spherical morphology and integrated structure around 130 nm under TEM detection (Fig. 1c). CII-RM@aCD4Tn had a hydrodynamic diameter of 165.7 nm with a Zeta potential of -16.1 mV (Fig. 1d,e). Release of CII and RM from CII-RM@aCD4Tn was quantified by ELISA and HPLC (Supplementary Fig. 5a). Encapsulation efficiency and drug loading capacity of CII and RM were demonstrated in Supplementary Fig. 5b, with CII-RM@aCD4Tn displaying sustained drug release over 24 h (Supplementary Fig. 5c). Long-term stability assessments exhibited that CII-RM@aCD4Tn retained structural integrity when stored in PBS or serum for up to 7 days (Supplementary Fig. 6a,b). Moreover, cryopreservation studies further confirmed their resilience, with no significant changes in particle size or biofunctional activity after 3 weeks of storage at -80℃. (Supplementary Fig. 6c,d).

2 CII-RM@aCD4Tn-mediated immunosuppression in DCs and CD4+ T cells
To determine the efficiency of Tn in enhancing the delivery of drugs (CII and RM) to DCs, FITC-labelled free drugs or CII-RM@aCD4Tn were incubated with bone marrow-derived dendritic cells (BMDC) on an orbital shaker at 40 rpm for 12 h. While all cells internalized CII and RM, drug uptake was 2.7-fold higher when the agents were encapsulated in Tn compared to the free form (Fig. 1f,g). The enhanced uptake by BMDCs are mainly attributable to more efficient ligand-mediated interactions between CII-RM@aCD4Tn and BMDCs. To assess antigen (CII) presentation on DC surface, FITC-labelled CII were incubated with BMDCs on an orbital shaker for 24 h. CII-RM@aCD4Tn facilitated antigen presentation, increasing surface displayed CII levels by 3.1-fold compared to free CII (Fig. 1h,i). To examine whether Tn-mediated antigen delivery facilitated processing and presentation via MHC II on the surface of DCs, Eα peptides, which can be detected by Yae antibody upon forming MHC II complex6, were employed in place of CII to generate Eα-RM@aCD4Tn. After co-culture of Eα-RM@aCD4Tn with BMDCs for 24 h, a substantial fraction of Eα peptide was presented on the cell surface via MHC II, resulting in a 5.8-fold increase compared to free Eα (Supplementary Fig. 7).

tDCs are crucial for maintaining immune homeostasis and preventing autoimmune responses20, 21, 25. BMDCs were incubated with various formulations for 48 h, followed by LPS stimulation for 24 h, and the expression of activation markers and cytokines was subsequently analyzed by flow cytometry (FC), ELISA and qRT-PCR. Matured BMDCs (mDC) displayed high expression levels of CD80, CD86, CD40 and MHC II, all of which were significantly reduced following CII-RM@aCD4Tn treatment (all P<0.0001) (Fig. 1j and Supplementary Fig. 8a). Interestingly, PD-L1 expression was markedly upregulated in BMDCs after CII-RM@aCD4Tn treatment compared to mDCs (P<0.0001) (Supplementary Fig. 8a). Furthermore, pro-inflammatory cytokine TNF and its corresponding mRNA level were highest in mDCs, whereas anti-inflammatory cytokine TGF-β and its mRNA expression were lowest, when compared to other groups (Fig. 1k and Supplementary Fig. 8b). Compared to mDCs, CII-RM@aCD4Tn treatment markedly suppressed TNF expression at both the protein and mRNA levels while significantly upregulating TGF-β (all P<0.0001) (Fig. 1k and Supplementary Fig. 8b).

iCD4 including Foxp3⁺ Treg cells and Foxp3⁻ suppressive CD4⁺ subsets, are essential for immune regulation and autoimmunity prevention, holding therapeutic promise for a range of conditions12, 18, 32, 33. To determine whether CII-RM@aCD4Tn could induce iCD4, CD4+ T cells were cultured with it for 48 h and subsequently activated with PMA for 6 h. FC analysis revealed increased expression of CTLA4 and PD-1 with reduced CD127 expression compared to active CD4+ T cells (aCD4) (all P<0.0001) (Fig. 2l and Supplementary Fig. 9a). Moreover, cells treated with CII-RM@aCD4Tn exhibited elevated TGF-β protein and mRNA levels, while TNF expression was significant reduced relative to aCD4 (all P<0.0001) (Fig. 2m and Supplementary Fig. 9b), suggesting successful induction of a immunosuppressive phenotype.

Together, these findings indicate that CII-RM@aCD4Tn promotes the differentiation of tDCs and iCD4, enhancing anti-inflammatory cytokine secretion while suppressing pro-inflammatory responses.

3 Bridging effects of CII-RM@aCD4Tn on CD4+ T cells and DCs
CD4 molecules on CD4+ T cells and CD80/86 co-stimulatory molecules on DCs represent stable surface biomarkers, making them suitable targets for CII-RM@aCD4Tn engagement (Fig. 2a). Equal numbers of CD4+ T cells and BMDCs were co-incubated for 1 h with either CII-RM@Tn or CII-RM@aCD4Tn. FC analysis of DC-CD4 conjugates showed that, in the presence of CII-RM@Tn, CD4⁺ T cells and BMDCs largely remained as single, unpaired cells, whereas CII-RM@aCD4Tn markedly increased the proportion of BMDC-CD4⁺ T cell pairs compared to CII-RM@Tn group (P<0.0001) (Fig. 2b). To directly evidence CTLA4-CD80/86 interactions between CII-RM@aCD4Tn and BMDCs, we selectively blocked CD80/86 on BMDCs with CTLA4 Ig (G2), CTLA4 on the NV surface with an anti-CTLA4 antibody (G3), or both binding partners simultaneously (G4) (Supplementary Fig. 10a). The frequency of DC-CD4 cell pairs decreased from 38.1% in the unblocked condition to ~8% when either CD80/86 or CTLA4 was blocked individually, and to ~6% when both were blocked (Supplementary Fig. 10b), demonstrating the effective interaction of CII-RM@aCD4Tn and BMDC mediated through CTLA4-CD80/86 axis.

To detect the in vivo gripping ability of CII-RM@aCD4Tn, inguinal LNs (iLN) from DBA/1 mice were collected for immunofluorescence staining after 24 h i.d injection at the flank. Cy5-CII-RM@aCD4Tn localized to CD11c+ DCs and also bound with CD4+ T cells (Fig. 2c). In addition, CII-RM@aCD4Tn promoted interactions between DCs and CD4+ T cells in paracortex of LNs (Fig. 2c). By contrast, CII-RM@Tn primarily resulted in DC-specific internalization without promoting DC-CD4 conjugation (Supplementary Fig. 11). To further identify the in vivo cellular targets, Cy5-CII-RM@aCD4Tn-associated cells were analyzed in iLN. Among Cy5⁺ CD45⁺ leukocytes, NVs were predominantly targeted to CD4+ T cells (44.8%) and CD11c⁺ DCs (31.3%), followed by CD19⁺ B cells (11.7%), whereas uptake by CD11b⁺ macrophages (7.9%) and Ly6G⁺ neutrophils (3.2%) was comparatively low (Supplementary Fig. 12a). When CD4+ T cells were co-cultured with purified APC subsets in the presence of CII-RM@aCD4Tn, CD11c⁺ DCs elicited the highest IL-10 levels in the medium (Supplementary Fig. 12b,c), implying that DCs are the dominant APC subset engaging the NVs to drive tolerogenic responses.

These data support the bispecific capacity of CII-RM@aCD4Tn to simultaneously engage CD4+ T cells and DCs realizing the effective cellular bridging both in vitro and in vivo.

4 Biodistribution and biosafety of CII-RM@aCD4Tn
To assess LN-targeting capability, CII-RM@aCD4Tn were administered via i.d. injection into the left or right flank of DBA/1 mice. Following unilateral injection, in vivo fluorescence imaging revealed that the Cy5-NVs localized exclusively to the ipsilateral iLN and axillary LN (aLN), with minimal signal detected in major organs (Fig. 2d,e and Supplementary Fig. 13). Following bilateral injections, the fluorescence intensity (FI) in iLNs and aLNs account for 80.6% of the total FI, which was significantly higher than unilateral injection (left P=0.0027, right P=0.0001) (Fig. 2f,g). These results indicate that administration at both flanks effectively delivers CII-RM@aCD4Tn to systemic LNs throughout the body, potentially exerting enhance immune modulation. In vivo imaging at different time points after i.d. administration revealed that Cy5-CII-RM@aCD4Tn remained localized near to the injection site for at least one week, whereas free drug (CII + RM) diffused quickly within 4 days (Fig. 2h,i). Quantitative analysis showed that the area under FI curve over one week in CII-RM@aCD4Tn group was 2.8-fold higher than that of free drug group (Supplementary Fig. 14), indicating prolonged local retention of the LNVs. Localization in iLN was further tracked over time. The iLN signal peaked on day 1 and then declined gradually (Fig. 2j,k). On day 7, ~13.1% of the maximal iLN FI remained, falling to near-background levels on day 9 (~3.3%) (Fig. 2k).

For biosafety assessment, male DBA/1 received i.d. injections of the respective formulations (Supplementary Fig. 15a). Blood biochemical analysis demonstrated no significant alterations in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin across all groups (Supplementary Fig. 15b), and routine haematological parameters remained within normal physiological ranges (Supplementary Fig. 15c). Furthermore, histological examination of major organs revealed no evidence of tissue damage or inflammation (Supplementary Fig. 15d). These results indicate no detectable systemic or organ toxicity, supporting the overall biocompatibility of the CII-RM@aCD4Tn nanosystem.

5 Antigen-specific immunosuppression mediated by the DC-CD4 bridging system
Given the induction of tDCs and iCD4 by CII-RM@aCD4Tn, we next examined the generation of Treg cells by this system. Considering the high responsiveness of CD4+ T cells from OT-II transgenic mice to chicken ovalbumin 323 to 339 peptides (OVA), we synthesized OVA loaded NVs (OVA-RM@aCD4Tn) and incubated them with OT-II splenocytes in vitro (Supplementary Fig. 16a). FC analysis revealed an increased proportion of CD25+ Foxp3+ Treg cells after OVA-RM@aCD4Tn treatment, with 1.5-fold elevation compared to OVA-RM@Tn group (Supplementary Fig. 16b,c). Moreover, OVA-RM@aCD4Tn treatment suppressed the level of the pro-inflammatory cytokine TNF and enhanced expression of the anti-inflammatory cytokine TGF-β compared to OVA group (both P<0.0001) (Supplementary Fig. 16d).

To clarify the extent of in vivo immunosuppression, different types of NVs were injected i.d. into OT-II mice at both flanks (Fig. 3a). Although the non-antigen containing formulations (G4 and G5) modestly increased the percentage of Treg cells compared to PBS, OVA-RM@aCD4Tn induced the highest proportions and absolute numbers of CD25+ Foxp3+ Treg cells in the LNs, with 9.7-fold and 1.3-fold higher percentages than PBS and OVA-RM@Tn, respectively (Fig. 3b,c and Supplementary Fig. 17). A similar pattern was observed in the spleen, where the OVA-RM@aCD4Tn increased the Treg cell proportion by 8.4-fold and 1.4-fold compared to PBS and OVA-RM@Tn, respectively (Fig. 3b,d and Supplementary Fig. 18). The immunosuppressive efficacy of this system was further evaluated in DBA/1 mice after i.d. injection of CII-loaded NVs (Supplementary Fig. 19a). Consistently, CII-RM@aCD4Tn yielded the highest numbers and proportions of Treg cells in both LNs and spleen among all treatment groups (Supplementary Fig. 19b,c). Taken together, Ag-RM@aCD4Tn platform robustly expands Treg cells in both LNs and spleen.

To determine whether the induced immunosuppression was antigen-specific, splenocytes isolated from OT-II or DBA/1 mice pre-immunized with different formulations were re-stimulated ex vivo with either OVA or CII, and IFN-γ-producing cells in splenocytes were quantified by ELISPOT (Supplementary Fig. 20a). In OT-II mice, OVA restimulation revealed a marked reduction in IFN-γ-secreting cells after pretreatment with OVA-RM@aCD4Tn (G1 vs G2, P<0.0001), corresponding to a 71.6% decrease (Fig. 3e,f). Pretreatment with CII-RM@aCD4Tn produced a 30.5% reduction, which was significantly weaker than that induced by OVA-RM@aCD4Tn (G2 vs G4, P<0.0001) (Fig. 3e,f). However, CII-RM@aCD4Tn also demonstrated a significant reduction in IFN-γ production compared to CII alone in OT-II mice (G3 vs G4, P=0.0001) (Fig. 3e,f), which is likely attributable to the intrinsic baseline immunosuppressive properties of the iCD4-derived NVs. Conversely, in DBA/1 mice, CII restimulation demonstrated that CII-RM@aCD4Tn produced significantly suppression of IFN-γ-producing cells than OVA-RM@aCD4Tn (G2 vs G4, P<0.0001) (Fig. 3g,h). Consistent with these findings, cytokine levels of TNF and TGF-β in the culture medium were also quantified by ELISA, and the antigen-specific suppression were likewise observed (Supplementary Fig. 20b,c). To directly detect antigen-specific Treg cells, I-Aᵇ OVA 323-339 tetramer was used. Because OT-II CD4⁺ T cells are inherently OVA reactive and exhibit robust baseline tetramer staining even without treatment, the assay was performed in DBA/1 mice. Upon OVA restimulation, OVA-RM@aCD4Tn pretreatment led to an obvious increase in I-Aᵇ OVA 323-339 tetramer⁺ Treg cells compared to free OVA (G1 vs G2, P=0.0027), whereas CII or CII-RM@aCD4Tn pretreatment failed to induce OVA-specific Treg cells (Supplementary Fig. 20d).

Moreover, the functional properties of CII-specific Treg cells were investigated (Supplementary Fig. 21a). CII-specific Treg cells markedly suppressed CII-induced BMDC activation, as evidenced by reduced CD80 and CD86 expression and decreased TNF, together with increased TGF-β levels in the supernatant (G3 vs G5, all P<0.0001) (Fig. 3i and Supplementary Fig. 21b). In contrast, OVA-specific Treg cells exerted only modest effects under the same CII restimulation conditions (Fig. 3i and Supplementary Fig. 21b). Following co-culture, Treg cells from both groups (G4 and G5) showed upregulation of CTLA4 (OVA-specific Treg cells with P=0.0002, CII-specific Treg cells with P<0.0001) and LAG-3 (both P<0.0001) expression compared to unstimulated states, and CTLA4 and LAG-3 expression in CII-specific Treg cells were 1.7-fold and 1.3-fold higher than in OVA-specific Treg cells (Supplementary Fig. 21c), suggesting their greater immunosuppressive activity. Collectively, these data demonstrate that Treg cells induced by Ag-RM@aCD4Tn confer antigen-specific immune tolerance that is engaged upon re-encounter with the cognate antigen.

To further elucidate the immunosuppressive mechanism mediated by OVA-RM@aCD4Tn, we performed transcriptomic analysis on CD4+ T cells isolated from the LNs of OT-II mice 4 days post-administration. Principal components analysis (PCA) revealed a high degree of similarity in gene expression profiles between OVA-RM@Tn and OVA-RM@aCD4Tn groups (Supplementary Fig. 22a). However, the magnitude of gene expression changes in OVA-RM@aCD4Tn group was more pronounced than in OVA-RM@Tn group when compared to OVA group (Supplementary Fig. 22b). In OVA-RM@aCD4Tn-treated mice, the cluster heatmap analysis illustrated a downregulation of pro-inflammatory mediators, including Il6, Tnf, Mmp9, Il1b and Il17a, alongside a upregulation of anti-inflammatory genes such as Il10, Foxp3, Tgfb1, Ctla4 and Il1rn (Fig. 3j), with these regulatory effects being more pronounced than those observed in the OVA-RM@Tn group. Gene ontology (GO) analysis of differentially expressed genes revealed enrichment of biological processes related to immune regulation and inflammatory response after OVA-RM@aCD4Tn intervention (Fig. 3k). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment further supported modulation of immune-related signaling, such as TGF-β, T cell receptor and TNF pathways (Supplementary Fig. 23). Taken together, OVA-RM@aCD4Tn exerted enhanced immunomodulatory program compared to OVA-RM@Tn.

6. Therapeutic and prophylactic efficacy of CII-RM@aCD4Tn in CIA mice
To evaluate the early therapeutic potential of this system, different nanoformulations were administered on day 22, 25 and 28 to treat collagen induced arthritis (CIA) mice (Fig. 4a). At the study endpoint, arthritis severity was assessed via blinded scoring of paw swelling and redness. Mice treated with CII-RM@aCD4Tn exhibited significantly reduced arthritis severity, with arthritis scores decreasing from 12.2 in PBS group to 1 (P<0.0001) (Fig. 4b), indicating substantial alleviation of joint inflammation and edema. When aCD4 was absent from the NV surface (G6), arthritis scores upgraded from 1 to 4.3 (P=0.0074) (Fig. 4b), consistent with an important contribution of DC-CD4 bridging by CII-RM@aCD4Tn. Additionally, whereas PBS group experienced pronounced body weight loss due to disease progression and severe swelling34, this effect was greatly attenuated in CII-RM@aCD4Tn-treated mice, which prevented 86.7% of the body weight loss observed in PBS group (P<0.0001) (Fig. 4c). Measurement of paw thickness further confirmed that CII-RM@aCD4Tn provided the most effective treatment outcome among all groups (Fig. 4d). We subsequently performed behavioral assessment to evaluate motor coordination and balance using a rotarod system. Mice treated with CII-RM@aCD4Tn showed significant improvements in multiple rotarod performance metrics across two training sessions, including fewer falls from the rotating rod and an increased rotating speed at the time of falling compared to PBS (all P<0.0001) (Fig. 4e).

We next assessed the pathological severity of the ankle joint through histological analysis. Hematoxylin and eosin (H&E)-stained sections of ankle joints from CII-RM@aCD4Tn-treated mice revealed nearly restored joint architecture, with a well-defined articular cavity and minimal synovitis or cartilage degeneration (Fig. 4f,g). In contrast, the PBS group showed severe pathological changes, including extensive inflammatory cell infiltration, synovial hyperplasia, pannus formation, and cartilage erosion (Fig. 4f,g). Pannus formation in the synovial intimal lining is a characteristic of the pathological feature of RA35. In immunohistochemical-stained sections, CII-RM@aCD4Tn-treated mice demonstrated the smallest IL-6 positive area in the joint and synovium among all groups, corresponding to a 76.8% reduction compared to PBS group (P<0.0001) (Fig. 4h,i). Masson staining revealed widespread synovial fibrosis in PBS group, whereas the CII-RM@aCD4Tn-treated group exhibited only 12.0% fibrosis area, representing a 94.2% reduction compared to PBS group (Fig. 4j,k). Moreover, Safranin O-Fast Green (SO-FG) staining revealed well-preserved cartilage architecture in CII-RM@aCD4Tn-treated group, with cartilage thickness of 135.5 μm, closely resembling that of healthy group (P>0.9999) (Fig. 4l,m). Since bone erosion is one of the key accountings for disease severity and poor prognosis36, three-dimensional micro-CT imaging was employed to evaluate bone integrity. The bone structures of ankle in PBS group displayed severe bone erosion, rough bone surfaces, and trabecular bone damage in tibia, fibula, and astragalus (Fig. 4n-p and Supplementary Fig. 24). In contrast, CII-RM@aCD4Tn-treated group presented improved bone structure, as reflected by significant improved micro-CT parameters in quantitative analysis of the region of interest (ROI) (G2 vs G7, all P<0.0001) (Fig. 4n-p and Supplementary Fig. 24), suggesting effective protection of bone damage. Overall, these results confirm that CII-RM@aCD4Tn effective alleviate inflammation, cartilage degradation, synovial fibrosis, and bone erosion in CIA mice model, demonstrating its therapeutic efficacy in mitigating RA progression and improving joint function.

RA is pathologically characterized by the infiltration of activated T cells and macrophages into the synovial membrane of multiple joints, where they secrete pro-inflammatory cytokines, including IFN-γ, IL-6, IL-1β and TNF37, 38. These cytokines perpetuate the inflammatory cascades, leading to synovial hyperplasia and subsequent bone erosion. Conversely, immunomodulatory cells, particularly Treg cells, help maintain immune homeostasis by secreting anti-inflammatory cytokines such as IL-10 and TGF-β15, 38. Immunofluorescence analysis revealed a marked increase in the proportion of Foxp3+ Treg cells in the synovium after CII-RM@aCD4Tn treatment (Fig. 5a-c). Moreover, Treg populations in LNs and spleen were further explored using FC analysis. Consistently, CII-RM@aCD4Tn markedly expanded the Treg cell population, with Treg cell numbers increasing 9.7-fold in LNs and 4.7-fold in spleen compared to PBS (Fig. 5d-g and Supplementary Fig. 25), indicating a systemic immunomodulatory effect. At the same time, levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF and IFN-γ) were significantly reduced in both serum and synovium (G2 vs G7, all P≤0.0001), while secretion of IL-10 and TGF-β was markedly upregulated (G2 vs G7, all P<0.0001) (Fig. 5h,i and Supplementary Fig. 26). These results suggest that CII-RM@aCD4Tn reprograms the inflammatory microenvironment towards an immunoregulatory state by promoting Treg cell generation and reducing proinflammatory cytokine levels, thereby helping to restore systemic immune homeostasis.

To assess late-phase therapeutic efficacy in CIA model, CII-RM@Tn or CII-RM@aCD4Tn were administered on day 27, 30 and 33, after mice had developed evident paw swelling and redness (Supplementary Fig. 27a). In line with the early-treatment findings, CII-RM@aCD4Tn treatment significantly alleviated arthritis score (P=0.0001), preserved body weight decline (P<0.0001), reduced paw swelling (P<0.0001) and improved motor ability (except for the numbers of falls in first training with P=0.0006, all others P<0.0001) compared to PBS group (Supplementary Fig. 27b-e). In the meanwhile, treatment with CII-RM@aCD4Tn also improved the system immune environment through decreasing pro-inflammatory cytokines (IL-6, IL-1β, TNF and IFN-γ) and increasing anti-inflammatory cytokines (IL-10 and TGF-β) in both serum and synovium (Supplementary Fig. 27f,g), and promoting the expansion of Treg cell populations in LNs and spleen (Supplementary Fig. 27h-k).

In order to investigate the preventive efficacy of CII-RM@aCD4Tn, mice received injections three and two weeks prior to CIA induction (Supplementary Fig. 28a), representing a preconditioning setting that is intrinsically more favorable than treatment of fully established disease. Following CII-RM@aCD4Tn treatment, mice illustrated normal morphology of paws without signs of redness or swelling, and their body weight recovered nearly to healthy levels (Supplementary Fig. 28b-d). Moreover, mice in CII-RM@aCD4Tn group demonstrated the highest motor ability among all treated groups (Supplementary Fig. 28e), indicating preserved joint function. Pro-inflammatory cytokines in CII-RM@aCD4Tn-treated mice decreased to healthy levels, accompanied by an increase in anti-inflammatory cytokines compared to PBS group (Supplementary Fig. 28f). In addition, both the numbers and proportions of Treg cells in LNs and spleen were the highest among all experimental groups (Supplementary Fig. 28g-j). These results highlight the prophylactic potential of the DC-CD4 bridging system in preventing RA progression, underscoring its feasibility as a vaccine-based immunomodulatory strategy.

7. Mechanistic validation and durable efficacy of CII-RM@aCD4Tn
To further clarify the underlying therapeutic mechanism of CII-RM@aCD4Tn, CIA mice were treated with different formulations (Fig. 6a). Free CII (G3) or free RM (G4) failed to control arthritis progression (Fig. 6a-f and Supplementary Fig. 29). To assess antigen specificity, we included an antigen-nonspecific group, OVA-RM@aCD4Tn (G7). OVA-RM@aCD4Tn showed markedly reduced therapeutic efficacy compared to CII-RM@aCD4Tn, as reflected by higher arthritis scores (P=0.0003) and synovitis scores (P<0.0001), and its overall performance was comparable to that of RM@aCD4Tn (G5) (Fig. 6a-f and Supplementary Fig. 29). When CII was administrated separately together with RM@aCD4Tn (G8), the efficacy remained inferior to that of co-loaded CII-RM@aCD4Tn (Fig. 6a-f and Supplementary Fig. 29), suggesting that simply co-injecting of free antigen with NVs is insufficient to induce strong antigen-specific tolerance. Furthermore, to dissect the functional contribution of CTLA4-mediated DC targeting, CTLA4 blocked NV formulation by neutralizing CTLA4 (G6) was prepared. Although this CTLA4 blocked formulation retained partial therapeutic activity, its efficacy was lower than that of CII-RM@aCD4Tn (G9) (Fig. 6a-f and Supplementary Fig. 29). Indeed, RM@aCD4Tn (G5), CII-RM@aCD4Tn lacking CTLA4 (G6), OVA-RM@aCD4Tn (G7), and CII + RM@aCD4Tn (G8) all exhibited significant therapeutic benefit compared to PBS (Fig. 6a-f and Supplementary Fig. 29).

Because non-intravenous routes such as i.d. injection are associated with an increased risk of anti-drug antibody (ADA) formation39-41, we next quantified serum anti-CII IgG levels by ELISA after three i.d. doses. Free CII elicited a robust humoral response, leading to markedly elevated anti-CII IgG titers (G3 vs G2, P<0.0001; G8 vs G2, P=0.0006) (Fig. 6g). In contrast, when CII was delivered in NV form, the antibody response was substantially attenuated (G6 vs G3, P<0.0001; G9 vs G3, P<0.0001) (Fig. 6g), indicating that NV encapsulation limits systemic immunogenicity.

In order to validate the therapeutic durability of CII-RM@aCD4Tn, a re-challenge study was investigated in CIA mice (Fig. 6h). Even without additional treatment (G3 and G4), mice previously treated with CII-RM@aCD4Tn maintained better disease control than those had received CII-RM@Tn, in which arthritis score increased from 3.7 (day 42) to 7.2 (day 57) (Fig. 6i). Upon redosing on day 43 (G5 and G6), CII-RM@aCD4Tn group conferred the greatest therapeutic benefit, with arthritis score reduced to 1.5, and CII-RM@Tn group displayed a partial improvement, with arthritis score decreasing from 7.2 to 5.0 (Fig. 6i). Changes in paw thickness, behavioral assessments and H&E staining of ankle joints demonstrated consistent trends with the arthritis score (Fig. 6j-m), further supporting the sustained efficacy of CII-RM@aCD4Tn in re-challenge CIA model.

8. Essential role of Foxp3⁺ Treg cells in CII-RM@aCD4Tn-mediated protection against CIA
To evaluate whether CII-RM@aCD4Tn induces a sustained endogenous regulatory response capable of suppressing disease pathogenesis, recipient mice were pretreated twice on day -14 and -21 before receiving arthritogenic cell transfer (Fig. 7a). Prophylactic administration of CII-RM@aCD4Tn largely prevented disease, whereas CII-RM@Tn conferred only moderate effects in attenuating arthritis score (P=0.0098) and paw thickness (P=0.1073), improving motor function (number of falls with P=0.0059; speed at falls with P=0.0205), and limiting bone erosion (P=0.0028) compared to CII-RM@aCD4Tn (Fig. 7b-e and Supplementary Fig. 30a). Histological analysis of ankle joints further confirmed the protective effects of CII-RM@aCD4Tn in this model (Fig. 7f,g and Supplementary Fig. 30b,c). In parallel, systemic immune modulation was improved following CII-RM@aCD4Tn treatment (Fig. 7h).

To investigate the role of Foxp3+ Treg cells in the immunomodulatory mechanism of CII-RM@aCD4Tn, we employed Foxp3+ Treg cell deficient mice as recipients. Following arthritogenic cell transfer, mice preconditioned with CII-RM@aCD4Tn exhibited only limited therapeutic benefit compared to PBS treated mice, with no significant difference compared to CII-RM@Tn groups (all P>0.05) (Fig. 8i-k and Supplementary Fig. 31). These findings collectively indicate that treatment with CII-RM@aCD4Tn induces a durable regulatory response mediated by Foxp3+ Treg cells that can restrain pathogenesis driven by the transferred arthritogenic cells.

To directly test whether Treg cells induced by CII-RM@aCD4Tn are responsible for its therapeutic efficacy, we performed adoptive Treg cells transfer in CIA mice (Fig. 7l). CD4+ CD25+ T cells that include Treg cells were isolated from the LNs and spleen of mice treated with i.d. injections of CII-RM@Tn or CII-RM@aCD4Tn. Cells were sorted based on CD4⁺ CD25⁺ expression alone, without Foxp3⁺ selection, to preserve therapeutic activity, as Foxp3⁺ cell sorting requires membrane permeabilization, which compromises viability and precludes successful adoptive transfer6, 42. When donor cells were derived from normal mice, CD4+ CD25+ T cells derived from CII-RM@aCD4Tn-treated mice could significantly lower arthritis score (P=0.0006) and paw thickness (P=0.0201) and improve motor ability (number of falls with P=0.0014; speed at falls with P=0.0189) compared to CII-RM@Tn-treated group (Fig. 7m,n and Supplementary Fig. 32a). Moreover, these mice exhibited increased numbers and proportions of Treg cells in both LNs and spleen (G2 vs G7, all P<0.0001) (Fig. 7o,p and Supplementary Fig. 32b,c), contributing to immune environment remodeling (Fig. 7q).

To further assess the role of CII-RM@aCD4Tn-mediated Foxp3+ Treg cells, Foxp3+ Treg cell deficient mice were used as donor mice. When adoptive Treg cells from Foxp3+ Treg cell deficient mice which were subjected to CII-RM@aCD4Tn, the therapeutic efficacy against RA was declined, producing effects similar to those observed in CII-RM@Tn group (all P>0.05) (Fig. 7r-t and Supplementary Fig. 33). These data suggest that CD4+ CD25+ Foxp3+ Treg cells induced by CII-RM@aCD4Tn are critical mediators of its immunomodulatory effects.

Methods
Reagents
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol)-2000-NHS (DSPE-PEG2000-NHS, DP-NHS) was purchased from Ponsure Biotech. Rapamycin (RM, Cat# S115842) was sourced from Aladdin. FITC-Rapamycin, FITC-NHS, Cy3-NHS and Cy5-NHS were obtained from Chongqing Yusi Pharmaceutical Technology. Phorbol 12-myristate 13-acetate (PMA, Cat# ab120297) was purchased from Abcam. DiO, DiR, DiI, DAPI, BCA Protein assay kit and Coomassie Blue Fast Staining Solution were all procured from Beyotime. Bovine type II collagen (Cat# 20022), Complete Freund’s Adjuvant (CFA, Cat# 7001), Incomplete Freund Adjuvant (IFA, Cat# 7002), type II collagen detection ELISA kit (Cat# 6018) and Mouse Anti-Bovine Type II Collagen IgG Antibody Assay Kit (Cat# 2032T) were purchased from Chondrex. ELISA kits for murine TNF (Cat# BMS607-3), TGF-β (Cat# BMS608-4), IL-10 (Cat# BMS614), IL-1β (Cat# BMS6002-2), IL-6 (Cat# KMC0061) and IFN-γ (Cat# KMC4021), as well as CountBright absolute counting beads (Cat# C36950) and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Cat# L34966), were purchased from Thermo Fisher Scientific. ELISA kits for murine IL-6 (Cat# JL20268-96RT) was obtained from Jonlnbio. ELISPOT kit for murine IFN-γ (Cat# 2210005) were obtained from Dakewe Biotech. ELISPOT kit for murine TNF (Cat# 430924) were purchased from Biolegend. Diphtheria toxin (Cat # D0564-1MG) was obtained from Sigma Aldrich. EasySep Mouse CD4+ CD25+ Regulatory T Cell Isolation (Cat# 18783), CD4+ T Cell Isolation (Cat# 19852), CD11c+ Selection (Cat# 18780), CD19+ Selection (Cat# 18954), CD11b+ Selection (Cat# 18970) and Neutrophil Enrichment (Cat# 19762) Kits plus the EasySep Magnet (Cat# 18000) were purchased from STEMCELL. CII 259-273, FITC-CII 259-273, OVA 323-339 and primers used for qRT-PCR were obtained from GL biochem.

Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and murine IL-2, IL-7 and TGF-β were acquired from Peprotech. Antibodies against murine CTLA4, CD3 and CD28 were purchased from Bio X Cell. Mouse anti-CD4 Fab fragment was obtained from Creative Biolabs. Recombinant CD4 protein was purchased from MedChemExpress. An anti-IL-6 antibody for immunohistochemistry was purchased from BioLegend. Antibodies against CD4, CD25, Foxp3 and CD11c for immunofluorescence staining were obtained from BioLegend. CD25-PE, CD25-APC, CD45-FITC, CTLA4-APC-Cy7, PD-1-PE, Foxp3-APC, Eα/MHC II-APC, CD80-PE, CD4-APC, CD11c-PE, CD40-APC, MHC II-PE, CD86-APC, CD127-APC, PD-L1-PE antibodies for flow cytometry (FC) assay were purchased from BioLegend. LAG-3-FITC antibodies for FC assay was obtained from Thermo Fisher. The I-Ab-APC monoclonal antibody for FC was obtained from Invitrogen. I-Ab OVA 323-339 Tetramer-PE for FC was obtained from MBL. The working dilutions or concentrations of antibodies used in this study were listed in Supplementary table 1.

Mice
Six- to eight-week-old female C57BL/6 (Stock# C000101) and male DBA/1 (Stock# C000105) mice were purchased from Cavens Experimental Animal Technology. C57BL/6Smoc-Igs2em1(CD2-TCRa(OT-II)-CD2-TCRb(OT-II))Smoc (OT-II, Stock# 004194) mice were purchased from the Jackson Laboratory. C57BL/6Smoc-Foxp3em3(IRES-DTREGFP)Smoc (Foxp3-IRES-DTRGFP, Stock# NM-KI-190046) mice were obtained from Shanghai Model Organisms Center. All animals were housed under a specific pathogen-free facility at 22±2℃ with a relative humidity of 45~55% on a 12-hour light/dark cycle. Experimental and control animals are maintained in the same SPF room under identical husbandry conditions, but are housed in separate cages. All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of Soochow University and conducted in strict accordance with relevant ethical guidelines and regulations.

Induction of CD4+ T cells into iCD4 in vitro
CD4+ T cells were isolated from the spleen and lymph nodes (LN) of mice via magnetic bead based negative separation. Briefly, the inguinal, axillary, and mesenteric LNs, along with the spleen were collected from CO2 euthanized mice. Obtained organs were mechanically dissociated through a 70 μm strainer, and the resulting cell suspensions were centrifuged at 4℃, 600 g for 5 min. After removing red blood cells (RBC) with ACK lysis buffer at room temperature (RT) for 3 min, the remaining cells were purified using the EasySep Mouse CD4+ T Cell Isolation Kit according to the manufacturer’s protocol.

Isolated CD4+ T cells were seeded in 6-well plates and activated with plate-bound antibodies against CD3 (1 μg ml-1) and CD28 (2 μg ml-1) in T cell culture medium, which was RPMI 1640 medium supplemented with 10 % (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin, 2 mM L-glutamine, 20 ng ml-1 IL-2 and 20 ng ml-1 IL-7. For iCD4 differentiation, TGF-β (2-20 ng ml-1) and RM (50 nM) were added59, 60. Cells were cultured for 4 days, and iCD4 induction was verified by FC (Aria IIEI, BD) to detect CD25, Foxp3, CTLA4, LAG-3, and PD-1.

Preparation and characterization of Te and Tn
Extracellular vesicles derived from iCD4 were designated as Te. In brief, induced iCD4 were cultured in 1640 medium containing 10% (v/v) exosome-free FBS and 1% (v/v) penicillin/streptomycin for 24 h. The supernatant was collected and centrifuged at 4℃, 500 g for 10 min to remove debris, followed by another centrifugation at 4℃, 3,000 g for 15 min. Te were pelleted by ultracentrifugation (OPTIMAL-90K, BECKMAN) at 4℃, 100,000 g for 2 h.

Nanovesicles (NV) derived from iCD4 were referred to as Tn. Differentiated iCD4 were centrifuged, washed three times with PBS, and then resuspended in PBS at 1 × 107 cells per milliliter. The suspensions were sequentially extruded through polycarbonate membranes with pore sizes of 5 μm, 1 μm, 400 nm and 200 nm via a liposome extruder (LF-1, Avestin), with 20 reciprocal passes for each pore size. The resulting NV suspensions were collected by ultracentrifugation at 4℃, 100,000 g for 2 h.

Te and Tn were quantified using the BCA Protein Assay Kit. To compare their LN targeting ability, Te and Tn with an equivalent amount of protein were labelled with DiR by co-incubation at 37℃ for 30 min, followed by intradermal (i.d.) injection into both flanks of DBA/1 mice. After 24 h, axillary lymph nodes (aLN) and inguinal lymph nodes (iLN) were isolated for fluorescent intensity (FI) detection by an IVIS imaging system (Lumina Series III, PerkinElmer). In addition, when Te or Tn were derived from an equal number of iCD4, LN targeting was also investigated by the same approach.

Preparation of CII-RM@Tn and CII-RM@aCD4Tn
To obtain CII-RM@Tn, iCD4 at a concentration of 1 × 107 cells per milliliter were mixed with CII (20 μg ml-1) and RM (4 μg ml-1), followed by sequential extrusion using the same protocol as for Tn preparation.

Anti-CD4 Fab fragment (aCD4) engineered CII-RM@aCD4Tn were constructed using a DSPE-PEG platform, which avidly inserts into the NV membranes as its hydrophobic segment61, 62. First, DSPE-PEG2000-aCD4 (DP-aCD4) was synthesized by reacting DP-NHS with aCD4 at various molar ratios of 0:1, 2:1, 4:1, 8:1, 12:1, 16:1 and 20:1, and the pH of the solution was adjusted to 8.5 by 1M NaHCO3. The reaction proceeded on a four-dimensional rotator at RT for 12 h, and the products were purified using 50 kDa ultrafiltration tube to remove unconjugated DP-NHS. Next, to shield CD4 protein on the surface of iCD4, the cells were preincubated with free aCD4 (30 μg per million T cells), and then exposed to DP-aCD4 (50 μg per million T cells) at 37℃ for 30 min. Free aCD4 and DP-aCD4 were removed after centrifugation at 4℃, 600 g for 5 min to collect aCD4 modified iCD4 (aCD4-iCD4). Finally, CII-RM@aCD4Tn were produced by sequential extrusion following the same steps as CII-RM@Tn.

Characterization of CII-RM@aCD4Tn
Protein profiles of iCD4 and Tn were analyzed using 10% SDS-PAGE followed by Coomassie blue staining. To evaluate the anchoring efficiency of DP-aCD4 to CD4+ T cells, Cy3-labelled aCD4 with varying DP molar modification ratios were incubated with CD4+ T cells at 37℃ for 30 min. After centrifugation, the FI of the supernatant was detected by a Microplate reader (CLARIOstar Plus, BMG LABTECH) to calculate the amount of DP-aCD4 inserted into the cell membrane. Cy3-labelled CII-RM@aCD4Tn were incubated with CD4+ T cells at 37℃ for 30 min, followed by FC detection to calculate the conjugation ability. Cy3-labelled CII-RM@Tn or CII-RM@aCD4Tn were incubated with CD4+ T cells for confocal imaging (AX R, Nikon). FITC-labelled RM, Cy3-labelled collagen, and Cy5-labelled aCD4 were employed to fabricate CII-RM@aCD4Tn, followed by confocal detection. Transmission electron microscopy (TEM) was used to assess the morphology of Tn, aCD4Tn, CII-RM@Tn, and CII-RM@aCD4Tn. A particle size analyzer (Zetasizer Lab, Malvern) was employed to measure the hydrodynamic size and zeta potential.

Various feeding ratios of CII (5, 10, 15, 20 μg ml-1) and RM (1, 2, 3, 4 μg ml-1) were tested when synthesizing CII-RM@aCD4Tn. Encapsulation efficiencies and loading capacities of CII were determined by Collagen ELISA kit according to the manufacturer’s protocol. RM content was confirmed by HPLC under the following conditions: column temperature 50℃, mobile phase 10 mM ammonium acetate and acetonitrile (25:75, v/v), flow rate 1.5 ml min-1, and injection volume 5 μl. Before measurement, NVs were disrupted with 0.1% Triton X-100 for 10 min. Drug release profiles were evaluated by dialyzing the samples in dialysis bags (MW cutoff 800 kDa) against PBS at 37℃, with aliquots of the external solution collected over 24 h for quantification.

NVs stability was first examined by measuring the particle size of Tn, aCD4Tn, CII-RM@Tn and CII-RM@aCD4Tn in PBS or 10% FBS at 4℃ over a week. To investigate storage stability, CII-RM@aCD4Tn were maintained at -80, -20, 4 and 25℃ for 0, 7, 14 and 21 days, followed by size measurements. The bioactivity of membrane proteins was tested by incubating stored samples with dye-labelled antibodies against CD25, CTLA4, PD-1, and recombinant CD4 at RT for 15 min, then measuring the FI of the supernatant after ultracentrifugation.

The uptake of CII-RM@aCD4Tn by bone marrow-derived dendritic cells (BMDC) was evaluated. BMDCs were generated from male DBA/1 mice by flushing bone marrow from the femur and tibia with PBS. RBCs were removed by ACK lysis buffer and then filtered through a 70 μm cell strainer to remove debris. The collected cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin, and 20 ng ml-1 GM-CSF to promote differentiation. After 3 days, fresh medium was added. On day 7, BMDCs were harvested and purified using a EasySep Mouse CD11c+ Selection Kit for subsequent experiments.

For NVs uptake and presentation evaluation, CII and RM were labelled with FITC. BMDCs (1 × 106 cells per well in a 6-well plate) were incubated with CII-RM@aCD4Tn or free drug (FITC-CII and FITC-RM) on an orbital shaker at 40 rpm for 12 or 24 h. After that, BMDCs were washed twice with PBS, and stained with DAPI for nuclei and DiI for membrane. Before confocal observation, cells were fixed with 4% paraformaldehyde (PFA) and resuspended in antifade mounting medium. To detect the Eα peptide on MHC II of BMDCs, free Eα plus RM or Eα-RM@aCD4Tn treated BMDCs for 24 h were stained with dye-labelled I-Ab monoclonal antibody, DAPI and DiI for confocal imaging.

Immunosuppression of CII-RM@aCD4Tn on DC and CD4+ T cells
To examine tolerogenic DCs (tDC), BMDCs were co-cultured with different NVs for 48 h and then activated with LPS (50 ng ml-1) for an additional 24 h. tDCs were induced by adding 10 ng ml-1 IL-10 and 10 ng ml-1 TGF-β into the medium for 72 h. Afterward, the expression levels of CD80, CD86, MHC II, CD40 and PD-L1 were detected by FC. The mRNA levels of TNF and TGF-β in the cells were evaluated with the qRT-PCR StepOnePlus Real-Time PCR system (Applied Biosystems, CA). The primers used for mRNA level analysis were listed in Supplementary table 2. In parallel, ELISA was conducted to measure the secretion levels of TNF and TGF-β from BMDCs.

For iCD4 analysis, CD4⁺ T cells were co-cultured with each NVs type for 48 h and stimulated with PMA (20 ng ml-1) for 6 h. Positive iCD4 were induced by supplying with 10 ng ml-1 IL-10 and 10 ng ml-1 TGF-β for 72 h. FC was used to detect CTLA4, CD127 and PD-1 expression, while qRT-PCR and ELISA were performed to measure TNF and TGF-β in cells and culture supernatants, respectively.

Bridging effect of CII-RM@aCD4Tn on CD4+ T cells and DCs
The DC-CD4 bridging ability of CII-RM@aCD4Tn was assessed by co-incubating BMDCs (1 × 106) and CD4⁺ T cells (1 × 106) with either CII-RM@Tn or CII-RM@aCD4Tn at 37 ℃ for 1 h. Cells were then fixed at RT for 30 min, and stained with dye-labelled antibodies against CD4 and CD11c to detect DC-CD4 cell pairs via FC.

To visualize DC-CD4 engagement of CII-RM@aCD4Tn in vivo, Cy5-labelled CII-RM@aCD4Tn were administered i.d. into male DBA/1 mice at both flanks. Mice were CO2 euthanized 24 h later, iLNs were harvested for cryosection, followed by staining with dye-labelled CD11c and CD4 antibodies. These sections were subjected to panoramic scanning (Pannoramic MID-250FLASH-DESK, 3DHISTECH). To precisely identify the cell populations targeted by NVs, iLNs were mechanically dissociated into single cell suspensions and then analyzed by FC for CD4, CD11c, CD11b, Ly6G and CD19.

To assess the contribution of different APC subsets to iCD4 induction, CD11c+ DCs, CD11b⁺ macrophages, Ly6G⁺ neutrophils and CD19⁺ B cells derived from male DBA/1 mice were co-cultured with CD4+ T cells (1 × 106) at a number ratio of 1:1 in the presence of CII-RM@aCD4Tn for 3 days. IL-10 levels in culture medium were quantified by ELISA.

To investigate the interaction mechanism between CII-RM@aCD4Tn and DCs, BMDCs were preincubated with CTLA4 Ig to block surface CD80/86, and CII-RM@aCD4Tn was pretreated with an anti-CTLA4 antibody to mask CTLA4 on the surface of NV. DC-CD4 cell pairs were then quantified by FC.

Biodistribution of CII-RM@aCD4Tn
Cy5-labelled CII-RM@aCD4Tn were injected i.d. into male DBA/1 mice either at one flank or both flanks with the same total dosage. Mice were CO2 euthanized 12 h later, and the heart, liver, spleen, lung, kidney, aLNs and iLNs were harvested for IVIS imaging. To track in vivo release, Cy5-labelled CII plus RM or CII-RM@aCD4Tn were injected i.d. at the right flank of male DBA/1 mice. The FI at the injected site was monitored for up to 7 days by IVIS. Concurrently, the FI in the ipsilateral iLN was monitored for up to 13 days

Safety evaluation of CII-RM@aCD4
For in vivo safety assessment, male DBA/1 mice were randomly assigned to six groups, and each group contained 6 mice. One group was healthy (G1), whereas the other five groups included 150 μg CII + 20 μg RM (G3), 1 mg Tn (G4), 1mg aCD4Tn (G5), 1.2 mg CII-RM@Tn (G6) and 1.2 mg CII-RM@aCD4Tn (G7). Each formulation was injected i.d. on both flanks of mice on day 0 and 3. On day 7, blood was collected from mice in each group for liver function tests and complete blood counts. After CO2 euthanizing mice, the heart, liver, spleen, lung and kidney were harvested, and analyzed by H&E staining to detect any pathological abnormalities.

Antigen-specific T cell induction in vitro and in vivo
Treg cell induction was initially explored in vitro by incubating various NVs with splenocytes. Briefly, splenocytes were harvested from OT-II transgenic mice, in which CD4+ T cells specifically respond to chicken OVA peptide. Therefore, OVA was used as the antigen to fabricate OVA-RM@Tn or OVA-RM@aCD4Tn. Splenocytes were suspended in T cell culture medium and plated in 6-well plates coated with CD3 (1 μg ml-1) and CD28 (2 μg ml-1) antibodies. Different NVs were added for co-incubation for 72 h, followed by OVA (50 μg ml-1) activation for 24 h. After that, cells were collected, and then analyzed by FC for CD45, CD4, CD25 and Foxp3. The levels of TNF and TGF-β in the medium were also quantified by ELISA.

For in vivo Treg cell induction, OVA encapsulated NVs were injected i.d. at both flanks of OT-II mice. Four days later, aLNs, iLNs and spleen were harvested for Treg cell analysis using FC after adding 2 μl counting beads and staining for CD45, CD4, CD25 and Foxp3. For Treg cell induction in wild-type mice, male DBA/1 mice received two i.d. injections of CII loaded NVs at weekly intervals. Seven days after the second dose, mice were immunized i.d. on both sides of the flank with a CII emulsion, prepared by homogenizing CII and CFA at 1:1 volume under ice-cold condition. After one week, aLNs, iLNs and spleen were collected for Treg cell analysis.

To assess antigen-specific tolerance, OT-II and DBA/1 mice were injected i.d. at both flanks with OVA or CII encapsulated NVs. After that, splenocytes were harvested for IFN-γ ELISPOT assays to evaluate the specific activation. Splenocytes were seeded into ELISPOT plates with 1 × 106 cells per well, and stimulated with 10 μg ml-1 OVA or 10 μg ml-1 CII respectively. After 24 h, supernatants were collected for TNF and TGF-β detection by using ELISA kits, and IFN-γ-secreting cells were quantified according to the manufacturer’s instructions. Furthermore, OVA-specific Treg cells in splenocytes from DBA/1 mice were analyzed by staining with I-Ab OVA 323-339 Tetramer.

For functional characterization of CII-specific Treg cells, DBA/1 mice were treated as described above, and CD4⁺ CD25⁺ T cells were isolated and cocultured with BMDCs in the presence of 50 ng ml-1 LPS and 10 μg ml-1 CII. After 24 h, culture supernatants were collected for the measurement of TNF and TGF-β levels, and CD80/CD86 expression on BMDCs and CTLA4 and LAG-3 on Treg cells were analyzed by FC.

RNA sequencing and differential gene enrichment analysis
OVA, OVA-RM@Tn or OVA-RM@aCD4Tn was injected i.d. into both flanks of OT-II mice. After 4 days, CD4+ T cells were isolated from iLNs and aLNs. Total RNA was extracted from the collected CD4+ T cells according to the manufacturer’s instructions. RNA libraries were constructed and pooled based on their effective concentrations. High-quality sequencing data were quantified at the transcript level using HTSeq. RNA sequencing was performed on the MGISEQ-T7 platform, and subsequent bioinformatic analyses were conducted using the online platform (https://www.bioinformatics.com.cn/).

Therapeutics efficacy of CII-RM@aCD4Tn in CIA mice
To establish the collagen induced arthritis (CIA) mouse model, male DBA/1 mice were injected i.d. with 100 μl CII emulsion (CII:CFA=1:1) at the tail base of both sides, followed 21 days later by a boost immunization with CII in IFA. Mice were randomly divided into seven groups, and each group contained 6 mice. One group served as a healthy (G1), whereas the other six groups included PBS (G2), 150 μg CII + 20 μg RM (G3), 1 mg Tn (G4), 1 mg aCD4Tn (G5), 1.2 mg CII-RM@Tn (G6) and 1.2 mg CII-RM@aCD4Tn (G7). Notably, the amounts of CII and RM delivered in G6 and G7 were equivalent to those in G3. Early therapeutic intervention was performed from day 22 to 28 (three injections total, every three days) by i.d. injection at both flanks, and the injected dose was 200 μl per mouse. For late treatment, mice exhibiting paw swelling at day 27 were administered three doses of CII-RM@Tn (G6) or CII-RM@aCD4Tn (G7) every three days from day 27 to 33.

Arthritis score and body weight were recorded every three days. Arthritis score was evaluated according to the following criteria: 0, no evidence of erythema and swelling; 1, erythema and mild swelling confined to the tarsals or ankle joint; 2, erythema and mild swelling extending from the ankle to the tarsals; 3, erythema and moderate swelling extending from the ankle to metatarsal joints; 4, erythema and severe swelling encompassing the ankle, foot and digits, or ankylosis of the limb. Each paw was scored individually, and the maximum score was 16 for each mouse63. On day 42, paw thickness was measured, and rotarod test was conducted. Serum and synovium levels of IL-6, IL-1β, TNF, IFN-γ, IL-10 and TGF-β were quantified using ELISA kits. The count and percentage of Treg cells in spleens and LNs were determined by FC.

Rotarod test
A computerized rotarod apparatus (ZL-200c, Bestcell) was used to assess motor coordination. Mice were initially habituated to the experimental setup by placing them on the stationary rod for a period of 2~3 min daily for two consecutive days, to minimize stress and ensure familiarity with the apparatus. On day 41, mice were placed on the rotating rod at a constant speed of 15 rpm for 3 min, and the number of times they fell off the rod was recorded. Additionally, the speed of rotation was gradually increased from 4 to 20 rpm over 3 min, and the rotating speed was recorded when mice fell. This procedure was repeated on day 42.

Histological and immunohistochemical analysis of inflamed joints
To evaluate RA-related pathological changes, including angiogenesis, synovitis, cartilage destruction and fibrosis, hind ankle joints were harvested at the endpoint and subjected to hematoxylin and eosin (H&E), safranin O-fast green (SO-FG), and Masson staining. H&E sections were scored by three pathologists according to three features of chronic synovitis: enlargement of lining cell layer, cellular density of synovial stroma and leukocytic infiltration. Each feature was semi-quantitatively evaluated on a scale from 0 (absent) to 3 (strong). The sum of these yielded the synovitis score, which was interpreted as follows: 0~1, no synovitis; 2~4, low-grade synovitis; 5~9, high-grade synovitis64. Cartilage thickness in the tibia and talus was measured in SO-FG sections by Image J software (version 1.53c). Fibrotic areas of synovium in Masson sections were quantified by Image J software.

Microcomputed tomography (micro-CT) (SkyScan 1276, Bruker) scans was performed on hind paws according to the following parameters: source current, 385 μA; source voltage, 65 kV; image pixel size, 17.76 µm; vertical object position, 109.456 mm; optical axis, 653 line; filter, Al 1 mm. To compare the therapeutic effects of the different groups, three-dimensional images were reconstructed using Mimics software (Materialize) and the bone volume/total volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), trabecular separation (Tb. Sp), structural model index (SMI), and bone mineral density (BMD) in the tibia were calculated by CTAn software (Skyscan).

Immunohistochemical analysis was also performed to observe the distribution of IL-6 in the synovium of ankle joints, and the IL-6 positive areas was quantified using Image J software. To visualize the distribution of Treg cells in synovium, the hind ankle joints were stained with dye-labelled CD4, CD25 and Foxp3 antibody, followed by Image J software analysis.

Mechanistic investigation of CII-RM@aCD4Tn
CIA mice were established as above method. Mice were randomly divided into nine groups, and each group contained 6 mice. One group served as a healthy (G1), whereas the other eight groups included PBS (G2), 150 μg CII (G3), 20 μg RM (G4), 1 mg RM@aCD4Tn (G5), 1.2 mg CII-RM@aCD4Tn (lacking CTLA4) (G6), 1.2 mg OVA-RM@aCD4Tn (G7), 150 μg CII + 1 mg RM@aCD4Tn (G8) and 1.2 mg CII-RM@aCD4Tn (G9). The amounts of CII and RM were identical across all groups. Notably, CTLA4 on the surface of Tn was blocked via anti-CTLA4 antibody to generate group 6. The NVs were injected on day 22, 25 and 28. Arthritis score and body weight were monitored every three days. Paw thickness measurement and rotarod test were conducted on day 42. Hind paws were collected for H&E staining. In addition, serum samples were obtained on day 42 for quantification of anti-CII IgG antibodies using an ELISA kit.

Re-challenge study of CII-RM@aCD4Tn in CIA mice
To investigate therapeutic durability, DBA/1 mice that had completed the initial three-dose treatment were re-challenged with CII antigen on day 42. Male DBA/1 mice were randomly assigned to six groups, and each group contained 6 mice. One group was healthy (G1), while the other five groups included PBS (G2), 1.2 mg CII-RM@Tn (G3), 1.2 mg CII-RM@aCD4Tn (G4), 1.2 mg CII-RM@Tn with redose (G5) and 1.2 mg CII-RM@aCD4Tn with redose (G6). Each dose of CII-RM@Tn or CII-RM@aCD4Tn contained 150 μg CII and 20 μg RM. On day 42, 100 μl CII emulsion (CII:IFA=1:1) was injected i.d. at both sides of the tail base. On day 43, mice in groups G5 and G6 received a single additional (redose) injection of the corresponding formulation. Arthritis scores and body weight were monitored every three days. Paw thickness and motor performance (rotarod test) were assessed on day 57, and hind paws were collected for histological analysis by H&E staining.

Prophylactic efficacy of CII-RM@aCD4Tn in CIA mice
To assess prophylactic potential, DBA/1 mice were administered NVs prior to CIA induction. Male DBA/1 mice were randomly assigned to seven groups, and each group contained 6 mice. One group was healthy (G1), while the other six groups included PBS (G2), 150 μg CII + 20 μg RM (G3), 1 mg Tn (G4), 1mg aCD4Tn (G5), 1.2 mg CII-RM@Tn (G6) and 1.2 mg CII-RM@aCD4Tn (G7). The amounts of CII and RM delivered in G6 and G7 were equivalent to those in G3. The NVs were injected twice on day -21 and day -14, followed by CIA induction on day 0 and day 21. Arthritis score and body weight were monitored every three days. Paw thickness measurement and rotarod test were conducted on day 42. The levels of IL-6, IL-1β, TNF, IFN-γ, IL-10 and TGF-β in serum and synovium were examined using ELISA kits. The numbers and percentage of Treg cells in spleen and LNs were detected by FC.

Adoptive transfer of arthritogenic cells
CIA model was established in female C57BL/6 mice through two rounds of collagen immunization, which was similar to that in male DBA/1. After 6 days of second immunization, splenocytes were collected and suspended in T cell culture medium. These cells were seeded in plates which were bound with CD3 (1 μg ml-1) and CD28 (2 μg ml-1) antibodies, and activated with 50 μg ml-1 of CII for 72 h to generate arthritogenic cells. Recipient mice received PBS, CII-RM@Tn or CII-RM@aCD4Tn on days -21 and -14 before accepting an i.v. injection of 2 × 10⁷ arthritogenic cells. Each group included 6 mice. Arthritis score, paw thickness and rotarod test were conducted. Right hind paws were collected for micro-CT and H&E, SO-FG and Masson section staining. The levels of IL-6, IL-1β, TNF, IFN-γ, IL-10 and TGF-β in serum and synovium were examined.

To confirm the functional role of Foxp3+ Treg cells, Foxp3-IRES-DTR mice were enrolled as recipient mice, allowing for the specific deletion of Foxp3+ Treg cells in vivo. These mice received 2 μg/kg diphtheria toxin daily by i.v. injection from days -24 to -22, followed by one dose per week until the end time point to ensure complete depletion of Foxp3+ Treg cells. Subsequently, these mice were pretreated with PBS, CII-RM@Tn or CII-RM@aCD4Tn on day -21 and day -14, and then transferred i.v. with 2 × 10⁷ arthritogenic cells. Arthritis score, paw thickness and rotarod test were performed.

Adoptive transfer of regulatory cells
Female C57BL/6 mice were administered i.d. with PBS, CII-RM@Tn or CII-RM@aCD4Tn on day -21 and day -14. Splenocytes were collected 6 days after the second injection, and suspended in T cell culture medium. These cells were stimulated with 50 μg ml-1 CII plus plate-bound anti-CD3 and anti-CD28 for 72 h to generate regulatory cells, and CD4+ CD25+ T cells were isolated using the EasySep Mouse CD4+ CD25+ T Cell Isolation Kit according to the manufacturer’s protocol. After that, 2 × 106 of CD4+ CD25+ T cells were transferred i.v. into recipient mice. One day later, recipient mice underwent CIA induction. Arthritis score, paw thickness and rotarod test were conducted. The levels of IL-6, IL-1β, TNF, IFN-γ, IL-10 and TGF-β in serum and synovium were examined. Treg cells in spleen and LNs were checked by FC.

To confirm the therapeutic role of Foxp3+ Treg cells, Foxp3-IRES-DTR mice were employed as donor mice. These mice were pretreated with PBS, CII-RM@Tn or CII-RM@aCD4Tn on day -21 and -14, and Foxp3+ Treg cells were depleted by three consecutive i.v. injections of 2 μg/kg diphtheria toxin from day -7 to -5. On day -3, splenocytes were harvested and stimulated with 50 μg ml-1 CII for 72 h. After that, isolated CD4+ CD25+ T cells were transferred i.v into recipient mice at 2 × 106 cells per mouse, followed by CIA induction. Arthritis score, paw thickness and rotarod test were performed.

Statistics and reproducibility
All data are presented as means ± SD., and statistical analysis was performed using GraphPad Prism software (version 10.1). Data comparisons were made by unpaired two-tailed t-test, one-way analysis of variance (ANOVA) with Tukey’s post hoc test or two-way ANOVA with Tukey’s post hoc test. All n values reported represent biologically independent replicates. For experiments presenting representative images, the number of independent repeats with similar results is consistent with the biologically independent replicates used for statistical analysis when quantification is provided; for representative image experiments without statistical analysis, experiments are repeated independently at least three times with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the present manuscript, the authors have detailed the general characteristics and initial functional studies with a regulatory CD4+ T cell (Treg)-based extrusion nanoparticle (NP). The primary premise for utilization of this method is to produce a NP that contains both CTLA4 (to targt CD80/CD86+ dendritic cells (DCs)) and anti-CD4 Fab on the surface of the NP (to target CD4+ T cells), while encapsuling rapamycin and antigen (Ag) within the NP. While the data are extensive for characterization of the NP and there are promising data for the functionality of the NP in vivo, there are critical controls and deficiencies within the data sets which preclude publication.\nComments:\n1. In the Abstract the authors imply that the generation of Tregs in vitro for use as a therapeutic is too intricate and that the in vivo induction of Treg in vivo is insufficient for the treatment of autoimmune disease. However, the present methodology first requires the induction and/or expansion of Tregs in vitro, and the goal of this NP therapy is to induce Ag-specific Tregs in vivo. This would appear to be the authors arguing against the translatability of their own therapy. Additionally, Figure 1a presents data showing that the in vitro induction of Tregs is only up to 27% of total T cells. This is an extremely low percentage for in vitro induction cultures. The contour plot presented also indicates that the analysis has compensation issues, as the double-positive FoxP3+/CD25+ population is a thin rocket. This same compensation issue is present in Figure 4b, 6e, and 7h.\n2. In the presentation of Tregs within the spleen and the lymph nodes, the authors use different dating for the presented contour plots. Why is this?\n3. In the present dosing scheme in vivo, the mice receive 3 doses that are separated by 3 days. While the treatment appears to be promising, do the authors know if mice can be redosed at later dates?\n4. Have the authors measured the level of NP-associated Ag antibody production? The injection of NP via the present route of administration is well known to induce significant ADA responses.\n5. The authors present data showing that both rapamycin and the loaded Ag are released from the NP within 12-24hrs in vitro. It would also be presumed that NP of the 150-200nm size with a negative surface charge would be phagocytosed by neutrophils and macrophages. As such, have the authors assessed if any other APC population is targeted by their NP?\n6. Beside DCs, activated monocyte/macrophages, and B cells will express CD80/CD86. This is particularly a confounder with the present hypothesis of only targeting DCs in the therapeutic setting. As an illustration of this, the data present in Figure 3c show that the vast majority of the CII-RM@aCD4n is not close to the CD11c+ stained cells within the lymph node. Do the authors know what other cell type the CII-RM@aCD4n may be interacting within the lymph nodes post injection?\n7. The authors state that bone marrow cells cultured in the presence of GMCSF are DCs. This is incorrect. While there will be a portion of the cells that are DCs, the cultures will be a mixed population of monocytes, macrophages, DCs, and some neutrophils. Did the authors complete any additional selective driving on the cell population or purification of the resultant cell prior to culture in the presence of their NP to support the claim that BMDCs were used in these studies were bonified BMDCs?\n8. In Figure 2g and 2h it is stated that CII-RM@aCD4Tn more readily targeted the Ag to cells as compared to free Ag. This is an artifact of the culture system. The BMDCs will be on the bottom of the well, and the free Ag will be even dispersed throughout the volume of the well. In contrast, the NP will also more readily settle to the bottom of the well, where the BMDCs are located. The present comment is not to refute the presented data, but instead the usable conclusion from this experiment is limited due to the model system used.\n9. What is the total dosage of rapamycin and Ag given per dose of CII-RM@aCD4Tn?\n10. In the disease course data presented in Figures 5 and 7, a critical treatment group is missing. The authors have not included an Ag-non-specific version of Ag-RM@aCD4Tn. Given the authors hypothesis that the use of CTLA4+ NP coated with anti-CD4Fab will specifically pull DCs and CD4+ T cells together, and the data from Supplementary Figure 5b showing that rapamycin is released from the NPs within 12-24hrs, the authors cannot determine if functional decrease in disease severity is truly Ag-specific or if this effect is merely due to more targeted release of rapamycin to CD4+ T cells.\n11. The authors completed tracking studies to determine the location of the NPs post dosing. Beside the draining lymph node, the data show that a significant amount of the NP-associated signal is present within the liver. Have the authors assessed T cell populations within this organ post dosing, as the liver is also held to be a tolerogenic site.\n12. Can the injection of exogenous Ag mixed with RM@aCD4Tn induce the same response as CII- RM@aCD4Tn? Meaning, does the Ag and NP need to be connected locationally for the functional activity? This would be critical for an in vivo setting; this is important given the Ag-release profile of RM@aCD4Tn (Supplementary Figure 5b) and the argument that Ag-specific tolerance is induced.\n13. The authors state that Ag-specific tolerance is induced, but the data used to support this claim are from OTII mice treated with OVA-RM@aCD4Tn and the ex vivo recall cultures compare the OVA ex vivo recall response to the CII ex vivo recall response. As the OTII T cell are TCR transgenic and cannot respond to CII in the absence or presence of OVA-RM@aCD4Tn, this in no way shows that OVA-RM@aCD4Tn treatment is Ag-specific.\n14. On line 288, the authors state that CII-RM@aCD4Tn induced an increase in Ag-specific Tregs. However, the authors did not use tracible TCR transgenic T cells or tetramer staining to support this conclusion. Here again, the authors also did not used an Ag-non-specific RM@aCD4Tn control treatment, as such the authors cannot claim that these cells are Ag-specific Tregs due to the treatment.\n15. In Figure 5a-c, the author claim that treatments G2-G5 made disease severity worse. However, the authors have not included a saline/PBS treatment group. What treatment group are the authors using as the comparator to make this claim?\n16. In Figure 6b and 6c, the percentage of area for FoxP3+ cells is ~0.7%, while the CD4+CD25+FoxP3+ area is ~2%. How is the area increasing by increasing the stringency of marker co-expression for quantifying the cells?\n17. Figure 1b presents a summary of the hypothesis. This should be either removed or placed at the end of the manuscript as part of the Discussion. With the present placement of the figure, this is attempting to lead the reader to make the same conclusion as the author without the reader having been able to objectively review the data first.\n18. Figure 7 and Extended Data Figure 8 are the same figure.\n19. The authors neglect to mention any of the multiple companies and academic laboratories that have Ag-specific therapeutics and Ag-specific NP-based therapeutics that are presently in clinical trials.", "reviewer_2": "This manuscript presents a nanoparticle platform based on Treg cell-derived membrane nanoparticles (Tn) decorated with anti-CD4 (aCD4) antibodies and encapsulated with an antigenic peptide (AP) and rapamycin (RM), designed to induce antigen-specific Tregs for the treatment of rheumatoid arthritis (RA). While the therapeutic potential of the platform is of interest, the manuscript falls short in demonstrating significant advancement over previously published technologies, and the mechanistic claims are not sufficiently supported by the data. Below are specific concerns and suggestions.\n1. This study failed to demonstrate advancement over prior work. A closely related study has previously been reported (Science Advances 8(47), eabo5284, 2022), demonstrating that nanoparticles co-loaded with antigenic peptide and rapamycin can effectively induce tolerogenic dendritic cells (tDCs) and antigen-specific Tregs for treatment of arthritis. The principal advancement claimed in the current study is the addition of CTLA-4 and anti-CD4 antibody to the nanoparticle surface. While some positive effects of anti-CD4 are demonstrated in the manusript, the role of CTLA-4 remains speculative due to the absence of proper in vivo controls. To substantiate the contribution of CTLA-4, the authors should include control nanoparticles lacking CTLA-4 but retaining anti-CD4 decoration. Furthermore, the above-mentioned study should be properly cited and discussed in the Introduction to clarify how the current work advances the field.\n2. The mechanistic claims were not experimentally supported. Figure 1b proposes that AP-RM@aCD4Tn interacts with CD80/86 on DCs via CTLA-4 to induce immune tolerance and promote tDC formation, mediated by both the surface-bound molecules and release of RM and AP. However, the manuscript lacks direct evidence supporting CTLA-4–CD80/86 interactions. Data presented in Figures 2g and 2h show internalization of Cll-RM@aCD4Tn (AP = collagen II) by DCs but do not address surface-level immunological synapse formation. Additionally, while Figure 1b depicts release of rapamycin from the nanoparticle, no data are presented to confirm this, and prior work (Science Advances 8(47), eabo5284, 2022) indicates that rapamycin is typically retained within lipid layers rather than being readily released. These mechanistic assertions require either experimental support or more cautious interpretation.\n3. This study lacks evidence for collagen II-specific Treg induction in vivo. While Figure 6 suggests in vivo Treg induction by Cll-RM@aCD4Tn, the manuscript does not demonstrate that these Tregs are specific to collagen II. This is a critical gap, given that collagen II antigen specificity is central to the therapeutic rationale for RA. The authors are encouraged to perform functional assays to confirm collagen II specificity; e.g., co-culturing isolated Tregs with DCs in the presence of collagen II, followed by proliferation or cytokine profiling assays.\n4. This approach concerns feasibility of clinical translation. The abstract highlights the current challenges associated with ex vivo manipulation of autologous Tregs for autoimmune disease treatment, and proposes Cll-RM@aCD4Tn nanoparticles as a more practical alternative. However, the preparation of such nanoparticles at clinically relevant scales may still require large-scale isolation and manipulation of Tregs, potentially undermining the proposed translational advantage. The authors should address this limitation and provide clarification regarding the scalability and feasibility of their approach.", "reviewer_3": "In this interesting manuscript, the authors report the development of DC-CD4 tolerogenic nanoparticle and apply these to varied mouse models of arthritis to demonstrate their their therapeutic potential and draw conclusions about their bio-distribution and mechanism of action. In general the work appears to be technically rigorous and the findings should be of interest to the readership of Nature Communications. However it would be helpful for the authors to consider the following points:\n1) In the introduction, the authors mention specific limitations to cell-based therapy approaches but that statement is not referenced. It would be useful to cite at least one definitive review on that topic.\n2) The authors tout their tolerogenic DC-CD4 particles as a nano-technology-based strategy that overcomes the limitations of cell-based therapy. However, as outlined in Figure 1, their method still relies on the extraction, manipulation, and re-infusion of cells. Importantly, use of extracellular vesicles in place was less effective. The authors should fully elaborate why their workflow generates a tolerogenic product with fewer limitations than competing methods (both cell based and completely synthetic nanoparticles, as described by Steve Miller and others). Moving beyond inbred mice, DC-CD4 particles could perhaps be manufactured in an autologous or non-autologous manner. Which approach would be the most promising to pursue?\n3) A clearer schematic explaining the sequential steps for making the nanoparticles is needed.\n4) The rationale for comparing Tn vs Te is not clear. Was this an original hope to develop a cell free therapy that did not come to fruition?\n5) It is not obvious why Tn mediated antigen delivery increases surface display of the CII peptide so dramatically. The authors should discuss the mechanism for that increase.\n6) Figure 2n showns mRNA levels of TNFa and TGFb. In the conclusion of this section, it is stated that secretion of anti-inflammatory cytokines is enhanced. Can TNFa and TGFb protein be measured in addition to mRNA levels?\n7) The meaning of the sentence in lines 202-203 is unclear. This should be revised.\n8) The data presented in Figure 3 E and F appears to lack a statistical analysis.\n9) In section 4 of the results, localization of NPs is followed for 7 days. Is localization in the lymph nodes still observed beyond 7 days? Knowing this may be important to discern whether the NPs could function as a one time treatment, or if recurrent treatment would be required.\n10) In section 5, it is unclear why key experiments were performed using Ova 323-339 rather than the CII peptide. Could similar results be obtained with the autoantigenic peptide?\n11) The KEGG pathway data discussed in section 6 does not seem that informative. Are there other published transcript signatures that might be better suited to this analysis?\n12) The fluorescence image in figure 6 A is difficult to read.\n13) in section 7, mice are sacrificed 14 days post treatment. If mice are kept for longer; does the disease come back?\n14) The promising results from different versions of the arthritis model are impressive. However, it must be mentioned that pre-conditioning and prevention settings are easier to mitigate than established disease.\n15) The adoptive transfer experiments are very good work and conclude the result section nicely.\n16) The paper would benefit from having a discussion section, where the discussion points requested above could be included, along with brief comments about further testing and potential translation of this technology.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_6": "The authors address an important problem, that of antigen-specific tolerance, by developing a bispecific nanoparticle delivering antigen peptide and rapamycin to induce tolerogenic DCs, inactive CD4s, and Tregs which leads to increased anti-inflammatory cytokine levels and decreased proinflammatory mediators. They show the colocalization of the nanoparticle with DCs and CD4 T cells, as well as increased and sustained drug localization and the animal experiments are good to support efficacy and immune response to treatment, and support a MOA at least partially related to Tregs induction/expansion. Yet, this manuscript lacks some of the necessary controls and often uses exaggerated or unsubstantiated language. In general there is no evidence for the antigen specificity of this nanoparticle. For example, in section 5, CII stimulation is not an appropriate comparison, as there is not expected to be a reaction; a better control would be a CII-RM@aCD4Tn with OVA stimulation. In subsequent experiments, another control should be RM@aCD4Tn, to look at the impact of the actual antigen peptide on efficacy and/or RM@aCD4Tn loaded with a different peptide than is being tested, and also rapamycin alone in animal experiments. Specific issues are listed below.\n1. Claiming antigen-specific tolerance is a high bar. As currently written, there is not strong enough justification for the use of “antigen-specific” to describe cells. Some more controls as listed above would be appropriate. In addition, some negative controls were not needed. At a minimum, the manuscript needs to be rewritten with accurate language for what they show (including removing antigen specificity until proven, quantifying results, and using precise language for the degree and specificity of changes).\n2. The structure and communication of results limit understanding and the ability to make precise data-driven conclusions.\na. Abstract: isolation and ex vivo manipulation of autologous Treg is listed as a challenge to overcome since it’s “intricate”. Can the authors be more precise about what needs to be improved and how their approach addressed this? The bispecific nanparticles are poorly described in the abstract making interpretation of results difficult. Include metrics of how Treg are measures and quantification of change instead of “potent” without justification. How were increased anti-inflammatory and reduced inflammatory cytokines measured, comparison groups (again, more precise language)?\nb. Throughout, results are claimed to be “robust” or “significant” without justifying this claim. Instead of robust, share x fold change or a p value including the comparison group. All results need to be quantified, comparisons being made need to be accurately described and data is shared without judgement – it’s robustness should be convincing to the reader when you share the actual values.\nc. The title needs to include murine indicating these are all results in mouse or state that it’s a model of RA therapy, not RA itself.\nd. Section titles should be either descriptive or declarative, not a mix.\ne. Discussion: This work adapts technology that is predominantly used in the cancer field in the context of autoimmune disease and shows promise in a mouse model of RA, with some supporting evidence for a Treg dependent MOA. Comparisons should be addressed in the discussion along with application of general concepts about nanoparticles use based on the authors findings. In addition, the discussion should frame all results within the context of the literature. Currently it’s written as a simple summary or findings.\n3. It is argued that Tregs play a role in antigen-specific tolerance induced by the nanoparticles. To make this argument, the authors need to justify the use of different markers of Treg in different experiments, show functional data, and consider Treg tracking experiments (i.e. GFP, adoptive transfer, etc) to directly link tolerance to Treg.\n4. Figure 1 makes claims without showing data. This information should be folded into figures where the hypotheses are tested or used to summarize results once data has proven points.\n5. Treg induction protocol is standard, but is not referenced or compared to others results inducing Treg in other contexts.\n6. CD80/86 is discussed and shown on DC, but in vivo these molecules are expressed on all antigen presenting cells. Either test on multiple cell types or describe experiments and conclusions more precisely.", "reviewer_7": "Manuscript 624613 addresses the topic of antigen-specific therapy for autoimmune disease by utilizing a nanoparticle approach in the setting of collagen-induced arthritis.\nThe experiments and data are extensive and largely supports the text and conclusions, but there are still a number of suggested improvements that would further increase clarity and context.\n1. There is no discussion in the manuscript only a short conclusion section. The presented data need to be compared to other efforts made in this very active field, both in experimental models and in the human disease itself.\n2. The conceptual in vitro data is based on DC, but in mice (or humans) there are other antigen-presenting cells that could participate in activating (or turning off) T cells, this needs to be at least discussed, and the sentence in the introduction (line 44) modified or clarified.\n3. The iCD4 nomenclature is not widely used and would benefit from being introduced and discussed in more detail. Is this a reflection of the Treg differentiation not incorporating a purification step? What is the frequency of Tregs generated? Are the batch-to-batch variation when performing the NP preparation?\n4. Chapter 7: the authors should be careful when to use the phrase ‘therapeutic’, the data presented in figure 5 is ‘preventing’ CIA not ‘treating’. This figure (5) could even be moved into the supplementary data set. In contrast the data in extending figure 7a which is ‘therapeutic’ could be moved in as a main figure.\n5. Figure 6a is not showing any triple positive cells!? This would be important to show.\n6. Extended figure 8 is identical to main figure 7. Probably a mix-up, please check!\n7. Information (methods) and data on the cell transfer experiment (main figure 8) is missing!", "reviewer_8": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Reply to comments from reviewers\nHigh-efficiency induction of antigen-specific regulatory T cells via DC-CD4 bispecific\ntolerogenic nanoparticles for rheumatoid arthritis therapy\n(Nature Communications, NCOMMS-25-47323-T)\nWe appreciate the valuable comments and suggestions from the eight reviewers,\nwhich improved the quality and clarity of the manuscript. We have revised the manuscript\nas suggested. Here, we describe the changes that we made:\nText in italic: comments from the reviewers.\nBlue letters: revised or added figures and text in the revised version.\nReviewer #1\nIn the present manuscript, the authors have detailed the general characteristics and initial\nfunctional studies with a regulatory CD4+ T cell (T )-based extrusion nanoparticle (NP).\nreg\nThe primary premise for utilization of this method is to produce a NP that contains both\nCTLA4 (to target CD80/CD86+ dendritic cells (DCs)) and anti-CD4 Fab on the surface of\nthe NP (to target CD4+ T cells), while encapsuling rapamycin and antigen (Ag) within the\nNP. While the data are extensive for characterization of the NP and there are promising\ndata for the functionality of the NP in vivo, there are critical controls and deficiencies within\nthe data sets which preclude publication.\nComments:\n1. In the Abstract the authors imply that the generation of T in vitro for use as a\nregs\ntherapeutic is too intricate and that the in vivo induction of T in vivo is insufficient for the\nreg\ntreatment of autoimmune disease. However, the present methodology first requires the\ninduction and/or expansion of T in vitro, and the goal of this NP therapy is to induce Ag-\nregs\nspecific T in vivo. This would appear to be the authors arguing against the translatability\nregs\nof their own therapy. Additionally, Figure 1a presents data showing that the in vitro induction\nof T is only up to 27% of total T cells. This is an extremely low percentage for in vitro\nregs\ninduction cultures. The contour plot presented also indicates that the analysis has\ncompensation issues, as the double-positive FoxP3+/CD25+ population is a thin rocket.\nThis same compensation issue is present in Figure 4b, 6e, and 7h.\n2. In the presentation of T within the spleen and the lymph nodes, the authors use\nregs\ndifferent dating for the presented contour plots. Why is this?\n3. In the present dosing scheme in vivo, the mice receive 3 doses that are separated\nby 3 days. While the treatment appears to be promising, do the authors know if mice can\nbe redosed at later dates?\n4. Have the authors measured the level of NP-associated Ag antibody production?\nThe injection of NP via the present route of administration is well known to induce\nsignificant ADA responses.\n5. The authors present data showing that both rapamycin and the loaded Ag are\nreleased from the NP within 12-24hrs in vitro. It would also be presumed that NP of the\n150-200nm size with a negative surface charge would be phagocytosed by neutrophils and\nmacrophages. As such, have the authors assessed if any other APC population is targeted\nby their NP?\n2\n6. Beside DCs, activated monocyte/macrophages, and B cells will express\nCD80/CD86. This is particularly a confounder with the present hypothesis of only targeting\nDCs in the therapeutic setting. As an illustration of this, the data present in Figure 3c show\nthat the vast majority of the CII-RM@aCD4n is not close to the CD11c+ stained cells within\nthe lymph node. Do the authors know what other cell type the CII-RM@aCD4n may be\ninteracting within the lymph nodes post injection?\n7. The authors state that bone marrow cells cultured in the presence of GMCSF are\nDCs. This is incorrect. While there will be a portion of the cells that are DCs, the cultures\nwill be a mixed population of monocytes, macrophages, DCs, and some neutrophils. Did\nthe authors complete any additional selective driving on the cell population or purification\nof the resultant cell prior to culture in the presence of their NP to support the claim that\nBMDCs were used in these studies were bonified BMDCs?\n8. In Figure 2g and 2h it is stated that CII-RM@aCD4Tn more readily targeted the Ag\nto cells as compared to free Ag. This is an artifact of the culture system. The BMDCs will\nbe on the bottom of the well, and the free Ag will be even dispersed throughout the volume\nof the well. In contrast, the NP will also more readily settle to the bottom of the well, where\nthe BMDCs are located. The present comment is not to refute the presented data, but\ninstead the usable conclusion from this experiment is limited due to the model system used.\n9. What is the total dosage of rapamycin and Ag given per dose of CII-RM@aCD4Tn?\n10. In the disease course data presented in Figures 5 and 7, a critical treatment group\nis missing. The authors have not included an Ag-non-specific version of Ag-RM@aCD4Tn.\nGiven the authors hypothesis that the use of CTLA4+ NP coated with anti-CD4Fab will\nspecifically pull DCs and CD4+ T cells together, and the data from Supplementary Figure\n5b showing that rapamycin is released from the NPs within 12-24hrs, the authors cannot\ndetermine if functional decrease in disease severity is truly Ag-specific or if this effect is\nmerely due to more targeted release of rapamycin to CD4+ T cells.\n11. The authors completed tracking studies to determine the location of the NPs post\ndosing. Beside the draining lymph node, the data show that a significant amount of the NP-\nassociated signal is present within the liver. Have the authors assessed T cell populations\nwithin this organ post dosing, as the liver is also held to be a tolerogenic site.\n12. Can the injection of exogenous Ag mixed with RM@aCD4Tn induce the same\nresponse as CII- RM@aCD4Tn? Meaning, does the Ag and NP need to be connected\nlocationally for the functional activity? This would be critical for an in vivo setting; this is\nimportant given the Ag-release profile of RM@aCD4Tn (Supplementary Figure 5b) and the\nargument that Ag-specific tolerance is induced.\n3\n13. The authors state that Ag-specific tolerance is induced, but the data used to\nsupport this claim are from OTII mice treated with OVA-RM@aCD4Tn and the ex vivo recall\ncultures compare the OVA ex vivo recall response to the CII ex vivo recall response. As\nthe OTII T cell are TCR transgenic and cannot respond to CII in the absence or presence\nof OVA-RM@aCD4Tn, this in no way shows that OVA-RM@aCD4Tn treatment is Ag-\nspecific.\n14. On line 288, the authors state that CII-RM@aCD4Tn induced an increase in Ag-\nspecific T . However, the authors did not use tracible TCR transgenic T cells or tetramer\nregs\nstaining to support this conclusion. Here again, the authors also did not used an Ag-non-\nspecific RM@aCD4Tn control treatment, as such the authors cannot claim that these cells\nare Ag-specific T due to the treatment.\nregs\n15. In Figure 5a-c, the author claim that treatments G2-G5 made disease severity\nworse. However, the authors have not included a saline/PBS treatment group. What\ntreatment group are the authors using as the comparator to make this claim?\n16. In Figure 6b and 6c, the percentage of area for Foxp3+ cells is ~0.7%, while the\nCD4+ CD25+ Foxp3+ area is ~2%. How is the area increasing by increasing the stringency\nof marker co-expression for quantifying the cells?\n17. Figure 1b presents a summary of the hypothesis. This should be either removed\nor placed at the end of the manuscript as part of the Discussion. With the present\nplacement of the figure, this is attempting to lead the reader to make the same conclusion\nas the author without the reader having been able to objectively review the data first.\n18. Figure 7 and Extended Data Figure 8 are the same figure.\n19. The authors neglect to mention any of the multiple companies and academic\nlaboratories that have Ag-specific therapeutics and Ag-specific NP-based therapeutics that\nare presently in clinical trials.\nReviewer #2\nThis manuscript presents a nanoparticle platform based on T cell-derived\nreg\nmembrane nanoparticles (Tn) decorated with anti-CD4 (aCD4) antibodies and\nencapsulated with an antigenic peptide (AP) and rapamycin (RM), designed to induce\nantigen-specific T for the treatment of rheumatoid arthritis (RA). While the therapeutic\nregs\npotential of the platform is of interest, the manuscript falls short in demonstrating significant\nadvancement over previously published technologies, and the mechanistic claims are not\n4\nsufficiently supported by the data. Below are specific concerns and suggestions.\n1. This study failed to demonstrate advancement over prior work. A closely related\nstudy has previously been reported (Science Advances 8(47), eabo5284, 2022),\ndemonstrating that nanoparticles co-loaded with antigenic peptide and rapamycin can\neffectively induce tolerogenic dendritic cells (tDCs) and antigen-specific T for treatment\nregs\nof arthritis. The principal advancement claimed in the current study is the addition of CTLA-\n4 and anti-CD4 antibody to the nanoparticle surface. While some positive effects of anti-\nCD4 are demonstrated in the manuscript, the role of CTLA-4 remains speculative due to\nthe absence of proper in vivo controls. To substantiate the contribution of CTLA-4, the\nauthors should include control nanoparticles lacking CTLA-4 but retaining anti-CD4\ndecoration. Furthermore, the above-mentioned study should be properly cited and\ndiscussed in the Introduction to clarify how the current work advances the field.\n2. The mechanistic claims were not experimentally supported. Figure 1b proposes that\nAP-RM@aCD4Tn interacts with CD80/86 on DCs via CTLA-4 to induce immune tolerance\nand promote tDC formation, mediated by both the surface-bound molecules and release\nof RM and AP. However, the manuscript lacks direct evidence supporting CTLA-4–\nCD80/86 interactions. Data presented in Figures 2g and 2h show internalization of Cll-\nRM@aCD4Tn (AP = collagen II) by DCs but do not address surface-level immunological\nsynapse formation. Additionally, while Figure 1b depicts release of rapamycin from the\nnanoparticle, no data are presented to confirm this, and prior work (Science Advances\n8(47), eabo5284, 2022) indicates that rapamycin is typically retained within lipid layers\nrather than being readily released. These mechanistic assertions require either\nexperimental support or more cautious interpretation.\n3. This study lacks evidence for collagen II-specific T induction in vivo. While Figure\nreg\n6 suggests in vivo T induction by Cll-RM@aCD4Tn, the manuscript does not\nreg\ndemonstrate that these T are specific to collagen II. This is a critical gap, given that\nregs\ncollagen II antigen specificity is central to the therapeutic rationale for RA. The authors are\nencouraged to perform functional assays to confirm collagen II specificity; e.g., co-culturing\nisolated T with DCs in the presence of collagen II, followed by proliferation or cytokine\nregs\nprofiling assays.\n4. This approach concerns feasibility of clinical translation. The abstract highlights the\ncurrent challenges associated with ex vivo manipulation of autologous T for\nregs\nautoimmune disease treatment, and proposes Cll-RM@aCD4Tn nanoparticles as a more\npractical alternative. However, the preparation of such nanoparticles at clinically relevant\nscales may still require large-scale isolation and manipulation of T , potentially\nregs\nundermining the proposed translational advantage. The authors should address this\n5\nlimitation and provide clarification regarding the scalability and feasibility of their approach.\nReviewer #3\nIn this interesting manuscript, the authors report the development of DC-CD4\ntolerogenic nanoparticle and apply these to varied mouse models of arthritis to\ndemonstrate their therapeutic potential and draw conclusions about their bio-distribution\nand mechanism of action. In general, the work appears to be technically rigorous and the\nfindings should be of interest to the readership of Nature Communications. However, it\nwould be helpful for the authors to consider the following points:\n1. In the introduction, the authors mention specific limitations to cell-based therapy\napproaches but that statement is not referenced. It would be useful to cite at least one\ndefinitive review on that topic.\n2. The authors tout their tolerogenic DC-CD4 particles as a nano-technology-based\nstrategy that overcomes the limitations of cell-based therapy. However, as outlined in\nFigure 1, their method still relies on the extraction, manipulation, and re-infusion of cells.\nImportantly, use of extracellular vesicles in place was less effective. The authors should\nfully elaborate why their workflow generates a tolerogenic product with fewer limitations\nthan competing methods (both cell based and completely synthetic nanoparticles, as\ndescribed by Steve Miller and others). Moving beyond inbred mice, DC-CD4 particles could\nperhaps be manufactured in an autologous or non-autologous manner. Which approach\nwould be the most promising to pursue?\n3. A clearer schematic explaining the sequential steps for making the nanoparticles is\nneeded.\n4. The rationale for comparing Tn vs Te is not clear. Was this an original hope to\ndevelop a cell free therapy that did not come to fruition?\n5. It is not obvious why Tn mediated antigen delivery increases surface display of the\nCII peptide so dramatically. The authors should discuss the mechanism for that increase.\n6. Figure 2n showns mRNA levels of TNFa and TGFb. In the conclusion of this section,\nit is stated that secretion of anti-inflammatory cytokines is enhanced. Can TNFa and TGFb\nprotein be measured in addition to mRNA levels?\n7. The meaning of the sentence in lines 202-203 is unclear. This should be revised.\n8. The data presented in Figure 3 E and F appears to lack a statistical analysis.\n6\n9. In section 4 of the results, localization of NPs is followed for 7 days. Is localization\nin the lymph nodes still observed beyond 7 days? Knowing this may be important to discern\nwhether the NPs could function as a one time treatment, or if recurrent treatment would be\nrequired.\n10. In section 5, it is unclear why key experiments were performed using Ova 323-339\nrather than the CII peptide. Could similar results be obtained with the autoantigenic peptide?\n11. The KEGG pathway data discussed in section 6 does not seem that informative.\nAre there other published transcript signatures that might be better suited to this analysis?\n12. The fluorescence image in figure 6 A is difficult to read.\n13. In section 7, mice are sacrificed 14 days post treatment. If mice are kept for longer;\ndoes the disease come back?\n14. The promising results from different versions of the arthritis model are impressive.\nHowever, it must be mentioned that pre-conditioning and prevention settings are easier to\nmitigate than established disease.\n15. The adoptive transfer experiments are very good work and conclude the result\nsection nicely.\n16. The paper would benefit from having a discussion section, where the discussion\npoints requested above could be included, along with brief comments about further testing\nand potential translation of this technology.\nReviewer #4\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #5\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nThe authors address an important problem, that of antigen-specific tolerance, by\ndeveloping a bispecific nanoparticle delivering antigen peptide and rapamycin to induce\ntolerogenic DCs, inactive CD4s, and T which leads to increased anti-inflammatory\nregs\ncytokine levels and decreased proinflammatory mediators. They show the colocalization\nof the nanoparticle with DCs and CD4 T cells, as well as increased and sustained drug\nlocalization and the animal experiments are good to support efficacy and immune response\nto treatment, and support a MOA at least partially related to T induction/expansion. Yet,\nregs\nthis manuscript lacks some of the necessary controls and often uses exaggerated or\nunsubstantiated language. In general, there is no evidence for the antigen specificity of this\nnanoparticle. For example, in section 5, CII stimulation is not an appropriate comparison,\nas there is not expected to be a reaction; a better control would be a CII-RM@aCD4Tn\nwith OVA stimulation. In subsequent experiments, another control should be RM@aCD4Tn,\nto look at the impact of the actual antigen peptide on efficacy and/or RM@aCD4Tn loaded\nwith a different peptide than is being tested, and also rapamycin alone in animal\nexperiments. Specific issues are listed below.\n1. (i) Claiming antigen-specific tolerance is a high bar. As currently written, there is not\nstrong enough justification for the use of “antigen-specific” to describe cells.\n(ii) Some more controls as listed above would be appropriate. In addition, some\nnegative controls were not needed.\n(iii) At a minimum, the manuscript needs to be rewritten with accurate language for\nwhat they show (including removing antigen specificity until proven, quantifying results,\nand using precise language for the degree and specificity of changes).\n2. The structure and communication of results limit understanding and the ability to\nmake precise data-driven conclusions.\na. Abstract: isolation and ex vivo manipulation of autologous T is listed as a\nreg\nchallenge to overcome since it’s “intricate”. Can the authors be more precise about what\nneeds to be improved and how their approach addressed this? The bispecific nanoparticles\nare poorly described in the abstract making interpretation of results difficult. Include metrics\nof how T are measures and quantification of change instead of “potent” without\nreg\njustification. How were increased anti-inflammatory and reduced inflammatory cytokines\nmeasured, comparison groups (again, more precise language)?\nb. Throughout, results are claimed to be “robust” or “significant” without justifying this\nclaim. Instead of robust, share x fold change or a p value including the comparison group.\nand data is shared without judgement – it’s robustness should be convincing to the reader\nwhen you share the actual values.\nc. The title needs to include murine indicating these are all results in mouse or state\nthat it’s a model of RA therapy, not RA itself.\nd. Section titles should be either descriptive or declarative, not a mix.\ne. Discussion: This work adapts technology that is predominantly used in the cancer\nfield in the context of autoimmune disease and shows promise in a mouse model of RA,\nwith some supporting evidence for a Treg dependent MOA. Comparisons should be\naddressed in the discussion along with application of general concepts about nanoparticles\nuse based on the authors findings. In addition, the discussion should frame all results within\nthe context of the literature. Currently it’s written as a simple summary or findings.\n3. It is argued that T play a role in antigen-specific tolerance induced by the\nregs\nnanoparticles. To make this argument, the authors need to justify the use of different\nmarkers of T in different experiments, show functional data, and consider T tracking\nreg reg\nexperiments (i.e. GFP, adoptive transfer, etc) to directly link tolerance to T .\nreg\n4. Figure 1 makes claims without showing data. This information should be folded into\nfigures where the hypotheses are tested or used to summarize results once data has\nproven points.\n5. T induction protocol is standard, but is not referenced or compared to others\nreg\nresults inducing T in other contexts.\nreg\n6. CD80/86 is discussed and shown on DC, but in vivo these molecules are expressed\non all antigen presenting cells. Either test on multiple cell types or describe experiments\nand conclusions more precisely.\nReviewer #7\nManuscript 624613 addresses the topic of antigen-specific therapy for autoimmune\ndisease by utilizing a nanoparticle approach in the setting of collagen-induced arthritis. The\nexperiments and data are extensive and largely supports the text and conclusions, but\nthere are still a number of suggested improvements that would further increase clarity and\ncontext.\n1. There is no discussion in the manuscript only a short conclusion section. The\npresented data need to be compared to other efforts made in this very active field, both in\n9\nexperimental models and in the human disease itself.\n2. The conceptual in vitro data is based on DC, but in mice (or humans) there are other\nantigen-presenting cells that could participate in activating (or turning off) T cells, this\nneeds to be at least discussed, and the sentence in the introduction (line 44) modified or\nclarified.\n3. The iCD4 nomenclature is not widely used and would benefit from being introduced\nand discussed in more detail. Is this a reflection of the T differentiation not incorporating\nreg\na purification step? What is the frequency of T generated? Are the batch-to-batch\nregs\nvariation when performing the NP preparation?\n4. Chapter 7: the authors should be careful when to use the phrase ‘therapeutic’, the\ndata presented in figure 5 is ‘preventing’ CIA not ‘treating’. This figure (5) could even be\nmoved into the supplementary data set. In contrast the data in extending figure 7a which\nis ‘therapeutic’ could be moved in as a main figure.\n5. Figure 6a is not showing any triple positive cells!? This would be important to show.\n6. Extended figure 8 is identical to main figure 7. Probably a mix-up, please check!\n7. Information (methods) and data on the cell transfer experiment (main figure 8) is\nmissing!\nReviewer #8\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #1\nIn the present manuscript, the authors have detailed the general characteristics and initial\nfunctional studies with a regulatory CD4+ T cell (T )-based extrusion nanoparticle (NP).\nreg\nThe primary premise for utilization of this method is to produce a NP that contains both\nCTLA4 (to target CD80/CD86+ dendritic cells (DCs)) and anti-CD4 Fab on the surface of\nthe NP (to target CD4+ T cells), while encapsuling rapamycin and antigen (Ag) within the\nNP. While the data are extensive for characterization of the NP and there are promising\ndata for the functionality of the NP in vivo, there are critical controls and deficiencies within\nthe data sets which preclude publication.\nComments:\nQ1: In the Abstract the authors imply that the generation of T in vitro for use as a\nregs\ntherapeutic is too intricate and that the in vivo induction of T in vivo is insufficient for the\nreg\ntreatment of autoimmune disease. However, the present methodology first requires the\ninduction and/or expansion of T in vitro, and the goal of this NP therapy is to induce Ag-\nregs\nspecific T in vivo. This would appear to be the authors arguing against the translatability\nregs\nof their own therapy. Additionally, Figure 1a presents data showing that the in vitro induction\nof T is only up to 27% of total T cells. This is an extremely low percentage for in vitro\nregs\ninduction cultures. The contour plot presented also indicates that the analysis has\ncompensation issues, as the double-positive FoxP3+/CD25+ population is a thin rocket.\nThis same compensation issue is present in Figure 4b, 6e, and 7h.\nA1: We thank the reviewer for this insightful comment and apologize for the confusing\nwording in the original Abstract. Our intention was to summarize the limitations of existing\nT -based therapeutic strategies. Conventional T therapies typically either (i) rely on\nreg reg\nprolonged ex vivo expansion of rare T (7-14 days in clinical practice) for adoptive\nregs\ntransfer, or (ii) use agents to modestly expand T in vivo, which often yields limited\nregs\nantigen specificity and durability. In contrast, our approach uses only a short-term ex vivo\ninduction step (4 days) to generate immunosuppressive CD4⁺ T cells (iCD4) with high\nCTLA4 expression for DC targeting, and the therapeutic effect itself is mediated by efficient\nin vivo induction of antigen-specific T after bispecific nanovesicles (NVs) administration.\nregs\nThus, the short ex vivo step serves to manufacture the iCD4-derived NV product, rather\nthan to provide the primary therapeutic T population.\nreg\nDue to our unclear phrasing, this point was not articulated properly. We have revised\nthe relevant sentence in the Abstract to read as follows (lines 6-7):\n11\n[Abstract section:\nHowever, current approaches either require prolonged ex vivo expansion of rare T\nregs\nor generate insufficient antigen-specific T response in vivo.]\nregs\nTo explicitly acknowledge the remaining need for iCD4 in our platform, we have\nrevised the Discussion to state:\n[Discussion section:\nAlthough the final NV formulation is acellular at the point of administration, its\nmanufacture still requires short-term, ex vivo-induced iCD4 as the source.]\nIn addition, we agree that the percentage of CD4+ CD25+ Foxp3⁺ T obtained under\nregs\nour in vitro induction setting is not high. During method optimization, we observed that\nCD4⁺ T cells upregulated CD25 upon activation, and increasing the concentration of TGF-\nβ indeed led to a gradual increase in Foxp3 expression together with higher levels of\nCTLA4 (Supplementary Fig. 1a-c). However, high TGF-β levels also resulted in markedly\nreduced cell yields (Supplementary Fig. 1d). Our aim in this study was not to generate a\nhighly purified Foxp3⁺ T population in vitro, but rather to obtain CD4⁺ T cells with\nreg\nsufficiently high CTLA4 expression to effectively engage CD80/86 on dendritic cells in the\nbispecific NV design. We therefore selected 10 ng/mL TGF-β as a condition that provided\nan appropriate balance between CTLA4 upregulation and cell recovery, and this condition\nwas used for all subsequent experiments. To more accurately reflect their phenotype, we\ndesignated these induced cells as immunosuppressive CD4⁺ T cells (iCD4) rather than\nT . Regarding batch-to-batch variation, repeated inductions under the same conditions\nregs\nconsistently showed stable upregulation of CTLA4 on iCD4 cells (Supplementary Fig. 1e).\nTo fully clarify this point, we have now included the complete data in Supplementary Fig.\n1.\nThe revised figures and accompanying text read as follows:\na, Representative FC contour plots illustrating CD25+ Foxp3+ T in CD4+ cells after\nregs\ninduction with varying concentrations of TGF-β. b, Percentage of CD25+ Foxp3+ T in\nregs\nCD4+ cells (n=3). c, MFI of CTLA4 after induction in CD4+ T cells (n=3). d, Cell expansion\nfold of iCD4 (n=3). e, MFI of CTLA4 in iCD4 across different batch at 10 ng mL-1 TGF-β\n(n=3). f, Representative FC histograms and quantification of PD-1 and LAG-3 after iCD4\ninduction (n=3). Data are presented as mean ± SD, statistical significance was analyzed\nby One-way ANOVA with Tukey’s post hoc test (b-e) or unpaired two-tailed t-test (f).\n[Methods section:\nInduction of CD4+ T cells into immunosuppressive CD4⁺ T cells (iCD4) in vitro\nFor iCD4 differentiation, TGF-β (2-20 ng ml-1) and RM (50 nM) were added59, 60.\nCells were cultured for 4 days, and iCD4 induction was verified by FC (Aria III, BD) to\ndetect CD25, Foxp3, CTLA4, LAG-3, and PD-1.\nReferences:\n59. Chen, W. et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+\n13\nregulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med.\n198, 1875-1886 (2003).\n60. Battaglia, M., Stabilini, A. & Roncarolo, M.G. Rapamycin selectively expands\nCD4+CD25+FoxP3+ regulatory T cells. Blood 105, 4743-4748 (2005).\nResults section:\niCD4 with stabled high CTLA4 express were successfully induced from CD4+ T cells\nisolated from spleen and LNs, showing approximately threefold expansion at 10 ng mL-1\nTGF-β (Supplementary Fig. 1a-e). Additionally, the key immunomodulatory markers PD-1\n(P<0.0001) and LAG-3 (P=0.0012) were significantly upregulated following stimulation\n(Supplementary Fig. 1f).]\nMoreover, we agree that the double-positive CD25⁺ Foxp3⁺population in the original\nfigures was affected by suboptimal compensation. We have now re-processed the FC data\nand have replaced all relevant plots, including those in Fig. 3b, Fig. 5d,e, Fig. 7o,\nSupplementary Fig. 27h,j, Supplementary Fig. 28g,h and Supplementary Fig. 32b, with\ncorrected versions. We appreciate the reviewer’s careful assessment, which has helped\nus improve the quality and accuracy of our FC analyses.\nQ2: In the presentation of T within the spleen and the lymph nodes, the authors use\nregs\ndifferent dating for the presented contour plots. Why is this?\nA2: We thank the reviewer for raising this point. In the original submission, we used\ntwo different contour plots for spleen and lymph node T to provide varied forms of data\nregs\npresentation. However, we agree that this may introduce unnecessary confusion for\nreaders. To address this issue and ensure consistency, we have now replaced all T\nreg\ncontour plots with uniformly processed Foxp3⁺ CD25⁺ double-positive gating in both spleen\nand lymph nodes. This revision provides a clearer and more coherent presentation of the\ndata. We appreciate the reviewer’s comment, which helped us improve clarity and data\npresentation.\nQ3: In the present dosing scheme in vivo, the mice receive 3 doses that are separated\nby 3 days. While the treatment appears to be promising, do the authors know if mice can\nbe redosed at later dates?\nto further validate the therapeutic durability of our system. To address this question, we\nperformed a re-challenge study in CIA mice. After completing the initial three-dose\ntreatment, mice were challenged with CII antigen on day 42, and disease progression was\nmonitored thereafter (Fig. 6h). Notably, even without additional treatment (G3 and G4),\nmice in CII-RM@aCD4Tn group maintained substantial disease control, whereas mice in\nCII-RM@Tn group exhibited clear disease exacerbation, with the arthritis score increasing\nfrom 3.7 (day 42) to 7.2 (day 57) (Fig. 6i). Upon administration of a single redose on day\n43, CII-RM@aCD4Tn group showed the most pronounced therapeutic benefit, with the\narthritis index reduced to 1.5 (Fig. 6i). In contrast, CII-RM@Tn group displayed a partial\nimprovement, with the arthritis index decreasing from 7.2 to 5.0 (Fig. 6i). Paw thickness,\nbehavioral assessments and H&E staining of ankle joints demonstrated consistent trends\nwith the arthritis score (Fig. 6j-m), further supporting the effectiveness of CII-RM@aCD4Tn\nin re-challenge CIA model.\nWe appreciate the reviewer’s insightful comment, which significantly strengthened our\nstudy. The added figures and accompanying text read as follows:\nFig. 6 | h, Experimental scheme of re-challenge on day 42. i, Arthritis score curve (n=6). j,\nPaw thickness on day 57 (n=6). k, Motor evaluation of CIA mice on day 57 using rotarod\nsystem (n=6). l,m, Representative H&E section images of ankle (l) and histopathological\nevaluation of synovitis score (m) (n=6).\n15\n[Methods section:\nRe-challenge study of CII-RM@aCD4Tn in CIA mice\nTo investigate therapeutic durability, DBA/1 mice that had completed the initial three-\ndose treatment were re-challenged with CII antigen on day 42. Male DBA/1 mice were\nrandomly assigned to six groups, and each group contained 6 mice. One group was healthy\n(G1), while the other five groups included PBS (G2), 1.2 mg CII-RM@Tn (G3), 1.2 mg CII-\nRM@aCD4Tn (G4), 1.2 mg CII-RM@Tn with redose (G5) and 1.2 mg CII-RM@aCD4Tn\nwith redose (G6). Each dose of CII-RM@Tn or CII-RM@aCD4Tn contained 150 μg CII and\n20 μg RM. On day 42, 100 μl CII emulsion (CII:IFA=1:1) was injected i.d. at both sides of\nthe tail base. On day 43, mice in groups G5 and G6 received a single additional (redose)\ninjection of the corresponding formulation. Arthritis scores and body weight were monitored\nevery three days. Paw thickness and motor performance (rotarod test) were assessed on\nday 57, and hind paws were collected for histological analysis by H&E staining.\nResults section:\nIn order to validate the therapeutic durability of Cll-RM@aCD4Tn, a re-challenge study\nwas investigated in CIA mice (Fig. 6h). Even without additional treatment (G3 and G4),\nmice previously treated with CII-RM@aCD4Tn maintained better disease control than\nthose had received CII-RM@Tn, in which arthritis score increased from 3.7 (day 42) to 7.2\n(day 57) (Fig. 6i). Upon redosing on day 43 (G5 and G6), CII-RM@aCD4Tn group\nconferred the greatest therapeutic benefit, with arthritis score reduced to 1.5, and CII-\nRM@Tn group displayed a partial improvement, with arthritis score decreasing from 7.2 to\n5.0 (Fig. 6i). Changes in paw thickness, behavioral assessments and H&E staining of ankle\njoints demonstrated consistent trends with the arthritis score (Fig. 6j-m), further supporting\nthe sustained efficacy of CII-RM@aCD4Tn in re-challenge CIA model.]\nQ4: Have the authors measured the level of NP-associated Ag antibody production?\nThe injection of NP via the present route of administration is well known to induce\nsignificant ADA responses.\nA4: We thank the reviewer for this important comment. Because intradermal\nadministration is prone to eliciting anti-drug antibody (ADA) responses and our NVs\nencapsulate CII, we specifically evaluated CII-specific humoral responses in CIA mice after\nthe three-dose treatment regimen (Fig. 6a). Serum anti-CII IgG titers were quantified by\nELISA. This analysis was performed within the same experimental cohort used to address\nReviewer #1 (Q4, Q10, Q12), Reviewer #2 (Q1), and Reviewer #6 (Q1).\nanti-CII IgG levels in groups G3 and G8 (Fig. 6f). In contrast, when CII was delivered in the\nNV formulation, anti-CII antibody responses were substantially attenuated in groups G6\nand G9 (Fig. 6f). We attribute this reduction to antigen encapsulation within the NVs, which\nlikely limits direct antigen exposure and systemic immunogenicity. These data indicate that\nNV-associated CII elicits significantly lower CII-specific antibody production than free CII\nunder identical dosing conditions.\nThe added figure and text read as follows:\nFig. 6 | a, Experimental scheme of CIA mice treatment with various formulations. g, The\nlevel of anti-CII IgG antibody in serum (n=6).\n[Results section:\nBecause non-intravenous routes such as i.d. injection are associated with an\nincreased risk of anti-drug antibody (ADA) formation39-41, we next quantified serum anti-CII\nIgG levels by ELISA after three i.d. doses. Free CII elicited a robust humoral response,\nleading to markedly elevated anti-CII IgG titers (G3 vs G2, P<0.0001; G8 vs G2, P=0.0006)\n(Fig. 6g). In contrast, when CII was delivered in NV form, the antibody response was\nsubstantially attenuated (G6 vs G3, P<0.0001; G9 vs G3, P<0.0001) (Fig. 6g), indicating\nthat NV encapsulation limits systemic immunogenicity.\nReferences\n39. Srinoulprasert, Y. et al. A Pilot Study of Differences in Antibody Responses of\nIntradermal and Intramuscular Injections of Botulinum Toxin Type A. Dermatol Ther\n(Heidelb) 15, 3347-3360 (2025).\n40. Schellekens, H. Bioequivalence and the immunogenicity of biopharmaceuticals.\nNat. Rev. Drug Discov. 1, 457-462 (2002).\n41. Valenzuela, F. & Flores, R. Immunogenicity to biological drugs in psoriasis and\npsoriatic arthritis. Clinics (Sao Paulo) 76, e3015 (2021).]\nreleased from the NP within 12-24hrs in vitro. It would also be presumed that NP of the\n150-200nm size with a negative surface charge would be phagocytosed by neutrophils and\nmacrophages. As such, have the authors assessed if any other APC population is targeted\nby their NP?\nA5: We thank the reviewer for this insightful question. To determine whether additional\nAPC populations targeted by NVs in vivo, we labeled Cll-RM@aCD4Tn with Cy5 and\nexamined Cy5⁺ APC subsets in the draining LNs 24 h after i.d. administration in CIA mice.\nAmong Cy5⁺ CD45⁺ cells, NVs were predominantly associated with CD11c⁺ DCs (31.3%),\nfollowed by CD19⁺ B cells (11.7%), whereas uptake by CD11b⁺ macrophages (7.9%) and\nLy6G⁺ neutrophils (3.2%) was comparatively low. These data support our conclusion that\nDCs represent the principal APC population engaging CII-RM@aCD4Tn, while other APC\nsubsets make a more modest contribution.\nThe added figure and text read as follows:\nSupplementary Fig. 12. a, Cll-RM@aCD4Tn targeted cells in the iLNs of CIA mice after\n24 h i.d. administration (n=5).\n[Methods section:\nBridging effect of CII-RM@aCD4Tn on CD4+ T cells and DCs\nTo precisely identify the cell populations targeted by NVs, iLNs were mechanically\ndissociated into single cell suspensions and then analyzed by FC for CD4, CD11c, CD11b,\nLy6G and CD19.\nResults section:\nTo further identify the in vivo cellular targets, Cy5-Cll-RM@aCD4Tn-associated cells\nwere analyzed in iLN. Among Cy5⁺ CD45⁺ leukocytes, NVs were predominantly targeted\nto CD4+ T cells (44.8%) and CD11c⁺ DCs (31.3%), followed by CD19⁺ B cells (11.7%),\n18\nwhereas uptake by CD11b⁺ macrophages (7.9%) and Ly6G⁺ neutrophils (3.2%) was\ncomparatively low (Supplementary Fig. 12a).]\nQ6: Beside DCs, activated monocyte/macrophages, and B cells will express\nCD80/CD86. This is particularly a confounder with the present hypothesis of only targeting\nDCs in the therapeutic setting. As an illustration of this, the data present in Figure 3c show\nthat the vast majority of the CII-RM@aCD4n is not close to the CD11c+ stained cells within\nthe lymph node. Do the authors know what other cell type the CII-RM@aCD4n may be\ninteracting within the lymph nodes post injection?\nA6: We appreciate the reviewer’s insightful comment. This question is closely related\nto Q5, and we address both using the same flow cytometric (FC) analysis. To delineate\nwhich APC or lymphocyte subsets interact with CII-RM@aCD4Tn in vivo, we analyzed\niLNs after 24 h i.d. injection in CIA mice. The results showed that the NVs were most\nfrequently associated with CD4⁺ T cells (44.8%) and CD11c⁺ dendritic cells (31.3%). A\nsmaller but detectable fraction associated with CD19⁺ B cells (11.7%), whereas uptake by\nCD11b⁺ macrophages (7.9%) and Ly6G⁺ neutrophils (3.2%) was relatively modest. These\ndata indicate that, in inflamed LNs, CII-RM@aCD4Tn primarily engages CD4⁺ T cells and\nDCs, with more limited interaction with other APC subsets. This pattern is consistent with\nthe DC-CD4 based bispecific design of the NVs and explains why only a proportion of\nparticles appear in close proximity to CD11c⁺ cells in the images shown in Fig. 3c (now\nrepresented as Fig. 2c).\nQ7: The authors state that bone marrow cells cultured in the presence of GMCSF are\nDCs. This is incorrect. While there will be a portion of the cells that are DCs, the cultures\nwill be a mixed population of monocytes, macrophages, DCs, and some neutrophils. Did\nthe authors complete any additional selective driving on the cell population or purification\nof the resultant cell prior to culture in the presence of their NP to support the claim that\nBMDCs were used in these studies were bonified BMDCs?\nA7: We thank the reviewer for this important point and fully agree that GM-CSF\ncultured bone marrow cells constitute a heterogeneous population rather than exclusively\nDCs. To ensure that bona fide BMDCs were used in our experiments, we performed an\nadditional purification step prior to NV incubation. Specifically, CD11c⁺ cells were isolated\nusing a CD11c microbead-based magnetic selection kit, and only the purified CD11c⁺\nfraction was used for subsequent in vitro studies.\nreads as follows:\n[Methods section:\nOn day 7, BMDCs were harvested and purified using a EasySep™ Mouse CD11c+\nSelection Kit for subsequent experiments.]\nQ8: In Figure 2g and 2h it is stated that CII-RM@aCD4Tn more readily targeted the\nAg to cells as compared to free Ag. This is an artifact of the culture system. The BMDCs will\nbe on the bottom of the well, and the free Ag will be even dispersed throughout the volume\nof the well. In contrast, the NP will also more readily settle to the bottom of the well, where\nthe BMDCs are located. The present comment is not to refute the presented data,\nbut instead the usable conclusion from this experiment is limited due to the model system\nused.\nA8: We thank the reviewer for this thoughtful comment. We agree that, in static culture,\nfree antigen is more likely to remain evenly dispersed in the medium, whereas NVs tend\nto settle more rapidly to the bottom of the well, potentially influencing the uptake efficiency\nby BMDCs. To address this concern, we re-performed the experiment under dynamic\nconditions by placing the 6-well plates on an orbital shaker at 40 rpm during incubation to\nminimize sedimentation effects (Reviewer Fig. 1). Under shaking conditions, uptake of free\nantigen by BMDCs indeed increased (approximately a 4-fold enhancement compared to\nstatic culture). However, the uptake remained lower than that observed with CII-\nRM@aCD4Tn (Fig. 1f,g). This result suggests that although sedimentation influences the\nabsolute uptake levels, the higher interaction efficiency of the NVs by BMDCs persists even\nwhen sedimentation is minimized. Thus, the enhanced antigen delivery observed with CII-\nRM@aCD4Tn reflects the intrinsically greater uptake efficiency of the NV formulation. The\nenhanced uptake by BMDCs is mainly attributable to more efficient ligand-mediated\ninteractions (CTLA4-CD80/86) between CII-RM@aCD4Tn and BMDCs (Supplementary\nFig. 10).\nReviewer Fig. 1 Uptake of BMDCs evaluated under orbital shaking conditions.\ndata in the main text to reflect the use of orbital shaking conditions in these experiments.\nThe added figures and accompanying text read as follows:\nFig.1 | f,g, Confocal images (f) and FC analysis (g) of in vitro BMDCs uptake of Cll-\nRM@aCD4Tn or free drug (n=5). Scale bars, 5 μm. h,i, Confocal images (h) and FC\nanalysis (i) exhibited in vitro BMDCs presentation of antigen (CII) (n=5). Scale bars, 5 μm.\n[Methods section:\nBMDCs (1 × 10⁶ cells per well in a 6-well plate) were incubated with CII-RM@aCD4Tn\nor free drug (CII and RM) on an orbital shaker at 40 rpm for 12 or 24 h.\nResults section:\nTo determine the efficiency of Tn in enhancing the delivery of drugs (CII and RM) to\nDCs, FITC-labelled free drugs or CII-RM@aCD4Tn were incubated with bone marrow-\nderived dendritic cells (BMDCs) on an orbital shaker at 40 rpm for 12 h. While all cells\ninternalized CII and RM, drug uptake was 2.7-fold higher when the agents were\nencapsulated in Tn compared to the free form (Fig. 1f,g). The enhanced uptake by BMDCs\nare mainly attributable to more efficient ligand-mediated interactions between CII-\nRM@aCD4Tn and BMDCs. To assess antigen (CII) presentation on DC surface, FITC-\nlabelled CII was incubated with BMDCs on an orbital shaker for 24 h. CII-RM@aCD4Tn\nfacilitated antigen presentation, increasing surface displayed CII levels by 3.1-fold\ncompared to free CII (Fig. 1h,i).]\nA9: Each dose of CII-RM@aCD4Tn contained 150 μg of CII and 20 μg of rapamycin\n(RM). For clarity, we have now included these dosing details in Methods sections of the\nrevised manuscript.\nThe revised texts read as follows:\n[Methods section:\nTherapeutics efficacy of Cll-RA@aCD4Tn on CIA mice\nOne group served as a healthy (G1), whereas the other six groups included RA (G2),\n150 μg CII + 20 μg RM (G3), 1 mg Tn (G4), 1 mg aCD4Tn (G5), 1.2 mg CII-RM@Tn (G6)\nand 1.2 mg CII-RM@aCD4Tn (G7). Notably, the amounts of CII and RM delivered in G6\nand G7 were equivalent to those in G3.\nMechanistic investigation of CII-RM@aCD4Tn\nOne group served as a healthy (G1), whereas the other eight groups included PBS\n(G2), 150 μg CII (G3), 20 μg RM (G4), 1 mg RM@aCD4Tn (G5), 1.2 mg CII-RM@aCD4Tn\n(lacking CTLA4) (G6), 1.2 mg OVA-RM@aCD4Tn (G7), 150 μg CII + 1 mg RM@aCD4Tn\n(G8) and 1.2 mg CII-RM@aCD4Tn (G9). The amounts of CII and RM were identical across\nall groups.\nProphylactic efficacy of CII-RM@aCD4Tn on CIA mice\nOne group was healthy (G1), while the other six groups included RA (G2), 150 μg CII\n+ 20 μg RM (G3), 1 mg Tn (G4), 1mg aCD4Tn (G5), 1.2 mg CII-RM@Tn (G6) and 1.2 mg\nCII-RM@aCD4Tn (G7). The amounts of CII and RM delivered in G6 and G7 were\nequivalent to those in G3.\nRe-challenge study of CII-RM@aCD4Tn in CIA mice\nOne group was healthy (G1), while the other five groups included PBS (G2), 1.2 mg\nCII-RM@Tn (G3), 1.2 mg CII-RM@aCD4Tn (G4), 1.2 mg CII-RM@Tn with redose (G5)\nand 1.2 mg CII-RM@aCD4Tn with redose (G6). Each dose of CII-RM@Tn or CII-\nRM@aCD4Tn contained 150 μg CII and 20 μg RM.]\nQ10: In the disease course data presented in Figures 5 and 7, a critical treatment group\nis missing. The authors have not included an Ag-non-specific version of Ag-\nRM@aCD4Tn. Given the authors hypothesis that the use of CTLA4+ NP coated with anti-\nCD4Fab will specifically pull DCs and CD4+ T cells together, and the data from\nSupplementary Figure 5b showing that rapamycin is released from the NPs within 12-24hrs,\n22\nthe authors cannot determine if functional decrease in disease severity is truly Ag-specific\nor if this effect is merely due to more targeted release of rapamycin to CD4+ T cells.\nA10: We thank the reviewer for highlighting this important point. We agree that the\noriginal study design lacked several critical control groups. To address this, we performed\nan additional set of animal experiments within the same cohort used to address Reviewer\n#1 (Q4, Q10, Q12), Reviewer #2 (Q1), and Reviewer #6 (Q1).\nTo determine whether the therapeutic effect of CII-RM@aCD4Tn is antigen-specific in\nthe CIA model, we included an antigen-nonspecific group, OVA-RM@aCD4Tn (G7). The\nresults showed that OVA-RM@aCD4Tn exhibited markedly reduced therapeutic efficacy,\nfailing to suppress paw swelling, restore motor function or alleviate paw inflammation, and\nits overall performance was comparable to RM@aCD4Tn (G5) (Fig. 6a-f and\nSupplementary Fig. 29). These findings demonstrate that antigen-nonspecific NVs do not\ngenerate meaningful therapeutic benefit, confirming the antigen specificity of CII-\nRM@aCD4Tn in CIA model.\nFurthermore, to dissect the functional contribution of CTLA4 in mediating DC targeting,\nwe prepared a formulation in which CTLA4 activity was blocked during NV preparation by\nusing an anti-CTLA4 antibody (G6), allowing the particles primarily to target CD4⁺ T cells.\nAlthough this CTLA4-blocked formulation retained partial therapeutic activity, its efficacy\nwas significantly lower than that of CII-RM@aCD4Tn (G9) (Fig. 6a-f and Supplementary\nFig. 27b-f). These data support the conclusion that functional CTLA4 engagement, and\nthus DC targeting, is critical for achieving the full therapeutic effect.\nCollectively, the newly added groups support that the reduction in disease severity\nresults from antigen-specific immune modulation, together with the coordinated dual\ntargeting of DCs and CD4⁺ T cells, rather than from CD4⁺ T cell delivery alone.\nThe added figures and accompanying text read as follows:\nscore curve (n=6). c, Paw thickness on day 42 (n=6). d, Motor evaluation of CIA mice on\nday 42 using rotarod system (n=6). e,f, Representative H&E section images of ankle (e)\nand histopathological evaluation of synovitis score (f) (n=6). g, The level of anti-CII IgG\nantibody in serum (n=6).\nSupplementary Fig. 29. Speed at falls of CIA mice on day 42 using rotarod system.\nData are presented as mean ± SD (n=6), statistical significance was analyzed by One-\nway ANOVA with Tukey’s post hoc test.\n[Methods section:\nMechanistic investigation of CII-RM@aCD4Tn\nCIA mice were established as above method. Mice were randomly divided into nine\ngroups, and each group contained 6 mice. One group served as a healthy (G1), whereas\n24\nthe other eight groups included PBS (G2), 150 μg CII (G3), 20 μg RM (G4), 1 mg\nRM@aCD4Tn (G5), 1.2 mg CII-RM@aCD4Tn (lacking CTLA4) (G6), 1.2 mg OVA-\nRM@aCD4Tn (G7), 150 μg CII + 1 mg RM@aCD4Tn (G8) and 1.2 mg CII-RM@aCD4Tn\n(G9). The amounts of CII and RM were identical across all groups. Notably, CTLA4 on the\nsurface of Tn was blocked via anti-CTLA4 antibody to generate group 6. The NVs were\ninjected on day 22, 25 and 28. Arthritis score and body weight were monitored every three\ndays. Paw thickness measurement and rotarod test were conducted on day 42. Hind paws\nwere collected for H&E staining. In addition, serum samples were obtained on day 42 for\nquantification of anti-CII IgG antibodies using an ELISA kit.\nResults section:\nTo further clarify the underlying therapeutic mechanism of Cll-RM@aCD4Tn, CIA mice\nwere treated with different formulations (Fig. 6a). Free CII (G3) or free RM (G4) failed to\ncontrol arthritis progression (Fig. 6a-f and Supplementary Fig. 29). To assess antigen\nspecificity, we included an antigen-nonspecific group, OVA-RM@aCD4Tn (G7). OVA-\nRM@aCD4Tn showed markedly reduced therapeutic efficacy compared to CII-\nRM@aCD4Tn, as reflected by higher arthritis scores (P=0.0003) and synovitis scores\n(P<0.0001), and its overall performance was comparable to that of RM@aCD4Tn (G5) (Fig.\n6a-f and Supplementary Fig. 29). When CII was administrated separately together with\nRM@aCD4Tn (G8), the efficacy remained inferior to that of co-loaded CII-RM@aCD4Tn\n(Fig. 6a-f and Supplementary Fig. 29), suggesting that simply co-injecting of free antigen\nwith NVs is insufficient to induce strong antigen-specific tolerance. Furthermore, to dissect\nthe functional contribution of CTLA4-mediated DC targeting, CTLA4 blocked NV\nformulation by neutralizing CTLA4 (G6) was prepared. Although this CTLA4 blocked\nformulation retained partial therapeutic activity, its efficacy was lower than that of CII-\nRM@aCD4Tn (G9) (Fig. 6a-f and Supplementary Fig. 29).]\nQ11: The authors completed tracking studies to determine the location of the NPs post\ndosing. Beside the draining lymph node, the data show that a significant amount of the NP-\nassociated signal is present within the liver. Have the authors assessed T cell populations\nwithin this organ post dosing, as the liver is also held to be a tolerogenic site.\nA11: We thank the reviewer for raising this important question. Although the IVIS\nimages appear to show a fluorescent signal in the liver, this does not reflect substantial NV\naccumulation. Intradermal (i.d.) administration is well known to preferentially direct NV into\nthe lymphatic system, resulting in limited entry into the bloodstream and consequently low\nexposure to hepatic tissues1-3. Consistent with this, immunofluorescence imaging of liver\n25\nsections showed no detectable Cll-RM@aCD4Tn signal (Reviewer Fig. 2), supporting the\nabsence of meaningful hepatic accumulation.\nReviewer Fig. 2 Immunofluorescence images of liver sections. Scale bars, 100 μm.\nBlue, DAPI; Green, CD4; Yellow, Cy5-Cll-RM@aCD4Tn.\nThe apparently elevated liver signal in IVIS analysis is largely explained by the use of\ntotal fluorescence intensity (FI), which is strongly influenced by organ size. Because the\nliver is considerably larger than other harvested organs, even background\nautofluorescence results in high Total FI values. Indeed, the PBS group exhibited a similar\nliver fluorescence intensity (~4.2 × 10⁹) compared to the CII-RM@aCD4Tn group (~4.9 ×\n10⁹), indicating that the observed signal is predominantly due to tissue size dependent\nbackground rather than NV distribution. To more accurately represent biodistribution, we\nreprocessed the organ fluorescence data by subtracting the PBS background from each\norgan, which clearly revealed that NV accumulation in the liver is minimal, whereas LNs\nremain the primary target organs following i.d. injection (Fig. 2d-g and Supplementary Fig.\n13).\nThe revised figures and accompanying text read as follows:\n12 h. e, Relative fluorescence intensity (FI) of main organs (n=5). f, The proportion of FI in\neach organ after both side injection (n=5). g, Relative FI of total LNs (n=5).\nSupplementary Fig. 13. The proportion of FI in each organ following unilateral\ninjection.\n[Results section:\nTo assess LN-targeting capability, Cll-RM@aCD4Tn was administered via i.d. injection\ninto the left or right flank of DBA/1 mice. Following unilateral injection, in vivo fluorescence\nimaging revealed that the Cy5-NVs localized exclusively to the ipsilateral inguinal and\naxillary LNs (aLN), with minimal signal detected in major organs (Fig. 2d,e and\nSupplementary Fig. 13). Following bilateral injections, the fluorescence intensity (FI) in\niLNs and aLNs account for 80.6% of the total FI, which was significantly higher than\nunilateral injection (left P=0.0027, right P=0.0001) (Fig. 2f,g). These results indicated that\nadministration at both flanks effectively delivers Cll-RM@aCD4Tn to systemic LNs\nthroughout the body, potentially exerting enhance immune modulation.]\nQ12: Can the injection of exogenous Ag mixed with RM@aCD4Tn induce the same\nresponse as CII- RM@aCD4Tn? Meaning, does the Ag and NP need to be connected\n27\nlocationally for the functional activity? This would be critical for an in vivo setting; this is\nimportant given the Ag-release profile of RM@aCD4Tn (Supplementary Figure 5b) and the\nargument that Ag-specific tolerance is induced.\nA12: We thank the reviewer for this important question. To determine whether free\nantigen mixed with RM@aCD4Tn could replicate the effect of antigen-loaded CII-\nRM@aCD4Tn, we added an extra group receiving free CII peptide plus RM@aCD4Tn (G8:\nCII + RM@aCD4Tn) and compared it with the antigen loaded group (G9: CII-RM@aCD4Tn)\n(Fig. 6a). This experiment was performed within the same cohort described in our\nresponses to Reviewer #1 (Q4, Q10, Q12), Reviewer #2 (Q1), and Reviewer #6 (Q1).\nThe results showed that the free antigen mixture (G8) exhibited reduced therapeutic\nefficacy, which was comparable to RM@aCD4Tn alone (G5), and markedly inferior to the\nco-loaded CII-RM@aCD4Tn group (G9) (Fig. 6a-f and Supplementary Fig. 29). This\nsuggests that simply mixing free antigen with RM@aCD4Tn is insufficient to induce\nantigen-specific tolerance. A plausible explanation is that free antigen undergoes systemic\ndispersion and poor uptake by DCs, whereas antigen load to RM@aCD4Tn ensures co-\ndelivery and coordinated engagement of both the antigen and the tolerogenic cues by the\nsame DCs. These findings clearly demonstrate that spatial co-localization of antigen with\nRM@aCD4Tn is required to achieve effective antigen-specific immune tolerance.\nThe added figures and accompanying text are shown in A10.\nQ13: The authors state that Ag-specific tolerance is induced, but the data used to\nsupport this claim are from OTII mice treated with OVA-RM@aCD4Tn and the ex vivo recall\ncultures compare the OVA ex vivo recall response to the CII ex vivo recall response. As\nthe OTII T cell are TCR transgenic and cannot respond to CII in the absence or presence\nof OVA-RM@aCD4Tn, this in no way shows that OVA-RM@aCD4Tn treatment is Ag-\nspecific.\nA13: We thank the reviewer for this insightful comment. We agree that OT-II CD4⁺ T\ncells, due to their fixed TCR specificity for OVA , cannot respond to CII, and therefore\n323-339\nthe original comparison to CII recall cultures does not appropriately demonstrate antigen-\nspecificity.\nTo directly address this concern, we performed an additional experiment using both\nOT-II and DBA/1 mice. Animals were pre-treated with OVA (G1), OVA-RM@aCD4Tn (G2),\nCII (G3), or CII-RM@aCD4Tn (G4). This analysis was performed within the same\nexperimental cohort used to address Reviewer #1 (Q13, Q14), Reviewer #2 (Q3),\nwith either OVA or CII, and IFN-γ-producing cells were quantified by ELISPOT. In OT-II\nmice, OVA restimulation showed a marked reduction in IFN-γ-secreting cells only when\nmice were pre-treated with OVA-RM@aCD4Tn (G1 vs G2), whereas pre-treatment with\nCII-RM@aCD4Tn had minimal effect (G2 vs. G4) (Fig. 3e,f). Conversely, in DBA/1 mice,\nCII restimulation demonstrated that CII-RM@aCD4Tn produced a significantly stronger\nsuppression of IFN-γ-producing cells compared with OVA-RM@aCD4Tn (G2 vs G4) (Fig.\n3g,h). Moreover, cytokine levels of TNF-α and TGF-β in the culture medium were also\nquantified, and the antigen-specific suppression were likewise observed (Supplementary\nFig. 20a-c).\nWe further use I-Aᵇ OVA tetramer to identify antigen-specific T which provide\n323-339 regs\ndirect evidence for antigen specificity. Because OT-II CD4⁺ T cells inherently recognize\nOVA and are readily stained by the OVA tetramer even without treatment, this\n323-339\napproach cannot be used to distinguish induced antigen-specific T in OT-II mice.\nregs\nTherefore, we performed the tetramer experiment in DBA/1 mice, where endogenous CD4⁺\nT cells do not pre-recognize OVA. Splenocytes from DBA/1 mice pre-treated with OVA-\nRM@aCD4Tn (G2) showed a significant increase in OVA tetramer⁺ T following\n323-339 regs\nOVA stimulation, compared with mice receiving free OVA (G1) (Supplementary Fig. 20d).\nIn contrast, mice pre-treated with CII or CII-RM@aCD4Tn did not show induction of OVA-\nspecific T upon OVA restimulation (G3 and G4) (Supplementary Fig. 20d). Taken\nregs\ntogether, these data demonstrate that the Ag-RM@aCD4Tn platform is capable of inducing\nantigen-specific Tregs in vivo, and that this induction strictly depends on loading the\ncognate antigen into the NVs.\nIn addition, the functional characterization of CII-specific T was investigated within\nregs\nthe same cohort described in our responses to Reviewer #1 (Q13), Reviewer #2 (Q3),\nand Reviewer #6 (Q1, Q3). DBA/1 mice were first intradermally pre-treated with either CII-\nRM@aCD4Tn or OVA-RM@aCD4Tn to induce CII- or OVA-specific T , respectively.\nregs\nCD4⁺CD25⁺ T were then isolated and co-cultured with BMDCs in the presence of LPS\nregs\nand CII (Supplementary Fig. 21a). When CII-specific T (from CII-RM@aCD4Tn treated\nregs\nmice) were added (G5), BMDC activation was markedly suppressed, as evidenced by\nsignificantly reduced surface expression of CD80 and CD86(Fig. 3i), a pronounced\ndecrease in the pro-inflammatory cytokine TNF-α, and a concomitant increase in the anti-\ninflammatory cytokine TGF-β in the culture supernatant (Supplementary Fig. 21b). These\nfindings indicate effective suppressive function of CII-specific T in the presence of CII.\nregs\nIn contrast, OVA-specific T (from OVA-RM@aCD4Tn-treated mice) (G4) exerted only\nregs\nmodest inhibitory effects on BMDC activation and cytokine production under the same CII\n29\nrestimulation conditions, which were substantially weaker than those observed for CII-\nspecific T (Fig. 3i and Supplementary Fig. 21b). Before and after co-culture, T were\nregs regs\nstained for the key functional molecules CTLA4 and LAG-3, which are widely associated\nwith enhanced T -mediated suppression. We observed that both OVA-specific (G4) and\nreg\nCII-specific T (G5) became activated upon co-culture with BMDCs, as evidenced by\nregs\nincreased CTLA4 and LAG-3 expression, but the activation was most pronounced in the\nCII-specific T group (Supplementary Fig. 21c). These results provide functional evidence\nreg\nthat CII-RM@aCD4Tn induces CII-specific T in vivo, whose suppressive activity is\nregs\npreferentially engaged in the presence of the cognate CII antigen.\nThe revised figures and accompanying text read as follows:\nFig. 3 | T generation and antigen-specific immunosuppressive by Ag-\nregs\nRM@aCD4Tn in vivo. e,f, Representative ELISPOT images (e) and quantification (f) of\nIFN-γ-secreting splenocytes from OT-II mice treated with the indicated formulations after\nstimulation with either OVA or CII. g,h, Representative ELISPOT images (g) and\n323–339\nquantification (h) of IFN-γ-secreting splenocytes derived from DBA/1 mice. i, Cll-specific\nT -mediated BMDC suppression in the presence of LPS and CII, as evaluated by CD80\nreg\nand CD86 expression on BMDC (n=5).\nof I-Ab OVA + T following antigen restimulation of splenocytes. a,\n323-339 regs\nExperimental scheme of antigen-specific tolerance experiment in OT-II and DBA/1 mice.\nb, The levels of TNF-α and TGF-β in splenocytes from OT-II mice following ex vivo\nstimulation with OVA or CII (n=5). c, The levels of TNF-α and TGF-β in splenocytes\n323-339\nfrom DBA/1 mice (n=5). d, Percentage of I-Ab OVA + T in the splenocytes from\n323-339 regs\nDBA/1 mice (n=5). Data are presented as mean ± SD, statistical significance was analyzed\nby One-way ANOVA with Tukey’s post hoc test.\nregs\nillustration of co-culture CII or OVA-specific T with BMDCs in the presence of LPS and\nregs\nCII. b, Cytokine concentration of TNF-α and TGF-β in the medium (n=5). Data are\npresented as mean ± SD, statistical significance was analyzed by One-way ANOVA with\nTukey’s post hoc test.\n[Methods section:\nAntigen-specific T induction in vitro and in vivo\nreg\nTo assess antigen-specific tolerance, OT-II and DBA/1 mice were injected i.d. at both\nflanks with OVA or CII encapsulated NVs. After that, splenocytes were harvested for IFN-\nγ ELISPOT assays to evaluate the specific activation. Splenocytes were seeded into\nELISPOT plates with 1 × 106 cells per well, and stimulated with 10 μg ml-1 OVA or 10 μg\nml-1 CII respectively. After 24 h, supernatants were collected for TNF-α and TGF-β\ndetection by using ELISA kits, and IFN-γ-secreting cells were quantified according to the\nmanufacturer’s instructions. Furthermore, OVA-specific T in splenocytes from DBA/1\nreg\nmice were analyzed by staining with I-Ab OVA Tetramer.\nFor functional characterization of CII-specific T , DBA/1 mice were treated as\nregs\n32\ndescribed above, and CD4⁺ CD25⁺ T were isolated and cocultured with BMDCs in the\nregs\npresence of 50 ng ml-1 LPS and 10 μg ml-1 CII. After 24 h, culture supernatants were\ncollected for the measurement of TNF-α and TGF-β levels, and CD80/CD86 expression on\nBMDCs and CTLA4 and LAG-3 on T were analyzed by FC.\nregs\nResults section:\nTo determine whether the induced immunosuppression was antigen-specific,\nsplenocytes isolated from OT-II or DBA/1 mice pre-immunized with different formulations\nwere re-stimulated ex vivo with either OVA or CII, and IFN-γ-producing cells in splenocytes\nwere quantified by ELISPOT (Supplementary Fig. 20a). In OT-II mice, OVA restimulation\nrevealed a marked reduction in IFN-γ-secreting cells after pretreatment with OVA-\nRM@aCD4Tn (G1 vs G2, P<0.0001), corresponding to a 71.6% decrease (Fig. 3e,f).\nPretreatment with CII-RM@aCD4Tn produced a 30.5% reduction, which was significantly\nweaker than that induced by OVA-RM@aCD4Tn (G2 vs G4, P<0.0001) (Fig. 3e,f).\nConversely, in DBA/1 mice, CII restimulation demonstrated that CII-RM@aCD4Tn\nproduced significantly suppression of IFN-γ-producing cells than OVA-RM@aCD4Tn (G2\nvs G4, P<0.0001) (Fig. 3g,h). Consistent with these findings, cytokine levels of TNF-α and\nTGF-β in the culture medium were also quantified by ELISA, and the antigen-specific\nsuppression were likewise observed (Supplementary Fig. 20b,c). To directly detect\nantigen-specific T , I-Aᵇ OVA tetramer was used. Because OT-II CD4⁺ T cells are\nregs 323-339\ninherently OVA reactive and exhibit robust baseline tetramer staining even without\ntreatment, the assay was performed in DBA/1 mice. Upon OVA restimulation, OVA-\nRM@aCD4Tn pretreatment led to an obvious increase in I-Aᵇ OVA tetramer⁺ T\n323-339 regs\ncompared to free OVA (G1 vs G2, P=0.0027), whereas CII or CII-RM@aCD4Tn\npretreatment failed to induce OVA-specific T (Supplementary Fig. 20d).\nregs\nMoreover, the functional properties of CII-specific T were investigated\nregs\n(Supplementary Fig. 21a). CII-specific T markedly suppressed CII-induced BMDC\nregs\nactivation, as evidenced by reduced CD80 and CD86 expression and decreased TNF-α,\ntogether with increased TGF-β levels in the supernatant (G3 vs G5, all P<0.0001) (Fig. 3i\nand Supplementary Fig. 21b). In contrast, OVA-specific T exerted only modest effects\nregs\nunder the same CII restimulation conditions (Fig. 3i and Supplementary Fig. 21b).\nFollowing co-culture, T from both groups (G4 and G5) showed upregulation of CTLA4\nregs\n(OVA-specific T with P=0.0002, CII-specific T with P<0.0001) and LAG-3 (both\nregs regs\nP<0.0001) expression compared to unstimulated states, and CTLA4 and LAG-3\nexpression in CII-specific T were 1.7-fold and 1.3-fold higher than in OVA-specific T\nregs regs\n(Supplementary Fig. 21c), suggesting their greater immunosuppressive activity.\nCollectively, these data demonstrate that T induced by Ag-RM@aCD4Tn confer\nregs\n33\nantigen-specific immune tolerance that is engaged upon re-encounter with the cognate\nantigen.]\nQ14: On line 288, the authors state that CII-RM@aCD4Tn induced an increase in Ag-\nspecific T . However, the authors did not use tracible TCR transgenic T cells or tetramer\nregs\nstaining to support this conclusion. Here again, the authors also did not used an Ag-non-\nspecific RM@aCD4Tn control treatment, as such the authors cannot claim that these cells\nare Ag-specific T due to the treatment.\nregs\nA14: We thank the reviewer for this valuable comment. We agree that using tetramer\nto identify antigen-specific T would provide direct evidence for antigen specificity. This\nregs\nexperiment was conducted as part of the studies described in our response to Reviewer\n#1 Q13:\nWe further use I-Aᵇ OVA tetramer to identify antigen-specific T which provide\n323-339 regs\ndirect evidence for antigen specificity. Because OT-II CD4⁺ T cells inherently recognize\nOVA and are readily stained by the OVA tetramer even without treatment, this\n323-339\napproach cannot be used to distinguish induced antigen-specific T in OT-II mice.\nregs\nTherefore, we performed the tetramer experiment in DBA/1 mice, where endogenous CD4⁺\nT cells do not pre-recognize OVA. Splenocytes from DBA/1 mice pre-treated with OVA-\nRM@aCD4Tn (G2) showed a significant increase in OVA tetramer⁺ T following\n323-339 regs\nOVA stimulation, compared with mice receiving free OVA (G1) (Supplementary Fig. 20d).\nIn contrast, mice pre-treated with CII or CII-RM@aCD4Tn did not show induction of OVA-\nspecific T upon OVA restimulation (G3 and G4) (Supplementary Fig. 20d). Taken\nregs\ntogether, these data demonstrate that the Ag-RM@aCD4Tn platform is capable of inducing\nantigen-specific Tregs in vivo, and that this induction strictly depends on loading the\ncognate antigen into the NVs.\nQ15: In Figure 5a-c, the author claim that treatments G2-G5 made disease severity\nworse. However, the authors have not included a saline/PBS treatment group. What\ntreatment group are the authors using as the comparator to make this claim?\nA15: We thank the reviewer for pointing this out. In our original labeling scheme, the\nPBS treated group was labeled as “RA (G2)” and served as the disease model. We agree\nthat this nomenclature is potentially confusing, as it does not explicitly indicate that this\ngroup received PBS. To improve clarity and consistency, we have revised the entire\nmanuscript to relabel this group as “PBS” instead of “RA.” All comparisons regarding\n34\ndisease severity have been updated accordingly.\nQ16: In Figure 6b and 6c, the percentage of area for Foxp3+ cells is ~0.7%, while the\nCD4+ CD25+ Foxp3+ area is ~2%. How is the area increasing by increasing the stringency\nof marker co-expression for quantifying the cells?\nA16: We thank the reviewer for carefully noting this inconsistency. We agree that, if\nthe signal represented strictly CD4⁺ CD25⁺ Foxp3⁺ triple-positive cells, the percentage\nshould not exceed that of Foxp3⁺ cells alone. The discrepancy arose from our original\nquantification approach: in the initial analysis, we calculated the sum of the areas positive\nfor CD4, CD25, or Foxp3 rather than the area corresponding specifically to triple-positive\ncells. This composite measure does not accurately reflect the frequency of Foxp3⁺ T .\nregs\nTo resolve this, we re-analyzed the sections and now quantify Foxp3⁺ area normalized\nto CD4⁺ area (Foxp3⁺ area/CD4⁺ area), which better reflects the proportion of Foxp3⁺ T\ncells within the CD4⁺ compartment.\nThe revised figures and accompanying text read as follows:\nFig. 5. | a, Immunofluorescent staining images for CD4+ CD25+ Foxp3+ T in the\nregs\nsynovium. Scale bars, 60 μm. Blue, DAPI; Red, CD4; Green, CD25; Yellow, Foxp3. b,\nFoxp3+ area in synovium (n=6). c, Foxp3+ area/CD4+ area in synovium (n=6).\n[Results section:\nImmunofluorescence analysis revealed a marked increase in the proportion of Foxp3+\nT in the synovium after CII-RM@aCD4Tn treatment (Fig. 5a-c).]\nregs\nQ17: Figure 1b presents a summary of the hypothesis. This should be either removed\n35\nor placed at the end of the manuscript as part of the Discussion. With the present\nplacement of the figure, this is attempting to lead the reader to make the same conclusion\nas the author without the reader having been able to objectively review the data first.\nA17: We thank the reviewer for this helpful suggestion. We agree that presenting the\nschematic hypothesis at the beginning may unduly influence interpretation before the\nexperimental data are shown. To avoid this and ensure an objective flow of information, we\nhave removed Figure 1b from its original position and relocated it to the end of the\nmanuscript.\nQ18: Figure 7 and Extended Data Figure 8 are the same figure.\nA18: We thank the reviewer for noting this mistake. Figure 7 and Extended Data Figure\n8 were inadvertently duplicated in the original submission. We have corrected this error\nand updated the figures to ensure that each panel now presents the appropriate data.\nQ19: The authors neglect to mention any of the multiple companies and academic\nlaboratories that have Ag-specific therapeutics and Ag-specific NP-based therapeutics that\nare presently in clinical trials.\nA19: We thank the reviewer for this important comment. In the revised Discussion,\nwe now explicitly acknowledge ongoing antigen-specific and T -directed clinical programs by\nreg\nciting representative trials (NCT04987307, NCT03451422, ISRCTN11038572,\nNCT04817774). In addition, we have added a dedicated paragraph summarizing antigen-\nbearing nanoparticle approaches that have entered clinical testing, with particular\nemphasis on the work led by Prof. Stephen D. Miller and colleagues on gliadin-loaded PLG\nnanoparticles (TAK-101), which provide clinical proof-of-concept for antigen-specific, NP-\nbased tolerance induction in celiac disease. These additions place our DC-CD4 bispecific\nNV platform in the broader context of antigen-specific and nanoparticle-based tolerogenic\ntherapies being developed by both academic and industrial groups.\nThe revised Discussion read as follows:\n[Discussion section:\nT -directed therapies, with more than 70 clinical trials underway, are advancing\nregs\nalong two major lines. One relies on ex vivo expansion and adoptive transfer of T ,\nregs\nspanning polyclonal, donor-reactive, and engineered CAR/TCR T 16,17. The other seeks\nregs\nto augment endogenous T in vivo using pharmacological apporaches43-45. Early clinical\nregs\n36\nstudies show encouraging safety profile and signals of activity in selected indications\n(NCT04987307, NCT03451422, ISRCTN11038572, NCT04817774). For example,\nautologous HLA-A2-specific CAR-T therapies such as TX200-TR101 are currently being\nreg\nevaluated in a phase I/IIa trial for the prevention of immune-mediated rejection in HLA-A2-\nmismatched kidney transplantation (NCT04817774). Yet broad deployment remains\nconstrained by limited persistence and the manufacturing burden of individualized cell\nproducts16,17. In parallel, antigen-bearing nanomedicines have emerged to induce or\nstabilize antigen-restricted tolerance in vivo. Foundational studies using polymeric or lipid\ncarriers that deliver autoantigen, either alone or in combination with a tolerogenic\nmodulator, can attenuate pathogenic immunity6,7,46-50. Notably, gliadin-loaded PLG\nnanoparticles (TAK-101) have shown gluten-specific tolerance in patients with celiac\ndisease48. Nonetheless, the optimal means of orchestrating precise immune cell\ninteractions within secondary lymphoid organs to reliably generate durable pools of\nantigen-specific T remains to be defined.\nregs\nReferences\n6. Sohn, H.S. et al. Tolerogenic nanoparticles induce type II collagen-specific\nregulatory T cells and ameliorate osteoarthritis. Sci. Adv. 8, eabo5284 (2022).\n7. Nguyen, T.L. et al. Immunosuppressive biomaterial-based therapeutic vaccine to\ntreat multiple sclerosis via re-establishing immune tolerance. Nat. Commun. 13,\n7449 (2022).\n16. Wang, L. et al. Regulatory T cells in homeostasis and disease: molecular\nmechanisms and therapeutic potential. Signal Transduct. Target. Ther. 10, 345\n(2025).\n17. Wardell, C.M., Boardman, D.A. & Levings, M.K. Harnessing the biology of\nregulatory T cells to treat disease. Nat. Rev. Drug Discov. 24, 93-111 (2025)\n43. Harris, F., Berdugo, Y.A. & Tree, T. IL-2-based approaches to Treg enhancement.\nClin. Exp. Immunol. 211, 149-163 (2023).\n44. Scheid, J.F. et al. Safety, pharmacokinetics, and pharmacodynamics of MK-6194,\nan IL-2 mutein designed to selectively activate regulatory T cells: single ascending\ndose and multiple ascending dose trial data. Immunohorizons 9 (2025).\n45. VanDyke, D. et al. Engineered human cytokine/antibody fusion proteins expand\nregulatory T cells and confer autoimmune disease protection. Cell Rep. 41, 111478\n(2022).\n37\n46. Hunter, Z. et al. A biodegradable nanoparticle platform for the induction of antigen-\nspecific immune tolerance for treatment of autoimmune disease. ACS Nano 8,\n2148-2160 (2014).\n47. Prasad, S. et al. Tolerogenic Ag-PLG nanoparticles induce tregs to suppress\nactivated diabetogenic CD4 and CD8 T cells. J. Autoimmun. 89, 112-124 (2018).\n48. Kelly, C.P. et al. TAK-101 Nanoparticles Induce Gluten-Specific Tolerance in Celiac\nDisease: A Randomized, Double-Blind, Placebo-Controlled Study.\nGastroenterology 161, 66-80.e68 (2021).\n49. Saunders, M.N. et al. Allergen-encapsulating nanoparticles reprogram pathogenic\nallergen-specific Th2 cells to suppress food allergy. Adv. Healthc. Mater. 14,\ne2400237 (2025).\n50. Brzezicka, K.A. et al. Suppression of autoimmune rheumatoid arthritis with hybrid\nnanoparticles that induce B and T cell tolerance to self-antigen. ACS Nano 16,\n20206-20221 (2022).]\nThis manuscript presents a nanoparticle platform based on T cell-derived\nreg\nmembrane nanoparticles (Tn) decorated with anti-CD4 (aCD4) antibodies and\nencapsulated with an antigenic peptide (AP) and rapamycin (RM), designed to induce\nantigen-specific T for the treatment of rheumatoid arthritis (RA). While the therapeutic\nregs\npotential of the platform is of interest, the manuscript falls short in demonstrating significant\nadvancement over previously published technologies, and the mechanistic claims are not\n4\nsufficiently supported by the data. Below are specific concerns and suggestions.\n1. This study failed to demonstrate advancement over prior work. A closely related\nstudy has previously been reported (Science Advances 8(47), eabo5284, 2022),\ndemonstrating that nanoparticles co-loaded with antigenic peptide and rapamycin can\neffectively induce tolerogenic dendritic cells (tDCs) and antigen-specific T for treatment\nregs\nof arthritis. The principal advancement claimed in the current study is the addition of CTLA-\n4 and anti-CD4 antibody to the nanoparticle surface. While some positive effects of anti-\nCD4 are demonstrated in the manuscript, the role of CTLA-4 remains speculative due to\nthe absence of proper in vivo controls. To substantiate the contribution of CTLA-4, the\nauthors should include control nanoparticles lacking CTLA-4 but retaining anti-CD4\ndecoration. Furthermore, the above-mentioned study should be properly cited and\ndiscussed in the Introduction to clarify how the current work advances the field.\nA1: We thank the reviewer for this important comment and fully agree that the Science\nAdvances study (Sci. Adv. 2022, 8, eabo5284) represents a key prior contribution in this\nfield. This study indeed inspired aspects of our conceptual framework and experimental\ndesign. We have now properly cited this work in the Introduction and discussed how our\nstudy builds upon and advances their findings. Specifically, we highlight that our major\ninnovation lies in actively targeting both DCs and CD4+ T cells, and promoting the physical\nformation of DC-CD4⁺ T cell synapse through simultaneous CTLA4-CD80/86 and anti-\nCD4-CD4 interactions. This DC-CD4 bispecific “bridging” strategy is distinct from prior\napproaches using antigen- and rapamycin-loaded LNPs, which do not directly enforce such\nspatially organized DC-CD4 contacts. The revised Introduction read as follows:\n[Introduction section:\nTo overcome these limitations, nanotechnology-based strategies have been explored\nto deliver antigens and immunosuppressive agents specifically to antigen presenting cells\n(APCs), particularly dendritic cells (DCs), thereby boosting the generation of antigen-\nspecific T in situ4, 6, 12, 25, 26. Among these approaches, Sohn et al. reported lipid\nregs\nnanoparticles (LNPs) co-loaded with type II collagen peptide and rapamycin (RM), which\n39\nreduced synovial inflammation and ameliorated osteoarthritis in mice, providing a\ncompelling proof-of-concept for NP-mediated antigen-specific tolerance in joint disease6.\nHowever, this strategy relies on passive uptake by DCs followed by intradermal (i.d.)\ninjection, without actively targeting or programming DC fate to coordinate crosstalk with\nother immune cells. Therefore, there is an urgent need to develop a targeted tolerogenic\nstrategy that actively reprograms DCs and amplifies their interactions with immune\npartners, driving more efficient and durable induction of antigen-specific T .\nregs\nReferences\n4. Liu, Y. et al. Generation of tolerogenic antigen-presenting cells in vivo via the\ndelivery of mRNA encoding PDL1 within lipid nanoparticles. Nat. Biomed. Eng.\n(2025).\n6. Sohn, H.S. et al. Tolerogenic nanoparticles induce type II collagen-specific\nregulatory T cells and ameliorate osteoarthritis. Sci. Adv. 8, eabo5284 (2022).\n12. Scotland, B.L. et al. Cell and biomaterial delivery strategies to induce immune\ntolerance. Adv. Drug Deliv. Rev. 203, 115141 (2023).\n25. Cifuentes-Rius, A., Desai, A., Yuen, D., Johnston, A.P.R. & Voelcker, N.H. Inducing\nimmune tolerance with dendritic cell-targeting nanomedicines. Nat. Nanotechnol.\n16, 37-46 (2021).\n26. Krienke, C. et al. A noninflammatory mRNA vaccine for treatment of experimental\nautoimmune encephalomyelitis. Science 371, 145-153 (2021).]\nTo directly address the reviewer’s concern regarding the functional contribution of\nCTLA4, we performed an in vivo experiment using an anti-CTLA4 neutralizing antibody to\nblock the CTLA4 molecules displayed on the NV surface (G6) (Fig. 6a). This experiment\nwas conducted within the same cohort used to address Reviewer #1 (Q4, Q10, Q12),\nReviewer #2 (Q1), and Reviewer #6 (Q1).\nThis design allowed us to specifically evaluate whether CTLA4 contributes to\ntherapeutic efficacy. The results showed that NVs containing only aCD4 retained partial\ntherapeutic benefit (Fig. 6a-f and Supplementary Fig. 29), supporting the contribution of\nCD4⁺ T cell targeting. However, their efficacy was significantly reduced compared with the\nfull CTLA4/aCD4 bispecific NVs (G9), demonstrating that both CTLA4 and aCD4 are\nrequired for optimal therapeutic activity. Together, these findings validate the functional role\nof CTLA4 in vivo and support the advancement of our bispecific strategy.\nFig. 6 | a, Experimental scheme of CIA mice treatment with various formulations. b, Arthritis\nscore curve (n=6). c, Paw thickness on day 42 (n=6). d, Motor evaluation of CIA mice on\nday 42 using rotarod system (n=6). e,f, Representative H&E section images of ankle (e)\nand histopathological evaluation of synovitis score (f) (n=6). g, The level of anti-CII IgG\nantibody in serum (n=6).\nSupplementary Fig. 29. Speed at falls of CIA mice on day 42 using rotarod system.\nData are presented as mean ± SD (n=6), statistical significance was analyzed by One-\nway ANOVA with Tukey’s post hoc test.\n[Methods section:\nMechanistic investigation of CII-RM@aCD4Tn\ngroups, and each group contained 6 mice. One group served as a healthy (G1), whereas\nthe other eight groups included PBS (G2), 150 μg CII (G3), 20 μg RM (G4), 1 mg\nRM@aCD4Tn (G5), 1.2 mg CII-RM@aCD4Tn (lacking CTLA4) (G6), 1.2 mg OVA-\nRM@aCD4Tn (G7), 150 μg CII + 1 mg RM@aCD4Tn (G8) and 1.2 mg CII-RM@aCD4Tn\n(G9). The amounts of CII and RM were identical across all groups. Notably, CTLA4 on the\nsurface of Tn was blocked via anti-CTLA4 antibody to generate group 6. The NVs were\ninjected on day 22, 25 and 28. Arthritis score and body weight were monitored every three\ndays. Paw thickness measurement and rotarod test were conducted on day 42. Hind paws\nwere collected for H&E staining. In addition, serum samples were obtained on day 42 for\nquantification of anti-CII IgG antibodies using an ELISA kit.\nResults section:\nTo further clarify the underlying therapeutic mechanism of Cll-RM@aCD4Tn, CIA mice\nwere treated with different formulations (Fig. 6a). Free CII (G3) or free RM (G4) failed to\ncontrol arthritis progression (Fig. 6a-f and Supplementary Fig. 29). To assess antigen\nspecificity, we included an antigen-nonspecific group, OVA-RM@aCD4Tn (G7). OVA-\nRM@aCD4Tn showed markedly reduced therapeutic efficacy compared to CII-\nRM@aCD4Tn, as reflected by higher arthritis scores (P=0.0003) and synovitis scores\n(P<0.0001), and its overall performance was comparable to that of RM@aCD4Tn (G5) (Fig.\n6a-f and Supplementary Fig. 29). When CII was administrated separately together with\nRM@aCD4Tn (G8), the efficacy remained inferior to that of co-loaded CII-RM@aCD4Tn\n(Fig. 6a-f and Supplementary Fig. 29), suggesting that simply co-injecting of free antigen\nwith NVs is insufficient to induce strong antigen-specific tolerance. Furthermore, to dissect\nthe functional contribution of CTLA4-mediated DC targeting, CTLA4 blocked NV\nformulation by neutralizing CTLA4 (G6) was prepared. Although this CTLA4 blocked\nformulation retained partial therapeutic activity, its efficacy was lower than that of CII-\nRM@aCD4Tn (G9) (Fig. 6a-f and Supplementary Fig. 29).]\nQ2: The mechanistic claims were not experimentally supported. Figure 1b proposes\nthat AP-RM@aCD4Tn interacts with CD80/86 on DCs via CTLA-4 to induce immune\ntolerance and promote tDC formation, mediated by both the surface-bound molecules and\nrelease of RM and AP. However, the manuscript lacks direct evidence supporting CTLA-\n4–CD80/86 interactions. Data presented in Figures 2g and 2h show internalization of Cll-\nRM@aCD4Tn (AP = collagen II) by DCs but do not address surface-level immunological\nsynapse formation.\ndata are presented to confirm this, and prior work (Science Advances 8(47), eabo5284,\n2022) indicates that rapamycin is typically retained within lipid layers rather than being\nreadily released. These mechanistic assertions require either experimental support or\nmore cautious interpretation.\nA2: We thank the reviewer for this insightful comment. We agree that our original\nsubmission did not provide direct experimental evidence for CTLA4-CD80/86 interactions\nat the DC-CD4 interface.\nTo address this, we performed additional blocking experiments to directly test the role\nof CTLA4-CD80/86 binding in NV-mediated DC-CD4 cell conjugate formation. CII-\nRM@aCD4Tn treatment promoted the formation of BMDC-CD4⁺ T cell conjugates (G1)\n(Supplementary Fig. 10a). We then selectively interfered with the proposed CTLA4-\nCD80/86 axis by: masking CD80/86 on BMDCs with CTLA4 Ig (G2), blocking CTLA4 on\nthe NV surface with an anti-CTLA4 antibody (G3), or simultaneously applying both blocking\nstrategies (Supplementary Fig. 10a). In the absence of blocking, approximately 38.1% of\nevents were BMDC-CD4⁺ T cell conjugates (Supplementary Fig. 10b). This frequency was\nmarkedly reduced to ~8% when either CD80/86 or CTLA4 was blocked individually, and\nfurther decreased to ~6% when both were blocked simultaneously (Supplementary Fig.\n10b). These results demonstrate that effective interaction of CII-RM@aCD4Tn and BMDC\nbased on CTLA4-CD80/86, thereby providing direct mechanistic support for the model\nproposed in Figure 1b (now represented as Fig. 8).\nThe added figures and accompanying text read as follows:\ninteraction. a, Schematic illustration of the blocking of CTLA4 on CII-RM@aCD4Tn or\nCD80/86 on BMDCs. Created with Biorender.com. b, FC analysis of DC-CD4 cell pairs\nafter CD80/86 or CTLA4 blocking (n=3). Data are presented as mean ± SD, statistical\nsignificance was analyzed by One-way ANOVA with Tukey’s post hoc test.\n[Methods section:\nBridging effect of CII-RM@aCD4Tn on CD4+ T cells and DCs\nTo investigate the interaction mechanism between CII-RM@aCD4Tn and DCs,\nBMDCs were preincubated with CTLA4 Ig to block surface CD80/86, and CII-\nRM@aCD4Tn was pretreated with an anti-CTLA4 antibody to mask CTLA4 on the surface\nof NV. DC-CD4 cell pairs were then quantified by FC.\nResults section:\nTo directly evidence CTLA4-CD80/86 interactions between CII-RM@aCD4Tn and\nBMDCs, we selectively blocked CD80/86 on BMDCs with CTLA4 Ig (G2), CTLA4 on the\nNV surface with an anti-CTLA4 antibody (G3), or both binding partners simultaneously (G4)\n(Supplementary Fig. 10a). The frequency of DC-CD4 cell pairs decreased from 38.1% in\nthe unblocked condition to ~8% when either CD80/86 or CTLA4 was blocked individually,\nand to ~6% when both were blocked (Supplementary Fig. 10b), demonstrating the effective\ninteraction of CII-RM@aCD4Tn and BMDC mediated through CTLA4-CD80/86 axis.]\nWe sincerely thank the reviewer for this insightful comment about rapamycin (RM)\nrelease.\nFirst, RM release from NVs is derived from direct experimental measurements, using\na standard dialysis method widely adopted for assessing hydrophobic drug release from\nlipid- or membrane-based nanocarriers. Released RM was quantified by high-performance\nliquid chromatography (HPLC), and we now included the corresponding data in\nSupplementary Fig. 5a and clarified the experimental procedures in results. The added\nfigures read as follows:\nHPLC spectrum and standard calibration curve of RM concentration versus peak area.\nSecond, the observed gradual, time-dependent increase in RM release from our Tn is\nconsistent with the behavior reported for other biomimetic or lipid bilayer-containing\nnanocarriers. For example, RM-loaded leukosomes, which similarly integrate cell-derived\nmembrane proteins into a phospholipid bilayer, exhibited a clear measurable release\nkinetics over 0-22 h, with about 85% RM release at 6 h (Reviewer Fig. 3a) (Circ Res 2020)4.\nLikewise, RM encapsulated within two-stage liposomal systems also showed sustained\n\nrelease using dialysis assays (Reviewer Fig. 3b) (Mater Today Bio 2024)5. Another study\nalso exhibited sustained RM release from liposome, with ~50% and 80% at 4 h and 24 h\n(Reviewer Fig. 3c) (Exp Ther Med 2015)6. These independent studies demonstrate that,\nalthough RM preferentially associates with lipid domains, it can still undergo controlled\ndiffusion and time-dependent release from membrane-based nanostructures.\nReviewer Fig. 3 RM release profile from lipid- or membrane-based nanocarriers in\npublish paper. a, Release curve from leukosomes4. b, Release curve from liposomes5. c,\nRelease curve from liposomes6. Q, accumulative release rate.\nWe fully agree with the reviewer that certain lipid formulations reported in the literature,\nincluding the Science Advances 2022 study, may retain RM more tightly within the\nhydrophobic layer. However, RM release is known to depend strongly on lipid composition,\n45\nmembrane fluidity, protein incorporation, and the presence of packing defects. In our Tn\nplatform, the incorporation of T cell membrane proteins results in a more fluid, protein-\nreg\nrich membrane compared with purely synthetic lipid systems, which likely introduces\npacking defects and facilitates the gradual outward diffusion of RM, consistent with the\nrelease kinetics we observed experimentally.\nWe hope these clarifications adequately address the reviewer’s concern.\nQ3: This study lacks evidence for collagen II-specific T induction in vivo. While\nreg\nFigure 6 suggests in vivo T induction by Cll-RM@aCD4Tn, the manuscript does not\nreg\ndemonstrate that these T are specific to collagen II. This is a critical gap, given that\nregs\ncollagen II antigen specificity is central to the therapeutic rationale for RA. The authors are\nencouraged to perform functional assays to confirm collagen II specificity; e.g., co-culturing\nisolated T with DCs in the presence of collagen II, followed by proliferation or cytokine\nregs\nprofiling assays.\nA3: We thank the reviewer for this important comment. We agree that collagen II (CII)\nantigen specificity is central to the therapeutic rationale and that our original submission\ndid not provide direct functional evidence for CII-specific T activity.\nreg\nTo address this, we performed additional functional assay as suggested, which was\ncarried out within the same experimental cohort used to respond to Reviewer #1 (Q13),\nReviewer #2 (Q3), and Reviewer #6 (Q1, Q3). DBA/1 mice were first intradermally pre-treated\ni.d. with either CII-RM@aCD4Tn or OVA-RM@aCD4Tn to induce CII- or OVA-specific T ,\nregs\nrespectively. CD4⁺ CD25⁺ T were then isolated and co-cultured with BMDCs in the\nregs\npresence of LPS and CII (Supplementary Fig. 21a). When CII-specific T (from CII-\nregs\nRM@aCD4Tn treated mice) were added (G5), BMDC activation was markedly suppressed,\nas evidenced by significantly reduced surface expression of CD80 and CD86 (Fig. 3i),\na pronounced decrease in the pro-inflammatory cytokine TNF-α, and a concomitant increase\nin the anti-inflammatory cytokine TGF-β in the culture supernatant (Supplementary Fig.\n21b). These findings indicate robust activation of CII-specific T and their potent\nregs\nsuppressive function in the presence of CII. In contrast, OVA-specific T (from OVA-\nregs\nRM@aCD4Tn-treated mice) (G4) exerted only modest inhibitory effects on BMDC\nactivation and cytokine production under the same CII restimulation conditions, which were\nweaker than those observed for CII-specific T (Fig. 3i and Supplementary Fig. 121b).\nregs\nBefore and after co-culture, T were stained for the key functional molecules CTLA4\nregs\nand LAG-3, which are widely associated with enhanced T -mediated suppression. We\nreg\nobserved that both OVA-specific (G4) and CII-specific T (G5) became activated upon\nregs\n46\nco-culture with BMDCs, as evidenced by increased CTLA4 and LAG-3 expression, but the\nactivation was most pronounced in the CII-specific T group (Supplementary Fig. 21c).\nreg\nTogether, these results provide functional evidence that CII-RM@aCD4Tn induces\nCII-specific T in vivo, whose suppressive activity is preferentially engaged in the\nregs\npresence of the cognate antigen.\nThe added figures as follows:\nFig. 3 | i, Cll-specific T -mediated BMDC suppression in the presence of LPS and CII, as\nreg\nevaluated by CD80 and CD86 expression on BMDC (n=5).\nregs\nillustration of co-culture CII or OVA-specific T with BMDCs in the presence of LPS and\nregs\nCII. b, Cytokine concentration of TNF-α and TGF-β in the medium (n=5). Data are\npresented as mean ± SD, statistical significance was analyzed by One-way ANOVA with\nTukey’s post hoc test.\n[Methods section:\nAntigen-specific T induction in vitro and in vivo\nreg\nFor functional characterization of CII-specific T , DBA/1 mice were treated as\nregs\n48\ndescribed above, and CD4⁺ CD25⁺ T were isolated and cocultured with BMDCs in the\nregs\npresence of 50 ng ml-1 LPS and 10 μg ml-1 CII. After 24 h, culture supernatants were\ncollected for the measurement of TNF-α and TGF-β levels, and CD80/CD86 expression on\nBMDCs and CTLA4 and LAG-3 on T were analyzed by FC.\nregs\nResults section:\nMoreover, the functional properties of CII-specific T were investigated\nregs\n(Supplementary Fig. 21a). CII-specific T markedly suppressed CII-induced BMDC\nregs\nactivation, as evidenced by reduced CD80 and CD86 expression and decreased TNF-α,\ntogether with increased TGF-β levels in the supernatant (G3 vs G5, all P<0.0001) (Fig. 3i\nand Supplementary Fig. 21b). In contrast, OVA-specific T exerted only modest effects\nregs\nunder the same CII restimulation conditions (Fig. 3i and Supplementary Fig. 21b).\nFollowing co-culture, T from both groups (G4 and G5) showed upregulation of CTLA4\nregs\n(OVA-specific T with P=0.0002, CII-specific T with P<0.0001) and LAG-3 (both\nregs regs\nP<0.0001) expression compared to unstimulated states, and CTLA4 and LAG-3\nexpression in CII-specific T were 1.7-fold and 1.3-fold higher than in OVA-specific T\nregs regs\n(Supplementary Fig. 21c), suggesting their greater immunosuppressive activity.\nCollectively, these data demonstrate that T induced by Ag-RM@aCD4Tn confer\nregs\n49\nantigen-specific immune tolerance that is engaged upon re-encounter with the cognate\nantigen.]\nQ4: This approach concerns feasibility of clinical translation. The abstract highlights\nthe current challenges associated with ex vivo manipulation of autologous T for\nregs\nautoimmune disease treatment, and proposes Cll-RM@aCD4Tn nanoparticles as a more\npractical alternative. However, the preparation of such nanoparticles at clinically relevant\nscales may still require large-scale isolation and manipulation of T , potentially\nregs\nundermining the proposed translational advantage. The authors should address this\nlimitation and provide clarification regarding the scalability and feasibility of their approach.\nA4: We thank the reviewer for raising this important point regarding clinical translation.\nWe agree that our current approach still involves an ex vivo step, as CD4⁺ T cells need to\nbe isolated and converted into immunosuppressive CD4⁺ T cells (iCD4) before NV\nfabrication, which shares certain similarities with conventional T -based therapies. To\nreg\navoid overstating the translational advantage in the Abstract, we have revised the\ncorresponding sentence to a more conservative and accurate description:\n[Abstract section:\nHowever, current approaches either require prolonged ex vivo expansion of rare T\nregs\nor generate insufficient antigen-specific T response in vivo.]\nregs\nRather than completely eliminating ex vivo manipulation, the main practical advantage\nof our strategy lies in simplifying and relaxing the requirements of the ex vivo process\ncompared to classical adoptive T therapy. Conventional T therapies typically require:\nreg reg\nisolation of a rare T subset at high purity and extensive ex vivo expansion (often over 1-\nreg\n2 weeks). In contrast, our approach uses abundant bulk CD4⁺ T cells as the starting\npopulation, followed by short-term in vitro induction (4 days), without the need for\nprolonged expansion of a rare T subset. This substantially reduces the complexity, time,\nreg\nand potential cost of cell preparation, making the process more compatible with existing\nCD4⁺ T cell isolation technologies already used in the clinic.\nWe fully acknowledge, however, that scalability and feasibility remain important\nchallenges for any cell-based nanomedicine. To address this, we have added a dedicated\nparagraph in the Discussion to explicitly discuss the limitations and future directions for\noptimizing the scalability, process integration, and potential automation of this platform for\nclinical translation:\n[Discussion section:\nAlthough the final NV formulation is acellular at the point of administration, its\nmanufacture still requires short-term, ex vivo-induced iCD4 as the source. Autologous\nsources may mitigate immunogenicity, whereas banked allogeneic material could facilitate\nstandardization and lower-cost manufacturing if residual allo-epitopes are minimized. In\naddition, we only focused on a single antigen in a classical CIA model, whereas human RA\ninvolves a diverse and evolving autoantigen repertoire, including post-translationally\nmodified epitopes (e.g., citrullinated proteins) and epitope spreading58. Future variants\nshould incorporate multi-antigen or patient-tailored payloads and be benchmarked in more\nheterogeneous, relapse-prone, or humanized models to better reflect clinical complexity.\nReferences:\n58. van Delft, M.A.M. & Huizinga, T.W.J. An overview of autoantibodies in rheumatoid\narthritis. J. Autoimmun 110, 102392 (2020).]\nIn this interesting manuscript, the authors report the development of DC-CD4\ntolerogenic nanoparticle and apply these to varied mouse models of arthritis to\ndemonstrate their therapeutic potential and draw conclusions about their bio-distribution\nand mechanism of action. In general, the work appears to be technically rigorous and the\nfindings should be of interest to the readership of Nature Communications. However, it\nwould be helpful for the authors to consider the following points:\nQ1: In the introduction, the authors mention specific limitations to cell-based therapy\napproaches but that statement is not referenced. It would be useful to cite at least one\ndefinitive review on that topic.\nA1: We thank the reviewer for this helpful suggestion. In the revised manuscript, we\nnow support this statement with citations to definitive reviews that systematically discuss\nthe practical limitations of T - and tolerogenic cell-based therapies. Specifically, we have\nreg\nadded references to:\n(i) Raffin et al. “T cell-based therapies: challenges and perspectives” (Nat Rev\nreg\nImmunol 2020)7 (cited as ref. 22 in the manuscript);\n(ii) Ferreira et al. “Next-generation regulatory T cell therapy” (Nat Rev Drug Discov\n2019)8 (cited as ref. 23 in the manuscript);\n(iii) Mosanya & Isaacs “Tolerising cellular therapies: what is their promise for\nautoimmune disease?” (Ann Rheum Dis 2019)9 (cited as ref. 24 in the manuscript).\nThe sentence in the Introduction has been retained but is now properly referenced to\nthese key reviews.\nQ2: The authors tout their tolerogenic DC-CD4 particles as a nano-technology-based\nstrategy that overcomes the limitations of cell-based therapy. However, as outlined in\nFigure 1, their method still relies on the extraction, manipulation, and re-infusion of cells.\nImportantly, use of extracellular vesicles in place was less effective. The authors should\nfully elaborate why their workflow generates a tolerogenic product with fewer limitations\nthan competing methods (both cell based and completely synthetic nanoparticles, as\ndescribed by Steve Miller and others). Moving beyond inbred mice, DC-CD4 particles could\nperhaps be manufactured in an autologous or non-autologous manner. Which approach\nwould be the most promising to pursue?\nA2: We thank the reviewer for this thoughtful and important comment. We fully agree\n51\nthat our current workflow still involves extraction, short-term manipulation, and re-use of\npatient-derived T cells, and therefore does not completely eliminate all steps associated\nwith cell-based therapy. In the revised manuscript, we have softened our wording and now\nclarify that our DC-CD4 bispecific NVs are intended to mitigate rather than fully abolish the\nlogistical limitations of conventional cell-based approaches.\nCompared with classical T adoptive therapy, our strategy may offer several practical\nreg\nadvantages while still requiring ex vivo step. First, we use bulk CD4⁺ T cells as the starting\npopulation rather than rare endogenous T and the induction period is relatively short (4\nregs,\ndays), which could reduce the need for prolonged, large-scale expansion to achieve\ntherapeutic cell numbers. Second, the final therapeutic entity is a cell-free nanovesicle\nformulation (Tn) rather than a live cell product, which may be more amenable to\nstandardized formulation, storage, and dosing than reinfusion of viable T . These\nregs\nfindings support our decision to focus on Tn as a more scalable tolerogenic modality, while\nacknowledging that an ex vivo step is still required.\nNotably, Miller and colleagues have conducted extensive pioneering work on antigen-\nspecific tolerogenic NPs. In 2014, they reported a biodegradable PLG NP system for the\ninduction of antigen-specific immune tolerance in multiple autoimmune disease models\n(ACS Nano 2014)10. In 2021, a gliadin-loaded nanoparticle formulation (TAK-101) was\nshown to induce gluten-specific tolerance in patients with celiac disease in a randomized,\ndouble-blind, placebo-controlled trial (Gastroenterology 2021)11. Most recently, they further\ndemonstrated that allergen-encapsulating NPs can reprogram pathogenic allergen-specific\nTh2 cells toward a suppressive phenotype and ameliorate food allergy (Adv Healthc Mater\n2025)12. We now explicitly cite these studies in the Discussion section. Our platform\nrepresents a hybrid design that seeks to leverage the complex endogenous inhibitory\nrepertoire of T . Tn inherently display a dense, physiologic array of immune checkpoint\nregs\nmolecules (e.g., CTLA4, PD-1, LAG-3) and a native lipid/protein membrane context that\nare difficult to fully recapitulate on purely synthetic carriers. By further decorating Tn with\nanti-CD4, we enable simultaneous CTLA-4-CD80/86 and aCD4-CD4 engagement, thereby\nenforcing a physical DC-CD4⁺ T cell synapse in addition to antigen and rapamycin co-\ndelivery. We have expanded the Discussion to more clearly contrast these mechanistic and\ntranslational trade-offs between our Tn system, conventional T therapies, and fully\nreg\nsynthetic antigen NPs.\nRegarding future translation beyond inbred mice, both autologous and non-autologous\nmanufacturing routes are theoretically possible. At the current stage, an autologous\napproach (using the patient’s own CD4⁺ T cells as the source) appears most\nstraightforward from an immunogenicity and regulatory standpoint. In the longer term, non-\n52\nautologous or “universal donor” sources might be attractive from a manufacturing\nperspective, especially if combined with strategies to minimize allo-immunogenic\ncomponents in the membrane.\nWe have now added relative paragraph in the Discussion explicitly outlining these\npotential paths and their respective advantages and limitations.\n[Discussion section:\nT -directed therapies, with more than 70 clinical trials underway, are advancing along\nregs\ntwo major lines. One relies on ex vivo expansion and adoptive transfer of T , spanning\nregs\npolyclonal, donor-reactive, and engineered CAR/TCR T The other seeks to augment\nregs\nendogenous T in vivo using pharmacological apporaches. Early clinical studies show\nregs\nencouraging safety profile and signals of activity in selected indications\n(NCT04987307, NCT03451422, ISRCTN11038572, NCT04817774). For example,\nautologous HLA-A2-specific CAR-T therapies such as TX200-TR101 are currently being\nreg\nevaluated in a phase I/IIa trial for the prevention of immune-mediated rejection in HLA-A2-\nmismatched kidney transplantation (NCT04817774). Yet broad deployment remains\nconstrained by limited persistence and the manufacturing burden of individualized cell\nproducts. In parallel, antigen-bearing nanomedicines have emerged to induce or\nstabilize antigen restricted tolerance in vivo. Foundational studies using polymeric or lipid\ncarriers that deliver autoantigen, either alone or in combination with a tolerogenic\nmodulator, can attenuate pathogenic immunity. Notably, gliadin-loaded PLG nanoparticles\n(TAK-101) have shown gluten-specific tolerance in patients with celiac disease.\nNonetheless, the optimal means of orchestrating precise immune cell interactions within\nsecondary lymphoid organs to reliably generate durable pools of antigen-specific T\nregs\nremains to be defined.\nOur work adapts a design principle widely used in oncology, in which multi-specific\nconstructs are used to organize cell-cell synapses, and repurposes it to promote\ntolerogenic rather than stimulatory outcomes. In cancer, bi-/tri-specifics and nano-\nengagers bring immune effectors into contact with targets to amplify activation. Here, we\ndevelop a DC-CD4 bridging nanosystem (Ag-RM@aCD4Tn) that increases the frequency\nof encounters between DCs and CD4⁺ T cells in LNs, where the T -derived NVs displays\nreg\ninhibitory molecules (e.g., CTLA4, PD-1, and LAG-3) and antigen/rapamycin payloads that\ntogether bias the interaction toward regulation rather than activation, leading to durable\nantigen-specific tolerance.\nSeveral NV principles from our data help explain the outstanding tolerant performance.\nFirst, Ag-RM@aCD4Tn enhance antigen presentation by DCs (Fig. 1h,i, and\nSupplementary Fig. 7). This effect likely reflects the synergistic contributions of enhanced\nprolongs contact (Supplementary Fig. 10) and promotes uptake (Fig. 1f,g), and intrinsically\nhigher internalization of particulate cargo compared with soluble antigen6. Second,\nintradermal (i.d.) administration favors convective transport via afferent lymphatics, thereby\nconcentrating particulate cargo within the draining LNs, where is the principal venues for\nAPC-T cell crosstalk. Consistent with this, our bispecific NVs achieved high LN delivery\nafter i.d. injection, with up to 80.6% of the injected dose detected in LNs (Fig. 2d-f). Third,\nin vivo cellular profiling in LNs indicated preferential association with CD4+ T cells (44.8%)\nand CD11c+ DCs (31.3%) (Supplementary Fig. 12a), further converting them into tolerance\nphenotype (Fig. 1j-m, Supplementary Fig. 8 and 9). Fourth, beyond co-encapsulating\nantigen peptide and rapamycin, the NVs actively organize DC-CD4 proximity through\naCD4-CD4 and CTLA4-CD80/86 interactions (Fig. 2a,b and Supplementary Fig. 4,10),\nleading to effective induction of T in vivo (Fig. 3a-d, Fig. 5a-g, Supplementary Fig. 27h-\nregs\nk and Supplementary Fig. 28g-j).\nAntigen specificity is a central requirement for tolerogenic interventions. We therefore\nimplemented complementary assays across models. Antigen-specific recall assays in OT-\nII and DBA/1 mice showed suppression only when the cognate antigen was carried by the\nNVs (Fig. 3e-h and Supplementary Fig. 20a-c). Tetramer staining in DBA/1 mice identified\nan increase in antigen-specific T after treatment (Supplementary Fig. 20d). Functional\nregs\nco-cultures demonstrated that CII-specific T more effectively reduced DC costimulatory\nregs\nmolecules and shifted cytokines than irrelevant-antigen T under collagen restimulation\nregs\n(Fig. 3i and Supplementary Fig. 21a,b). In CIA mice, therapeutic differences between\nheterologous-antigen and cognate-antigen formulations further supported antigen\ndependence (Fig. 6 a-f, G7 vs G9). These converging data support an antigen-specific T -\nreg\ndependent mechanism of DC-CD4 bridging nanosystem.\nTherapeutic timing is another key determinant of precision immunomodulation.\nWithin the same disease model, we systematically evaluated prophylactic (Supplementary\nFig. 28), early intervention (Fig. 4,5), and late treatment regimens (Supplementary Fig. 27),\nand incorporated a re-challenge setting to probe durability (Fig. 6a-g). Pre-conditioning and\nearly-intervention are intrinsically more tractable than treating established disease.\nNevertheless, the bispecific NVs improved disease outcomes across all regimens, and the\nre-challenge study indicated sustained disease control. Collectively, the data support a\nmodel in which bispecific NVs seed a durable pool of antigen-specific T in LNs and\nregs\nspleen that restrains effector priming upon antigen re-encounter, enabling both induction\nand maintenance of remission across flare-remission cycles seen in patients with RA.\nmanufacture still requires short-term, ex vivo-induced iCD4 as the source. Autologous\nsources may mitigate immunogenicity, whereas banked allogeneic material could facilitate\nstandardization and lower-cost manufacturing if residual allo-epitopes are minimized. In\naddition, we only focused on a single antigen in a classical CIA model, whereas human RA\ninvolves a diverse and evolving autoantigen repertoire, including post-translationally\nmodified epitopes (e.g., citrullinated proteins) and epitope spreading. Future variants\nshould incorporate multi-antigen or patient-tailored payloads and be benchmarked in more\nheterogeneous, relapse-prone, or humanized models to better reflect clinical complexity.\nNote: References are not included in this version.]\nQ3: A clearer schematic explaining the sequential steps for making the nanoparticles\nis needed.\nA3: We thank the reviewer for this helpful suggestion. In the revised manuscript, we\nhave redrawn the schematic to more clearly illustrate the sequential steps involved in NV\npreparation. The new figure now explicitly depicts:\n(i) isolation of CD4⁺ T cells form spleen and LNs, initially expressing low levels of\nimmune inhibitory molecules and CTLA4, with these molecules clearly labeled on the cell\nsurface; (ii) their ex vivo conversion immunosuppressive CD4⁺ T cells (iCD4) with high\nlevels of immune inhibitory molecules and CTLA4; (iii) insertion of anti-CD4 (aCD4) into\nthe iCD4 membrane to generate aCD4-iCD4, with aCD4 explicitly labeled on the cell\nsurface, and (iv) co-extrusion of aCD4- iCD4, antigen peptide (AP) and rapamycin (RM) to\ngenerate AP-RM@aCD4Tn. In addition, we have used braces to group related\ncomponents and steps, thereby providing a more intuitive stepwise overview of the NV\nfabrication process.\nThe revised figure as follows:\nFig.1 | a, Schematic illustration of the fabrication process of DC-CD4 bispecific tolerogenic\nNVs (AP-RM@aCD4Tn). Created with Biorender.com.\ndevelop a cell free therapy that did not come to fruition?\nA4: We thank the reviewer for this thoughtful comment. Our initial goal was indeed to\ndevelop immunosuppressive CD4⁺ T cells (iCD4)-derived exosomes (Te) as a tolerogenic\ntherapy. However, we found that the practical yield of Te was too low for scalable\napplication. In head-to-head comparisons, at an equivalent injected protein dose, Tn\n(iCD4-derived nanovesicles) and Te exhibited comparable LN targeting efficiency\n(Supplementary Fig. 2b), while the protein yield of Tn was 4.8-fold higher than that of Te\n(Supplementary Fig. 2c). Thus, both Te and Tn function as acellular formulations, but Tn\noffers a substantially more favorable production profile. Based on these data, we decided\nto focus on Tn for all subsequent experiments. To better convey this rationale, we have\nreorganized the corresponding figure set and revised the related description in the Results\nsection.\nThe revised figures and accompanying text read as follows:\nSupplementary Fig. 2. Comparison of Te and Tn on LN-targeting. a, Experimental\nscheme for testing LN-targeting in DBA/1 mice. b, Fluorescent images and intensity of\naxillary lymph node (aLN) and inguinal lymph node (iLN) from mice treated with same\nprotein amounts of Te or Tn (n=5). c, Protein amount of Te and Tn derived from same\nnumbers of iCD4 (n=3). d, Fluorescent images and intensity of aLN and iLN from mice\ntreated with Te or Tn derived from same numbers of iCD4 (n=5). Data are presented as\nmean ± SD, statistical significance was analyzed by unpaired two-tailed t-test.\n56\n[Results section:\nFor CII-RM@aCD4Tn preparation, NVs derived from iCD4 (referred to as Tn) were\nprepared through sequential extrusion, while Te were exosomes isolated from iCD4. LN\ntargeting of Tn and Te were compared (Supplementary Fig. 2a). After i.d. injection for 24 h,\nTn and Te illustrated comparable LN-targeting efficiency (P=0.7395) when the injected\nprotein amounts were matched (Supplementary Fig. 2b). Notably, Tn displayed higher\nproduction efficiency, yielding 4.8-folder more protein amounts than Te (Supplementary Fig.\n2c). When normalized to the same number of parent cells, Te showed compromised LN-\ntargeting efficacy compared to Tn (P<0.0001) (Supplementary Fig. 2d), underscoring the\nadvantages of Tn in this context.]\nQ5: It is not obvious why Tn mediated antigen delivery increases surface display of\nthe CII peptide so dramatically. The authors should discuss the mechanism for that\nincrease.\nA5: We thank the reviewer for this important question. Although we did not directly\ndissect each step of antigen processing in this study, we believe the markedly enhanced\nsurface display of CII peptide after Tn-mediated delivery most likely arises from a\ncombination of the following mechanisms:\nFirst, CII-RM@aCD4Tn presents iCD4-derived membrane enriched in\ninhibitory/adhesion molecules (e.g., CTLA4 Supplementary Fig. 1c), which can strengthen\nphysical interactions with DCs via CTLA4-CD80/86 engagement. Consistent with this\nconcept, we verified the functional CTLA4-CD80/86 interaction in our Supplementary Fig.\n10 experiments. Enhanced and prolonged NV-DC contact would be expected to increase\nDC uptake of the antigen cargo, thereby elevating the amount of peptide available for\nprocessing and subsequent MHC-II loading, ultimately resulting in higher surface\npresentation.\nSecond, NV delivery generally improves antigen uptake by DCs relative to soluble\nantigen, a phenomenon that has been widely observed in antigen delivery systems and is\nconsistent with prior work (e.g., Tolerogenic nanoparticles induce type II collagen–specific\nregulatory T cells and ameliorate osteoarthritis, Science advance, 2022)13. Increased\nintracellular antigen availability would in turn be expected to increase peptide-MHC\ncomplex formation and surface display.\nTaken together, we propose that iCD4 membrane-mediated DC engagement (via\nCTLA4-CD80/86) and improved particulate uptake synergistically increase antigen delivery\n57\ninto DCs, thereby amplifying peptide loading and surface presentation. We have added a\ndiscussion of these potential mechanisms in the revised manuscript.\n[Discussion section:\nSeveral NV principles from our data help explain the outstanding tolerant performance.\nFirst, Ag-RM@aCD4Tn enhance antigen presentation by DCs (Fig. 1h,i, and\nSupplementary Fig. 7). This effect likely reflects the synergistic contributions of enhanced\nDC engagement driven by the iCD4 membrane interface (e.g., CTLA4-CD80/86), which\nprolongs contact (Supplementary Fig. 10) and promotes uptake (Fig. 1f,g), and intrinsically\nhigher internalization of particulate cargo compared with soluble antigen6.\nReferences\n6. Sohn, H.S. et al. Tolerogenic nanoparticles induce type II collagen-specific\nregulatory T cells and ameliorate osteoarthritis. Sci. Adv. 8, eabo5284 (2022).]\nQ6: Figure 2n showns mRNA levels of TNFa and TGFb. In the conclusion of this\nsection, it is stated that secretion of anti-inflammatory cytokines is enhanced. Can TNFa\nand TGFb protein be measured in addition to mRNA levels?\nA6: We thank the reviewer for this constructive suggestion. In our study, TNF-α and\nTGF-β protein levels were indeed quantified in the culture supernatants (previously shown\nin Extended Data Fig. 2b). To better align the data presentation with the conclusion that\nanti-inflammatory cytokine secretion is enhanced, we have now moved the protein data for\nTNF-α and TGF-β into the main figure (Fig. 1k and m) and relocated the corresponding\nmRNA results to the Supplementary Information (Supplementary Fig. 8b and\nSupplementary Fig. 9b).\nQ7: The meaning of the sentence in lines 202-203 is unclear. This should be revised.\nA7: We thank the reviewer for pointing out this lack of clarity. The original sentence in\nlines 202-203 (now represented in lines 194-197) was ambiguously phrased. We have now\nrevised it to more precisely describe the flow cytometry (FC) based conjugate analysis as\nfollows:\n[Results section:\nFC analysis of DC-CD4 conjugates showed that, in the presence of CII-RM@Tn,\nCD4⁺ T cells and BMDCs largely remained as single, unpaired cells, whereas CII-\nRM@aCD4Tn markedly increased the proportion of BMDC-CD4⁺ T cell pairs compared to\nCII-RM@Tn group (P<0.0001) (Fig. 2b).]\nQ8: The data presented in Figure 3 E and F appears to lack a statistical analysis.\nA8: We thank the reviewer for pointing this out. In original Fig. 3e,f (now presented as\nFig. 2e-g and Supplementary Fig. 13 in the revised manuscript), we have now performed\nstatistical analysis of the relative FI in LNs between the different injection methods. We\nreanalyzed the dataset and converted it into a bar graph summarizing the overall relative\nFI of LNs for unilateral versus bilateral injection (now presented as Fig. 2g). This\npresentation, together with the added statistical comparison, more clearly demonstrates\nthat bilateral (both-flank) injection results in higher total relative LN FI than unilateral\ninjection.\nThe revised figures and accompanying text read as follows:\nFig. 2 | DC-CD4 bridging ability and biodistribution of CII-RM@aCD4Tn. e, Relative\nfluorescence intensity (FI) of main organs (n=5). f, The proportion of FI in each organ after\nboth side injection (n=5). g, Relative FI of total LNs (n=5).\nSupplementary Fig. 13. The proportion of FI in each organ following unilateral\ninjection.\n[Results section:\nTo assess LN-targeting capability, Cll-RM@aCD4Tn was administered via i.d. injection\ninto the left or right flank of DBA/1 mice. Following unilateral injection, in vivo fluorescence\nimaging revealed that the Cy5-NVs localized exclusively to the ipsilateral inguinal and\naxillary LNs (aLN), with minimal signal detected in major organs (Fig. 2d,e and\niLNs and aLNs account for 80.6% of the total FI, which was significantly higher than\nunilateral injection (left P=0.0027, right P=0.0001) (Fig. 2f,g). These results indicated that\nadministration at both flanks effectively delivers Cll-RM@aCD4Tn to systemic LNs\nthroughout the body, potentially exerting enhance immune modulation.]\nQ9: In section 4 of the results, localization of NPs is followed for 7 days. Is localization\nin the lymph nodes still observed beyond 7 days? Knowing this may be important to discern\nwhether the NPs could function as a one time treatment, or if recurrent treatment would be\nrequired.\nA9: We thank the reviewer for this thoughtful comment. To address whether disease\nrelapses after treatment cessation, we performed a re-challenge study in CIA mice. We\nmonitored the biodistribution of Cy5-labeled CII-RM@aCD4Tn over an extended time\ncourse by IVIS imaging of the ipsilateral iLNs at different time points after i.d. injection. We\nfound that the fluorescence signal in the iLNs lagged behind that at the injection site,\npeaked at day 1, and then gradually declined (Fig. 2j). Importantly, approximately 13.1%\nof the maximal iLN fluorescence intensity was still detectable on day 7, whereas by day 9\nthe signal had dropped to near-background levels (~3.3%) (Fig. 2k).\nThese data indicate that NV accumulation in the draining LNs is largely confined to\nthe first week after administration, suggesting that the major immunomodulatory effects\nare exerted during this window. We have now included these extended biodistribution\nresults in Fig. 2j,k.\nThe added figures and accompanying text read as follows:\nFig. 2 | CD4 T-DC bridging ability and biodistribution of CII-RM@aCD4Tn. g,\nFluorescence image of iLN at indicated time points up to 15 days after i.d. infusing Cy5-\nCII-RM@aCD4Tn (n=3). k, Relative FI curve of iLN (n=3).\n[Methods section:\nBiodistribution of CII-RM@aCD4Tn\nConcurrently, the FI in the ipsilateral iLN was monitored for up to 13 days.\n2j,k). On day 7, ~13.1% of the maximal iLN FI remained, falling to near-background levels\non day 9 (~3.3%) (Fig. 2k).]\nQ10: In section 5, it is unclear why key experiments were performed using Ova 323-\n339 rather than the CII peptide. Could similar results be obtained with the autoantigenic\npeptide?\nA10: We thank the reviewer for this important comment. We initially chose OVA\n323-339\nas a model antigen because OT-II CD4⁺ T cells carry a transgenic TCR that specifically\nrecognizes this epitope, allowing highly controlled and sensitive analysis of antigen-specific\nT cell and T responses. This system is widely used to dissect mechanistic aspects of\nreg\nantigen-specific tolerance, as it enables us to distinguish cognate from non-cognate\nresponses with high precision14-16.\nTo directly address the reviewer’s question and to determine whether similar effects\ncan be achieved with the autoantigenic peptide, we performed additional experiments in\nboth OT-II and DBA/1 mice. Animals were pre-treated with OVA (G1), OVA-RM@aCD4Tn (G2),\nCII (G3), or CII-RM@aCD4Tn (G4). Splenocytes were then restimulated ex vivo with\neither OVA or CII, and IFN-γ-producing cells were quantified by ELISPOT. In OT-II mice,\nOVA restimulation showed a marked reduction in IFN-γ-secreting cells only when mice\nwere pre-treated with OVA-RM@aCD4Tn (G1 vs G2), whereas pre-treatment with CII-\nRM@aCD4Tn had minimal effect (G2 vs G4) (Fig. 3e,f). Conversely, in DBA/1 mice, CII\nrestimulation demonstrated that CII-RM@aCD4Tn produced a significantly stronger\nsuppression of IFN-γ-producing cells compared with OVA-RM@aCD4Tn (G2 vs G4) (Fig.\n3g,h). Moreover, cytokine levels of TNF-α and TGF-β in the culture medium were also\nquantified, and the antigen-specific suppression were likewise observed (Supplementary\nFig. 20a-c).\nWe further use I-Aᵇ OVA tetramer to identify antigen-specific T which provide\n323-339 regs\ndirect evidence for antigen specificity. Because OT-II CD4⁺ T cells inherently recognize\nOVA and are readily stained by the OVA tetramer even without treatment, this\n323-339\napproach cannot be used to distinguish induced antigen-specific T in OT-II mice.\nregs\nTherefore, we performed the tetramer experiment in DBA/1 mice, where endogenous CD4⁺\nT cells do not pre-recognize OVA. Splenocytes from DBA/1 mice pre-treated with OVA-\nRM@aCD4Tn (G2) showed a significant increase in OVA tetramer⁺ T following\n323-339 regs\nOVA stimulation, compared with mice receiving free OVA (G1) (Supplementary Fig. 20d).\nIn contrast, mice pre-treated with CII or CII-RM@aCD4Tn did not show induction of OVA-\nspecific T upon OVA restimulation (G3 and G4) (Supplementary Fig. 20d).\nregs\nThese results clearly demonstrate that tolerance induction by Ag-RM@aCD4Tn is\nantigen-specific, as only cognate antigen re-stimulation produce a robust suppressive\neffect.\nThe added figures and accompanying text read as follows:\nFig. 3 | T generation and antigen-specific immunosuppressive by Ag-\nregs\nRM@aCD4Tn in vivo. e,f, Representative ELISPOT images (e) and quantification (f) of\nIFN-γ-secreting splenocytes from OT-II mice treated with the indicated formulations after\nstimulation with either OVA or CII. g,h, Representative ELISPOT images (g) and\n323–339\nquantification (h) of IFN-γ-secreting splenocytes derived from DBA/1 mice. i, Cll-specific\nT -mediated BMDC suppression in the presence of LPS and CII, as evaluated by CD80\nreg\nand CD86 expression on BMDC (n=5).\nof I-Ab OVA + T following antigen restimulation of splenocytes. a,\n323-339 regs\nExperimental scheme of antigen-specific tolerance experiment in OT-II and DBA/1 mice.\nb, The levels of TNF-α and TGF-β in splenocytes from OT-II mice following ex vivo\nstimulation with OVA or CII (n=5). c, The levels of TNF-α and TGF-β in splenocytes\n323-339\nfrom DBA/1 mice (n=5). d, Percentage of I-Ab OVA + T in the splenocytes from\n323-339 regs\nDBA/1 mice (n=5). Data are presented as mean ± SD, statistical significance was analyzed\nby One-way ANOVA with Tukey’s post hoc test.\n[Methods section:\nAntigen-specific T induction in vitro and in vivo\nreg\nTo assess antigen-specific tolerance, OT-II and DBA/1 mice were injected i.d. at both\nflanks with OVA or CII encapsulated NVs. After that, splenocytes were harvested for IFN-\nγ ELISPOT assays to evaluate the specific activation. Splenocytes were seeded into\nELISPOT plates with 1 × 106 cells per well, and stimulated with 10 μg ml-1 OVA or 10 μg\nml-1 CII respectively. After 24 h, supernatants were collected for TNF-α and TGF-β\ndetection by using ELISA kits, and IFN-γ-secreting cells were quantified according to the\nmanufacturer’s instructions. Furthermore, OVA-specific T in splenocytes from DBA/1\nreg\nmice were analyzed by staining with I-Ab OVA Tetramer.\nResults section:\nTo determine whether the induced immunosuppression was antigen-specific,\nsplenocytes isolated from OT-II or DBA/1 mice pre-immunized with different formulations\nwere re-stimulated ex vivo with either OVA or CII, and IFN-γ-producing cells in splenocytes\nwere quantified by ELISPOT (Supplementary Fig. 20a). In OT-II mice, OVA restimulation\nrevealed a marked reduction in IFN-γ-secreting cells after pretreatment with OVA-\nRM@aCD4Tn (G1 vs G2, P<0.0001), corresponding to a 71.6% decrease (Fig. 3e,f).\nPretreatment with CII-RM@aCD4Tn produced a 30.5% reduction, which was significantly\nweaker than that induced by OVA-RM@aCD4Tn (G2 vs G4, P<0.0001) (Fig. 3e,f).\nConversely, in DBA/1 mice, CII restimulation demonstrated that CII-RM@aCD4Tn\nproduced significantly suppression of IFN-γ-producing cells than OVA-RM@aCD4Tn (G2\nvs G4, P<0.0001) (Fig. 3g,h). Consistent with these findings, cytokine levels of TNF-α and\nTGF-β in the culture medium were also quantified by ELISA, and the antigen-specific\nsuppression were likewise observed (Supplementary Fig. 20b,c). To directly detect\nantigen-specific T , I-Aᵇ OVA tetramer was used. Because OT-II CD4⁺ T cells are\nregs 323-339\ninherently OVA reactive and exhibit robust baseline tetramer staining even without\ntreatment, the assay was performed in DBA/1 mice. Upon OVA restimulation, OVA-\nRM@aCD4Tn pretreatment led to an obvious increase in I-Aᵇ OVA tetramer⁺ T\n323-339 regs\ncompared to free OVA (G1 vs G2, P=0.0027), whereas CII or CII-RM@aCD4Tn\npretreatment failed to induce OVA-specific T (Supplementary Fig. 20d).\nregs\nQ11: The KEGG pathway data discussed in section 6 does not seem that informative.\nAre there other published transcript signatures that might be better suited to this analysis?\nA11: We thank the reviewer for this insightful comment. We agree that KEGG pathway\nenrichment analysis provides a broad, pathway-level overview and is not optimally suited\nto capture transcriptional programs specifically associated with immune tolerance\ninduction.\nIn response to this suggestion, we have revised both the organization and emphasis\nof the transcriptomic analysis. Specifically, the KEGG pathway enrichment results have\nbeen moved to the Supplementary Information (Supplementary Fig. 23), where they are\npresented as supportive data. In the revised main manuscript, we place greater emphasis\non Gene Ontology (GO) analysis and gene-level expression changes, which more directly\nreflect immune regulatory and tolerogenic biological processes.\nImportantly, the biological conclusions of the study do not rely on pathway-level\nenrichment per se, but rather on the coordinated regulation of canonical tolerance-\nassociated genes (including Foxp3, Il10, Tgfb1, Ctla4, and Il1rn), the suppression of pro-\ninflammatory mediators (Il6, Tnf, and Il17a), together with extensive in vivo functional\nvalidation (Fig.3 a-i and Supplementary Fig. 19-21). The KEGG analysis remains\nconsistent with these observations and is therefore retained as complementary information.\nWe believe that this revision improves the clarity and biological relevance of the\ntranscriptomic section while preserving the completeness and transparency of the analysis.\nWe appreciate the reviewer’s suggestion, which has helped us refine the presentation of\nthese data.\nThe revised text read as follows:\n[Results section:\nTo further elucidate the immunosuppressive mechanism mediated by OVA-\nRM@aCD4Tn, we performed transcriptomic analysis on CD4+ T cells isolated from the LNs\nof OT-II mice 4 days post-administration. Principal components analysis (PCA) revealed a\nhigh degree of similarity in gene expression profiles between OVA-RM@Tn and OVA-\nRM@aCD4Tn groups (Supplementary Fig. 22a). However, the magnitude of gene\n64\nexpression changes in OVA-RM@aCD4Tn group was more pronounced than in OVA-\nRM@Tn group when compared to OVA group (Supplementary Fig. 22b). In OVA-\nRM@aCD4Tn-treated mice, the cluster heatmap analysis illustrated a downregulation of\npro-inflammatory mediators, including Il6, Tnf, Mmp9, Il1b and Il17a, alongside a\nupregulation of anti-inflammatory genes such as Il10, Foxp3, Tgfb1, Ctla4 and Il1rn (Fig.\n3j), with these regulatory effects being more pronounced than those observed in the OVA-\nRM@Tn group. Gene ontology (GO) analysis of differentially expressed genes revealed\nenrichment of biological processes related to immune regulation and inflammatory\nresponse after OVA-RM@aCD4Tn intervention (Fig. 3k). Kyoto Encyclopedia of Genes\nand Genomes (KEGG) pathway enrichment further supported modulation of immune-\nrelated signaling, such as TGF-β, T cell receptor and TNF pathways (Supplementary Fig.\n23). Taken together, OVA-RM@aCD4Tn exerted enhanced immunomodulatory program\ncompared to OVA-RM@Tn.]\nQ12: The fluorescence image in figure 6 A is difficult to read.\nA12: We thank the reviewer for this helpful comment. In the revised manuscript, we\nhave re-analyzed the sections and updated Fig. 6a accordingly. First, we increased the\nmagnification and added higher-resolution images so that individual positive cells can be\nmore clearly discerned. Second, we now quantify the immunofluorescence as Foxp3⁺ area\nnormalized to CD4⁺ area (Foxp3⁺ area/CD4⁺ area), which more accurately reflects the\nproportion of Foxp3⁺ T cells within the CD4⁺ compartment.\nThe revised figures and accompanying text read as follows:\nFig. 5. | a, Immunofluorescent staining images for CD4+ CD25+ Foxp3+ T in the\nregs\nsynovium. Scale bars, 60 μm. Blue, DAPI; Red, CD4; Green, CD25; Yellow, Foxp3. b,\nFoxp3+ area in synovium (n=6). c, Foxp3+ area/CD4+ area in synovium (n=6).\n65\n[Results section:\nImmunofluorescence analysis revealed a marked increase in the proportion of Foxp3+\nT in the synovium after CII-RM@aCD4Tn treatment (Fig. 5a-c).]\nregs\nQ13: In section 7, mice are sacrificed 14 days post treatment. If mice are kept for\nlonger; does the disease come back?\nA13: We thank the reviewer for this important question. To address whether disease\nrelapses after treatment cessation, we performed a re-challenge study in CIA mice.\nAfter completion of the initial three-dose regimen, mice were re-challenged with CII\nantigen on day 42 and disease progression was subsequently monitored (Fig. 6h). Notably,\neven without additional treatment (G3 and G4), mice in CII-RM@aCD4Tn group\nmaintained substantial disease control, whereas mice in CII-RM@Tn group exhibited clear\ndisease exacerbation, with the arthritis score increasing from 3.7 (day 42) to 7.2 (day 57)\n(Fig. 6i). Upon administration of a single redose on day 43, CII-RM@aCD4Tn group\nshowed the most pronounced therapeutic benefit, with the arthritis index reduced to 1.5\n(Fig. 6i). In contrast, CII-RM@Tn group displayed a partial improvement, with the arthritis\nindex decreasing from 7.2 to 5.0 (Fig. 6i). Paw thickness, behavioral assessments and\nH&E staining of ankle joints demonstrated consistent trends with the arthritis score (Fig.\n6j-m), further supporting the effectiveness of CII-RM@aCD4Tn in re-challenge CIA model.\nWe appreciate the reviewer’s insightful comment, which significantly strengthened our\nstudy. The added figures and accompanying text read as follows:\nPaw thickness on day 57 (n=6). k, Motor evaluation of CIA mice on day 57 using rotarod\nsystem (n=6). l,m, Representative H&E section images of ankle (l) and histopathological\nevaluation of synovitis score (m) (n=6).\n[Methods section:\nRe-challenge study of CII-RM@aCD4Tn in CIA mice\nTo investigate therapeutic durability, DBA/1 mice that had completed the initial three-\ndose treatment were re-challenged with CII antigen on day 42. Male DBA/1 mice were\nrandomly assigned to six groups, and each group contained 6 mice. One group was healthy\n(G1), while the other five groups included PBS (G2), 1.2 mg CII-RM@Tn (G3), 1.2 mg CII-\nRM@aCD4Tn (G4), 1.2 mg CII-RM@Tn with redose (G5) and 1.2 mg CII-RM@aCD4Tn\nwith redose (G6). Each dose of CII-RM@Tn or CII-RM@aCD4Tn contained 150 μg CII and\n20 μg RM. On day 42, 100 μl CII emulsion (CII:IFA=1:1) was injected i.d. at both sides of\nthe tail base. On day 43, mice in groups G5 and G6 received a single additional (redose)\ninjection of the corresponding formulation. Arthritis scores and body weight were monitored\nevery three days. Paw thickness and motor performance (rotarod test) were assessed on\nday 57, and hind paws were collected for histological analysis by H&E staining.\nResults section:\nIn order to validate the therapeutic durability of Cll-RM@aCD4Tn, a re-challenge study\nwas investigated in CIA mice (Fig. 6h). Even without additional treatment (G3 and G4),\nmice previously treated with CII-RM@aCD4Tn maintained better disease control than\nthose had received CII-RM@Tn, in which arthritis score increased from 3.7 (day 42) to 7.2\n(day 57) (Fig. 6i). Upon redosing on day 43 (G5 and G6), CII-RM@aCD4Tn group\nconferred the greatest therapeutic benefit, with arthritis score reduced to 1.5, and CII-\nRM@Tn group displayed a partial improvement, with arthritis score decreasing from 7.2 to\n5.0 (Fig. 6i). Changes in paw thickness, behavioral assessments and H&E staining of ankle\njoints demonstrated consistent trends with the arthritis score (Fig. 6j-m), further supporting\nthe sustained efficacy of CII-RM@aCD4Tn in re-challenge CIA model.]\nQ14: The promising results from different versions of the arthritis model are impressive.\nHowever, it must be mentioned that pre-conditioning and prevention settings are easier to\nmitigate than established disease.\nA14: We thank the reviewer for this thoughtful comment. We fully agree that pre-\nconditioning and prevention settings are generally easier to mitigate than established\ndisease, and that this distinction is important when interpreting the therapeutic efficacy of\ntolerance-inducing strategies. In our study, we therefore evaluated CII-RM@aCD4Tn in\nfour different CIA settings: (i) an early treatment regimen, in which nanoformulations were\nadministered on days 22, 25, and 28 after CIA induction; (ii) a late-phase treatment regimen,\nin which CII-RM@Tn or CII-RM@aCD4Tn was administered on days 27, 30, and 33, after\nmice had developed evident paw swelling and redness; (iii) a preventive regimen, in which\nCII-RM@aCD4Tn was given three and two weeks prior to CIA induction; and (iv) a re-\nchallenge model, in which mice that had completed the initial three-dose treatment were\nre-challenged with CII on day 42 and followed with or without a single redose.\nTo better reflect this limitation, we have added a statement in the preventive Results\nand Discussion explicitly noting that prevention settings are intrinsically easier to control\nthan fully established disease. Importantly, as addressed in our response to Q13, we have\nalso established a relapsing arthritis model, in which the durability of this approach is\nfurther validated.\n[Results section:\nEarly therapeutic\nTo evaluate the early therapeutic potential of this system, different nanoformulations\nwere administered on day 22, 25 and 28 to treat collagen induced arthritis (CIA) mice (Fig.\n4a).\nLate-phase therapeutic\nTo assess late-phase therapeutic efficacy in CIA model, CII-RM@Tn or CII-\nRM@aCD4Tn was administered on day 27, 30 and 33, after mice had developed evident\npaw swelling and redness (Supplementary Fig. 27a)\nPreventive efficacy\nIn order to investigate the preventive efficacy of CII-RM@aCD4Tn, mice received\ninjections three and two weeks prior to CIA induction (Supplementary Fig. 28a),\nrepresenting a preconditioning setting that is intrinsically more favorable than treatment of\nfully established disease.\nRe-challenge efficacy\nIn order to validate the therapeutic durability of Cll-RM@aCD4Tn, a re-challenge study\nwas investigated in CIA mice (Fig. 6h).\nDiscussion section:\nTherapeutic timing is another key determinant of precision immunomodulation55.\nWithin the same disease model, we systematically evaluated prophylactic (Supplementary\nFig. 28), early intervention (Fig. 4,5), and late treatment regimens (Supplementary Fig. 27),\nand incorporated a re-challenge setting to probe durability (Fig. 6a-g). Pre-conditioning and\nearly-intervention are intrinsically more tractable than treating established disease.\nNevertheless, the bispecific NVs improved disease outcomes across all regimens, and the\nre-challenge study indicated sustained disease control. Collectively, the data support a\nmodel in which bispecific NVs seed a durable pool of antigen-specific T in LNs and\nregs\nspleen that restrains effector priming upon antigen re-encounter, enabling both induction\nand maintenance of remission across flare-remission cycles seen in patients with RA56, 57.\nReferences\n55. Liu, H. & Liang, Q. Single-cell multi-omics-based immune temporal network\nresolution in sepsis: unravelling molecular mechanisms and precise therapeutic\ntargets. Front Immunol. 16, 1616794 (2025).\n56. Di Matteo, A., Bathon, J.M. & Emery, P. Rheumatoid arthritis. The Lancet 402,\n2019-2033 (2023).\n57. Emery, P. et al. Longterm drug survival of tumor necrosis factor inhibitors in patients\nwith rheumatoid arthritis. J. Rheumatol. 47, 493-501 (2020).]\nsection nicely.\nA15: We thank the reviewer for this encouraging comment. We agree that the adoptive\ntransfer experiments provide key functional evidence supporting the central role of CII-\nRM@aCD4Tn induced Foxp3⁺ T in mediating the therapeutic effects.\nregs\nQ16: The paper would benefit from having a discussion section, where the discussion\npoints requested above could be included, along with brief comments about further testing\nand potential translation of this technology.\nA16: We thank the reviewer for this constructive suggestion. In response, we have\nadded a dedicated Discussion section to the revised manuscript.\nIn the Discussion, we first place our work within the broader landscape of T -directed\nreg\ntherapies and antigen-bearing nanomedicines, highlighting ongoing clinical efforts and\ntheir limitations in persistence, manufacturability, and antigen specificity. We then explain\nhow our DC-CD4 bridging nanovesicle platform adapts multi-specific synapse-organizing\nprinciples from oncology to promote tolerogenic, rather than stimulatory outcomes, and\noutline key mechanistic features underlying its activity, including enhanced DC antigen\npresentation, efficient LN targeting, preferential engagement of DCs and CD4⁺ T cells, and\nenforced DC-CD4 proximity. We next summarize convergent evidence for an antigen-\nspecific, T -dependent mechanism from recall assays, tetramer staining, functional T\nreg reg\nco-cultures, and cognate versus heterologous antigen comparisons in CIA. We also\ndiscuss the impact of therapeutic timing across prophylactic, early-intervention, late-\ntreatment, and re-challenge regimens, supporting the notion that the platform seeds\ndurable antigen-specific T pools that restrain effector responses upon re-encounter.\nreg\nFinally, we acknowledge key limitations, such as reliance on ex vivo induced iCD4 as\nsource and evaluation in a single-antigen CIA model, and outline future directions toward\nmulti-antigen or patient-tailored formulations, testing in more complex and relapsing\nmodels, and further development of this modular, antigen-specific tolerance strategy for\ntranslation.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nThe authors address an important problem, that of antigen-specific tolerance, by\ndeveloping a bispecific nanoparticle delivering antigen peptide and rapamycin to induce\ntolerogenic DCs, inactive CD4s, and T which leads to increased anti-inflammatory\nregs\ncytokine levels and decreased proinflammatory mediators. They show the colocalization\nof the nanoparticle with DCs and CD4 T cells, as well as increased and sustained drug\nlocalization and the animal experiments are good to support efficacy and immune response\nto treatment, and support a MOA at least partially related to T induction/expansion. Yet,\nregs\nthis manuscript lacks some of the necessary controls and often uses exaggerated or\nunsubstantiated language. In general, there is no evidence for the antigen specificity of this\nnanoparticle. For example, in section 5, CII stimulation is not an appropriate comparison,\nas there is not expected to be a reaction; a better control would be a CII-RM@aCD4Tn\nwith OVA stimulation. In subsequent experiments, another control should be RM@aCD4Tn,\nto look at the impact of the actual antigen peptide on efficacy and/or RM@aCD4Tn loaded\nwith a different peptide than is being tested, and also rapamycin alone in animal\nexperiments. Specific issues are listed below.\nQ1: (i) Claiming antigen-specific tolerance is a high bar. As currently written, there is\nnot strong enough justification for the use of “antigen-specific” to describe cells.\n(ii) Some more controls as listed above would be appropriate. In addition, some\nnegative controls were not needed.\n(iii) At a minimum, the manuscript needs to be rewritten with accurate language for\nwhat they show (including removing antigen specificity until proven, quantifying results,\nand using precise language for the degree and specificity of changes).\nA1: (i) We thank the reviewer for this important comment and agree that claiming\nantigen-specific tolerance sets a high bar and requires rigorous experimental support.\nTo more directly address this concern, we performed additional experiments in both\nOT-II and DBA/1 mice. Animals were pre-treated with OVA (G1), OVA-RM@aCD4Tn (G2),\nCII (G3), or CII-RM@aCD4Tn (G4). This analysis was performed within the same\nexperimental cohort used to address Reviewer #1 (Q13, Q14), Reviewer #2 (Q3),\nReviewer #3 (Q10), and Reviewer #6 (Q1). Splenocytes were then re-stimulated ex vivo\nwith either OVA or CII, and IFN-γ-producing cells were quantified by ELISPOT. In OT-II\nmice, OVA restimulation revealed a marked reduction in IFN-γ-secreting cells after\npretreatment with OVA-RM@aCD4Tn (G1 vs G2, P<0.0001), corresponding to a 71.6%\ndecrease (Fig. 3e,f). Pretreatment with CII-RM@aCD4Tn produced a 30.5% reduction,\nwhich was significantly weaker than that induced by OVA-RM@aCD4Tn (P<0.0001) (Fig.\n73\n3e,f). Conversely, in DBA/1 mice, CII restimulation demonstrated that CII-RM@aCD4Tn\nproduced stronger suppression of IFN-γ-producing cells than OVA-RM@aCD4Tn\n(P<0.0001) (Fig. 3g,h). Moreover, cytokine levels of TNF-α and TGF-β in the culture\nmedium were also quantified, and the antigen-specific suppression were likewise observed\n(Supplementary Fig. 20a-c).\nWe further use I-Aᵇ OVA tetramer to identify antigen-specific T which provide\n323-339 regs\ndirect evidence for antigen specificity. Because OT-II CD4⁺ T cells inherently recognize\nOVA and are readily stained by the OVA tetramer even without treatment, this\n323-339\napproach cannot be used to distinguish induced antigen-specific T in OT-II mice.\nregs\nTherefore, we performed the tetramer experiment in DBA/1 mice, where endogenous CD4⁺\nT cells do not pre-recognize OVA. Splenocytes from DBA/1 mice pre-treated with OVA-\nRM@aCD4Tn (G2) showed an increase in OVA tetramer⁺ T following OVA\n323-339 regs\nstimulation, compared with mice receiving free OVA (G1) (P=0.0027) (Supplementary Fig.\n20d). In contrast, mice pre-treated with CII or CII-RM@aCD4Tn did not show induction of\nOVA-specific T upon OVA restimulation (G3 and G4) (Supplementary Fig. 20d). Taken\nregs\ntogether, these data demonstrate that the Ag-RM@aCD4Tn platform is capable of inducing\nantigen-specific T in vivo, and that this induction strictly depends on loading the cognate\nregs\nantigen into the NVs.\nIn addition, the functional characterization of CII-specific T was investigated within\nregs\nthe same cohort described in our responses to Reviewer #1 (Q13), Reviewer #2 (Q3),\nand Reviewer #6 (Q1, Q3). DBA/1 mice were first intradermally pre-treated with either CII-\nRM@aCD4Tn or OVA-RM@aCD4Tn to induce CII- or OVA-specific T , respectively.\nregs\nCD4⁺CD25⁺ T were then isolated and co-cultured with BMDCs in the presence of LPS\nregs\nand CII (Supplementary Fig. 21a). When CII-specific T (from CII-RM@aCD4Tn treated\nregs\nmice) were added (G5), BMDC activation was markedly suppressed, as evidenced by\nsignificantly reduced surface expression of CD80 and CD86 (G3 vs G5, both P<0.0001)\n(Fig. 3i), a pronounced decrease in the pro-inflammatory cytokine TNF-α, and a\nconcomitant increase in the anti-inflammatory cytokine TGF-β in the culture supernatant\n(G3 vs G5, both P<0.0001) (Supplementary Fig. 21b). These findings indicate effective\nsuppressive function of CII-specific T in the presence of CII. In contrast, OVA-specific\nregs\nT (from OVA-RM@aCD4Tn-treated mice) (G4) exerted only modest inhibitory effects\nregs\non BMDC activation and cytokine production under the same CII restimulation conditions,\nwhich were weaker than those observed for CII-specific T (Fig. 3i and Supplementary\nregs\nFig. 21b). Before and after co-culture, T were stained for the key functional molecules\nregs\nCTLA4 and LAG-3, which are widely associated with enhanced T -mediated suppression.\nreg\nFollowing co-culture, T from both groups (G4 and G5) showed upregulation of CTLA4\nregs\n74\nand LAG-3 expression compared to unstimulated states (all P<0.0001), and CTLA4 and\nLAG-3 expression in CII-specific T were 1.7-fold and 1.3-fold higher than in OVA-\nregs\nspecific T (Supplementary Fig. 21c), suggesting their greater immunosuppressive\nregs\nactivity. These results provide functional evidence that CII-RM@aCD4Tn induces CII-\nspecific T in vivo, whose suppressive activity is preferentially engaged in the presence\nregs\nof the cognate CII antigen.\nThe revised figures and accompanying text read as follows:\nFig. 3 | T generation and antigen-specific immunosuppressive by Ag-\nregs\nRM@aCD4Tn in vivo. e,f, Representative ELISPOT images (e) and quantification (f) of\nIFN-γ-secreting splenocytes from OT-II mice treated with the indicated formulations after\nstimulation with either OVA or CII. g,h, Representative ELISPOT images (g) and\n323–339\nquantification (h) of IFN-γ-secreting splenocytes derived from DBA/1 mice. i, Cll-specific\nT -mediated BMDC suppression in the presence of LPS and CII, as evaluated by CD80\nreg\nand CD86 expression on BMDC (n=5).\nof I-Ab OVA + T following antigen restimulation of splenocytes. a,\n323-339 regs\nExperimental scheme of antigen-specific tolerance experiment in OT-II and DBA/1 mice.\nb, The levels of TNF-α and TGF-β in splenocytes from OT-II mice following ex vivo\nstimulation with OVA or CII (n=5). c, The levels of TNF-α and TGF-β in splenocytes\n323-339\nfrom DBA/1 mice (n=5). d, Percentage of I-Ab OVA + T in the splenocytes from\n323-339 regs\nDBA/1 mice (n=5). Data are presented as mean ± SD, statistical significance was analyzed\nby One-way ANOVA with Tukey’s post hoc test.\nregs\nillustration of co-culture CII or OVA-specific T with BMDCs in the presence of LPS and\nregs\nCII. b, Cytokine concentration of TNF-α and TGF-β in the medium (n=5). Data are\npresented as mean ± SD, statistical significance was analyzed by One-way ANOVA with\nTukey’s post hoc test.\n[Methods section:\nAntigen-specific T induction in vitro and in vivo\nreg\nTo assess antigen-specific tolerance, OT-II and DBA/1 mice were injected i.d. at both\nflanks with OVA or CII encapsulated NVs. After that, splenocytes were harvested for IFN-\nγ ELISPOT assays to evaluate the specific activation. Splenocytes were seeded into\nELISPOT plates with 1 × 106 cells per well, and stimulated with 10 μg ml-1 OVA or 10 μg\nml-1 CII respectively. After 24 h, supernatants were collected for TNF-α and TGF-β\ndetection by using ELISA kits, and IFN-γ-secreting cells were quantified according to the\nmanufacturer’s instructions. Furthermore, OVA-specific T in splenocytes from DBA/1\nreg\nmice were analyzed by staining with I-Ab OVA Tetramer.\nFor functional characterization of CII-specific T , DBA/1 mice were treated as\nregs\n77\ndescribed above, and CD4⁺ CD25⁺ T were isolated and cocultured with BMDCs in the\nregs\npresence of 50 ng ml-1 LPS and 10 μg ml-1 CII. After 24 h, culture supernatants were\ncollected for the measurement of TNF-α and TGF-β levels, and CD80/CD86 expression on\nBMDCs and CTLA4 and LAG-3 on T were analyzed by FC.\nregs\nResults section:\nTo determine whether the induced immunosuppression was antigen-specific,\nsplenocytes isolated from OT-II or DBA/1 mice pre-immunized with different formulations\nwere re-stimulated ex vivo with either OVA or CII, and IFN-γ-producing cells in splenocytes\nwere quantified by ELISPOT (Supplementary Fig. 20a). In OT-II mice, OVA restimulation\nrevealed a marked reduction in IFN-γ-secreting cells after pretreatment with OVA-\nRM@aCD4Tn (G1 vs G2, P<0.0001), corresponding to a 71.6% decrease (Fig. 3e,f).\nPretreatment with CII-RM@aCD4Tn produced a 30.5% reduction, which was significantly\nweaker than that induced by OVA-RM@aCD4Tn (G2 vs G4, P<0.0001) (Fig. 3e,f).\nConversely, in DBA/1 mice, CII restimulation demonstrated that CII-RM@aCD4Tn\nproduced significantly suppression of IFN-γ-producing cells than OVA-RM@aCD4Tn (G2\nvs G4, P<0.0001) (Fig. 3g,h). Consistent with these findings, cytokine levels of TNF-α and\nTGF-β in the culture medium were also quantified by ELISA, and the antigen-specific\nsuppression were likewise observed (Supplementary Fig. 20b,c). To directly detect\nantigen-specific T , I-Aᵇ OVA tetramer was used. Because OT-II CD4⁺ T cells are\nregs 323-339\ninherently OVA reactive and exhibit robust baseline tetramer staining even without\ntreatment, the assay was performed in DBA/1 mice. Upon OVA restimulation, OVA-\nRM@aCD4Tn pretreatment led to an obvious increase in I-Aᵇ OVA tetramer⁺ T\n323-339 regs\ncompared to free OVA (G1 vs G2, P=0.0027), whereas CII or CII-RM@aCD4Tn\npretreatment failed to induce OVA-specific T (Supplementary Fig. 20d).\nregs\nMoreover, the functional properties of CII-specific T were investigated\nregs\n(Supplementary Fig. 21a). CII-specific T markedly suppressed CII-induced BMDC\nregs\nactivation, as evidenced by reduced CD80 and CD86 expression and decreased TNF-α,\ntogether with increased TGF-β levels in the supernatant (G3 vs G5, all P<0.0001) (Fig. 3i\nand Supplementary Fig. 21b). In contrast, OVA-specific T exerted only modest effects\nregs\nunder the same CII restimulation conditions (Fig. 3i and Supplementary Fig. 21b).\nFollowing co-culture, T from both groups (G4 and G5) showed upregulation of CTLA4\nregs\n(OVA-specific T with P=0.0002, CII-specific T with P<0.0001) and LAG-3 (both\nregs regs\nP<0.0001) expression compared to unstimulated states, and CTLA4 and LAG-3\nexpression in CII-specific T were 1.7-fold and 1.3-fold higher than in OVA-specific T\nregs regs\n(Supplementary Fig. 21c), suggesting their greater immunosuppressive activity.\nCollectively, these data demonstrate that T induced by Ag-RM@aCD4Tn confer\nregs\n78\nantigen-specific immune tolerance that is engaged upon re-encounter with the cognate\nantigen.]\n(ii) We performed an additional CIA efficacy study to dissect the contribution of each\ncomponent and to include the requested controls, which was carried out within the same\ncohort used to address Reviewer #1 (Q4, Q10, Q12), Reviewer #2 (Q1), and Reviewer\n#6 (Q1).\nIn this experiment, we added groups receiving free rapamycin (G4: RM, 20 μg),\nrapamycin-loaded bispecific NVs without antigen (G5: RM@aCD4Tn), and bispecific NVs\nloaded with an irrelevant antigen peptide (G7: OVA-RM@aCD4Tn) (Fig. 6a).The results\nshowed that free RM (G4) failed to ameliorate disease, indicating that systemic rapamycin\nalone at this dose is insufficient to treat CIA (Fig. 6a-f and Supplementary Fig. 29).\nRM@aCD4Tn (G5) produced a moderate therapeutic effect, reducing paw swelling and\nlowering the arthritis score to 5.5 (Fig. 6b), suggesting that T membrane derived\nreg\ninhibitory molecules plus rapamycin can confer partial immunosuppression even in the\nabsence of antigen. However, OVA-RM@aCD4Tn (G7) showed efficacy similar to\nRM@aCD4Tn and was markedly inferior to CII-RM@aCD4Tn (Fig. 6a-f and\nSupplementary Fig. 29), demonstrating that loading an irrelevant antigen does not\nreproduce the robust protection observed with the cognate CII peptide.\nTogether, these new controls support the conclusion that ① rapamycin alone is not\nsufficient, ② the T membrane anchored with aCD4 contributes to baseline tolerogenic\nreg\nactivity, and ③ the full therapeutic benefit of CII-RM@aCD4Tn critically depends on\nincorporation of the disease relevant antigen, consistent with antigen-specific tolerance.\nThe added figures and accompanying text read as follows:\nscore curve (n=6). c, Paw thickness on day 42 (n=6). d, Motor evaluation of CIA mice on\nday 42 using rotarod system (n=6). e,f, Representative H&E section images of ankle (e)\nand histopathological evaluation of synovitis score (f) (n=6). g, The level of anti-CII IgG\nantibody in serum (n=6).\nSupplementary Fig. 29. Speed at falls of CIA mice on day 42 using rotarod system.\nData are presented as mean ± SD (n=6), statistical significance was analyzed by One-\nway ANOVA with Tukey’s post hoc test.\n[Methods section:\nMechanistic investigation of CII-RM@aCD4Tn\nCIA mice were established as above method. Mice were randomly divided into nine\ngroups, and each group contained 6 mice. One group served as a healthy (G1), whereas\n80\nthe other eight groups included PBS (G2), 150 μg CII (G3), 20 μg RM (G4), 1 mg\nRM@aCD4Tn (G5), 1.2 mg CII-RM@aCD4Tn (lacking CTLA4) (G6), 1.2 mg OVA-\nRM@aCD4Tn (G7), 150 μg CII + 1 mg RM@aCD4Tn (G8) and 1.2 mg CII-RM@aCD4Tn\n(G9). The amounts of CII and RM were identical across all groups. Notably, CTLA4 on the\nsurface of Tn was blocked via anti-CTLA4 antibody to generate group 6. The NVs were\ninjected on day 22, 25 and 28. Arthritis score and body weight were monitored every three\ndays. Paw thickness measurement and rotarod test were conducted on day 42. Hind paws\nwere collected for H&E staining. In addition, serum samples were obtained on day 42 for\nquantification of anti-CII IgG antibodies using an ELISA kit.\nResults section:\nTo further clarify the underlying therapeutic mechanism of Cll-RM@aCD4Tn, CIA mice\nwere treated with different formulations (Fig. 6a). Free CII (G3) or free RM (G4) failed to\ncontrol arthritis progression (Fig. 6a-f and Supplementary Fig. 29). To assess antigen\nspecificity, we included an antigen-nonspecific group, OVA-RM@aCD4Tn (G7). OVA-\nRM@aCD4Tn showed markedly reduced therapeutic efficacy compared to CII-\nRM@aCD4Tn, as reflected by higher arthritis scores (P=0.0003) and synovitis scores\n(P<0.0001), and its overall performance was comparable to that of RM@aCD4Tn (G5) (Fig.\n6a-f and Supplementary Fig. 29). When CII was administrated separately together with\nRM@aCD4Tn (G8), the efficacy remained inferior to that of co-loaded CII-RM@aCD4Tn\n(Fig. 6a-f and Supplementary Fig. 29), suggesting that simply co-injecting of free antigen\nwith NVs is insufficient to induce strong antigen-specific tolerance. Furthermore, to dissect\nthe functional contribution of CTLA4-mediated DC targeting, CTLA4 blocked NV\nformulation by neutralizing CTLA4 (G6) was prepared. Although this CTLA4 blocked\nformulation retained partial therapeutic activity, its efficacy was lower than that of CII-\nRM@aCD4Tn (G9) (Fig. 6a-f and Supplementary Fig. 29).]\n(iii) We fully agree that, in the original manuscript, some of the wording was stronger\nthan what was directly supported by the data. In the revised version, we have addressed\nthis concern at two levels.\nFirst, we performed additional experiments to more rigorously test antigen specificity,\nincluding ① OT-II and DBA/1 cross-restimulation ELISPOT assays with relevant and\nirrelevant antigen (Fig. 3e,h), ② analysis of cytokine production (TNF-α, TGF-β) in these\nsettings (Supplementary Fig. 20b,c), ③ I-Aᵇ OVA tetramer staining for antigen-\n323-339\nspecific T after OVA-RM@aCD4Tn administration in DBA/1 (Supplementary Fig. 20d),\nregs\nand ④ functional characterization of collagen II-specific T (Fig. 3i and Supplementary\nregs\nof Ag-RM@aCD4Tn are antigen dependent. Accordingly, we now retain the term “antigen-\nspecific” only in sections where this is experimentally demonstrated.\nSecond, we have carefully revised the wording throughout the manuscript to ensure\nthat all statements are quantitatively aligned with the data. In particular, ① we have\nreplaced qualitative terms such as “strong,” “dramatic,” or “complete” with more precise\ndescriptions that include fold-changes or statistical significance; ② we clearly distinguish\nbetween general tolerogenic or immunomodulatory effects and strictly antigen-specific\neffects; and ③ we avoid overinterpretation in instances. All figure legends and Results text\nhave been updated to use more precise language.\nWe hope that these additional data and textual revisions adequately address the\nreviewer’s concern and ensure that the manuscript now uses language that accurately\nreflects the strength and scope of the experimental evidence.\nQ2: The structure and communication of results limit understanding and the ability to\nmake precise data-driven conclusions.\na. Abstract: isolation and ex vivo manipulation of autologous T is listed as a\nreg\nchallenge to overcome since it’s “intricate”. Can the authors be more precise about what\nneeds to be improved and how their approach addressed this? The bispecific nanoparticles\nare poorly described in the abstract making interpretation of results difficult. Include metrics\nof how T are measures and quantification of change instead of “potent” without\nreg\njustification. How were increased anti-inflammatory and reduced inflammatory cytokines\nmeasured, comparison groups (again, more precise language)?\nb. Throughout, results are claimed to be “robust” or “significant” without justifying this\nclaim. Instead of robust, share x fold change or a p value including the comparison group.\nand data is shared without judgement – it’s robustness should be convincing to the reader\nwhen you share the actual values.\nc. The title needs to include murine indicating these are all results in mouse or state\nthat it’s a model of RA therapy, not RA itself.\nd. Section titles should be either descriptive or declarative, not a mix.\ne. Discussion: This work adapts technology that is predominantly used in the cancer\nfield in the context of autoimmune disease and shows promise in a mouse model of RA,\nwith some supporting evidence for a Treg dependent MOA. Comparisons should be\naddressed in the discussion along with application of general concepts about nanoparticles\nuse based on the authors findings. In addition, the discussion should frame all results within\nthe context of the literature. Currently it’s written as a simple summary or findings.\nA2: We thank the reviewer for these thoughtful suggestions. In the revised manuscript,\nwe have carefully addressed all of the points raised: the Abstract has been rewritten to\nmore precisely describe the limitations of current T approaches, clearly outline our\nreg\nbispecific nanovesicles platform, and replace qualitative terms with quantitative descriptors;\nqualitative terms such as “robust” or “significant” are now used only when accompanied by\nexplicit numerical data and/or P values; the title has been modified to clearly indicate that\nthe work is conducted in a murine RA model; section headings have been standardized to\na consistent and descriptive style; and the Discussion has been substantially revised to\ncompare our findings with related nanoparticle and tolerance studies and to better situate\nour results within the broader literature.\nQ3: It is argued that T play a role in antigen-specific tolerance induced by the\nregs\nnanoparticles. To make this argument, the authors need to justify the use of different\nmarkers of T in different experiments, show functional data, and consider T tracking\nreg reg\nexperiments (i.e. GFP, adoptive transfer, etc) to directly link tolerance to T .\nreg\nA3: We thank the reviewer for this important comment. In our study, T were\nregs\nconsistently defined phenotypically as CD4⁺ CD25⁺ Foxp3⁺ cells, and, in selected\nexperiments, we additionally analyzed functional/activation markers to better characterize\ntheir suppressive potential. To provide more direct functional support for the role of T in\nregs\nantigen-specific tolerance, we performed co-culture experiments in which OVA-specific or\nCII-specific T were incubated with BMDCs in the presence of CII (Supplementary Fig.\nregs\n21a). This experiment was conducted to address Reviewer #1 (Q13), Reviewer #2 (Q3),\nand Reviewer #6 (Q1, Q3).\nBefore and after co-culture, T were stained for the key functional molecules CTLA4\nregs\nand LAG-3, which are widely associated with enhanced T -mediated suppression.\nreg\nFollowing co-culture, T from both groups (G4 and G5) showed upregulation of CTLA4\nregs\nand LAG-3 expression compared to unstimulated states (all P<0.0001), and CTLA4 and\nLAG-3 expression in CII-specific T were 1.7-fold and 1.3-fold higher than in OVA-\nregs\nspecific T (Supplementary Fig. 21c), suggesting their greater immunosuppressive\nregs\nactivity. These data provide functional evidence that CII-RM@aCD4Tn induces antigen-\nspecific T that are preferentially activated and exert stronger regulatory effects in the\nregs\n83\npresence of CII.\nRegarding the reviewer’s suggestion to perform T tracking experiments (e.g.,\nreg\nadoptive transfer) to directly link tolerance to T , we had conducted adoptive transfer\nregs\nstudies in CIA mice in original manuscript (Fig. 7l-t). However, due to a figure assembly\nerror, Figure 7 and Extended Data Figure 8 were inadvertently duplicated in the initial\nsubmission. We have now corrected this mistake and updated the figures so that each\npanel displays the appropriate adoptive transfer data.\nCD4⁺ CD25⁺ T cells, enriched for T , were isolated from the LNs and spleen of donor\nregs\nmice pretreated with CII-RM@Tn or CII-RM@aCD4Tn. These cells were sorted based on\nCD4⁺ CD25⁺ expression without Foxp3 gating to preserve viability, as Foxp3 staining\nrequires membrane permeabilization and precludes successful adoptive transfer. Transfer\nof CD4⁺ CD25⁺ cells from CII-RM@aCD4Tn treated donors significantly reduced arthritis\nscores (P=0.0006) and paw thickness (P=0.0201) and improved motor performance\n(number of falls with P=0.0014; speed at falls with P=0.0189) in CIA recipients compared\nto cells from CII-RM@Tn treated donors (Fig. 7m,n and Supplementary Fig. 32a). This was\naccompanied by an increased frequency and number of T in recipient LNs and spleen\nregs\n(G2 vs G7, all P<0.0001), as well as a more tolerogenic cytokine milieu (elevated IL-\n10/TGF-β and reduced pro-inflammatory cytokines) (Fig. 7o-q and Supplementary Fig.\n32b,c).\nTo more specifically assess the contribution of Foxp3⁺ T , we further used Foxp3-\nregs\nIRES-DTR mice as donors. In these mice, Foxp3⁺ T were selectively depleted by\nregs\ndiphtheria toxin treatment before cell isolation. When CD4⁺CD25⁺ cells from Foxp3⁺ T\nreg\ndepleted, CII-RM@aCD4Tn treated donors were adoptively transferred, the therapeutic\nbenefit against CIA was markedly diminished and became comparable to that observed\nwith CII-RM@Tn-derived cells (Fig. 7r-t and Supplementary Fig. 33). These findings\ndemonstrate that the protective effect of adoptively transferred CD4⁺CD25⁺ cells critically\ndepends on the presence of Foxp3⁺ T .\nregs\nregs\ninducing systemic immunomodulation. l, Experimental scheme of regulatory cell\ntransfer experiment. m, Arthritis score curve of normal mice subjected to regulatory cell\ntransfer followed by CIA induction (n=6). n, Paw thickness on day 42 (n=6). o,\nRepresentative contour plots of FC illustrating Foxp3+ T in CD25+ cells in LNs. p,\nregs\nPercentage of Foxp3+ in CD25+ cells, CD25+ Foxp3+ in CD4+ cells and numbers of T in\nregs\nLNs (n=6). q, Heatmap of pro- and anti-inflammatory cytokines in serum and synovium\n(n=6). r, Arthritis score curve of CIA mice pre-transplanted with regulatory cells derived\nfrom Foxp3+ T deficient donor. s, Paw thickness on day 42 (n=6). t, Motor evaluation of\nreg\nfall numbers in mice (n=6).\nQ4: Figure 1 makes claims without showing data. This information should be folded\ninto figures where the hypotheses are tested or used to summarize results once data has\nproven points.\nA4: We thank the reviewer for this helpful suggestion. We agree that a schematic\nfigure should not present before the supporting data are shown In the revised manuscript,\nwe have therefore relocated the original Fig. 1b to the end of the Discussion as a summary\nschematic (Fig. 8), where it now serves to integrate and illustrate the findings after the\nexperimental evidence has been presented.\nreg\nresults inducing T in other contexts.\nreg\nA5: We thank the reviewer for this comment. We agree that our T induction protocol\nreg\nfollows a standard in vitro approach and should be clearly referenced and placed in the\ncontext of previous work. In the revised manuscript, we have now cited relevant studies\nthat use similar conditions to generate Foxp3⁺ induced T .\nregs\nThe cited papers as follows:\n(i) Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+ CD25-naive T\ncells to CD4+ CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3.\nJ Exp Med. 2003;198(12):1875-1886. doi:10.1084/jem.2003015217 (cited as 59 in the\nmanuscript);\n(ii) Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+\nCD25+ FoxP3+ regulatory T cells. Blood. 2005;105(12):4743-4748. doi:10.1182/blood-\n2004-10-393218 (cited as 60 in the manuscript).\nQ6: CD80/86 is discussed and shown on DC, but in vivo these molecules are\nexpressed on all antigen presenting cells. Either test on multiple cell types or describe\nexperiments and conclusions more precisely.\nA6: We thank the reviewer for this helpful comment. We agree that CD80/86\nexpression is not restricted to DCs, but is also found on other APC such as B cells and\nmacrophages.\nTo directly examine which APC populations interact with CII-RM@aCD4Tn in vivo, we\ni.d. injected Cy5-labelled CII-RM@aCD4Tn and analyzed Cy5⁺ APC subsets in the\ndraining LNs by FC. Among Cy5⁺ CD45⁺ cells, NVs were predominantly associated with\nCD11c⁺ DCs (31.3%), followed by CD19⁺ B cells (11.7%), whereas uptake by CD11b⁺\nmacrophages (7.9%) and Ly6G⁺ neutrophils (3.2%) was comparatively low. These data\nsupport our conclusion that DCs represent the principal APC population engaging CII-\nRM@aCD4Tn, while other APC subsets make a more modest contribution.\nThe added figure and text read as follows:\n24 h i.d. administration (n=5).\n[Methods section:\nBridging effect of CII-RM@aCD4Tn on CD4+ T cells and DCs\nTo precisely identify the cell populations targeted by NVs, iLNs were mechanically\ndissociated into single cell suspensions and then analyzed by FC for CD4, CD11c, CD11b,\nLy6G and CD19.\nResults section:\nTo further identify the in vivo cellular targets, Cy5-Cll-RM@aCD4Tn-associated cells\nwere analyzed in iLN. Among Cy5⁺ CD45⁺ leukocytes, NVs were predominantly targeted\nto CD4+ T cells (44.8%) and CD11c⁺ DCs (31.3%), followed by CD19⁺ B cells (11.7%),\nwhereas uptake by CD11b⁺ macrophages (7.9%) and Ly6G⁺ neutrophils (3.2%) was\ncomparatively low (Supplementary Fig. 12a).]\nManuscript 624613 addresses the topic of antigen-specific therapy for autoimmune\ndisease by utilizing a nanoparticle approach in the setting of collagen-induced arthritis. The\nexperiments and data are extensive and largely supports the text and conclusions, but\nthere are still a number of suggested improvements that would further increase clarity and\ncontext.\nQ1: There is no discussion in the manuscript only a short conclusion section. The\npresented data need to be compared to other efforts made in this very active field, both in\nexperimental models and in the human disease itself.\nA1: We thank the reviewer for this important comment. We agree that a dedicated\nDiscussion section is essential to place our findings in the context of previous work in this\nrapidly evolving field. In the revised manuscript, we have added a comprehensive\nDiscussion section.\nIn the Discussion, we first place our work within the broader landscape of T -directed\nreg\ntherapies and antigen-bearing nanomedicines, highlighting ongoing clinical efforts and\ntheir limitations in persistence, manufacturability, and antigen specificity. We then explain\nhow our DC-CD4 bridging nanovesicle platform adapts multi-specific synapse-organizing\nprinciples from oncology to promote tolerogenic, rather than stimulatory outcomes, and\noutline key mechanistic features underlying its activity, including enhanced DC antigen\npresentation, efficient LN targeting, preferential engagement of DCs and CD4⁺ T cells, and\nenforced DC-CD4 proximity. We next summarize convergent evidence for an antigen-\nspecific, T -dependent mechanism from recall assays, tetramer staining, functional T\nreg reg\nco-cultures, and cognate versus heterologous antigen comparisons in CIA. We also\ndiscuss the impact of therapeutic timing across prophylactic, early-intervention, late-\ntreatment, and re-challenge regimens, supporting the notion that the platform seeds\ndurable antigen-specific T pools that restrain effector responses upon re-encounter.\nreg\nFinally, we acknowledge key limitations, such as reliance on ex vivo induced iCD4 as\nsource and evaluation in a single-antigen CIA model, and outline future directions toward\nmulti-antigen or patient-tailored formulations, testing in more complex and relapsing\nmodels, and further development of this modular, antigen-specific tolerance strategy for\ntranslation.\nWe hope that this expanded Discussion provides a clearer framework for how our work\nfits into and extends current efforts in both experimental models and human disease.\nother antigen-presenting cells that could participate in activating (or turning off) T cells, this\nneeds to be at least discussed, and the sentence in the introduction (line 44) modified or\nclarified.\nA2: We thank the reviewer for this important comment. We fully agree that DCs are\nnot the only APCs and that other APC subsets can also participate in activating or tolerizing\nT cells. To clarify which APC populations interact with our NVs in vivo, we i.d. injected Cy5-\nlabelled CII-RM@aCD4Tn and analyzed Cy5⁺ APC subsets in the draining LNs by FC.\nAmong Cy5⁺ CD45⁺ cells, NVs were predominantly associated with CD11c⁺ DCs (31.3%),\nfollowed by CD19⁺ B cells (11.7%), whereas uptake by CD11b⁺ macrophages (7.9%) and\nLy6G⁺ neutrophils (3.2%) was comparatively low (Supplementary Fig. 12a). These data\nsupport our conclusion that DCs represent the principal, although not exclusive, APC\npopulation engaging CII-RM@aCD4Tn.\nMoreover, we directly compared the ability of different APC subsets to induce an\nimmunosuppressive CD4⁺ T cell response in the presence of CII-RM@aCD4Tn. Purified\nCD11c⁺ DCs, CD11b⁺ macrophages, CD19⁺ B cells and Ly6G⁺ neutrophils were each co-\ncultured with CD4⁺ T cells for 3 days in the presence of CII-RM@aCD4Tn, and IL-10 levels\nin the supernatant were measured (Supplementary Fig. 12b). CD11c⁺ DCs induced the\nhighest IL-10 production under CII-RM@aCD4Tn treatment as compared with\nmacrophages, B cells and neutrophils induced only modest or minimal increases in IL-10\n(Supplementary Fig. 12c). These results further indicate that, in the context of our bispecific\nNVs, DCs are the dominant APC subset driving tolerogenic CD4⁺ T-cell responses.\nThe added figure and text read as follows:\ncontribution to tolerogenic responses. a, Cll-RM@aCD4Tn targeted cells in the iLNs of\nCIA mice after 24 h i.d. administration (n=5). b, Schematic illustration of co-culture different\nAPCs with CD4⁺ T cells in the presence of CII-RM@aCD4Tn for 3 days. Created with\nBiorender.com. c, IL-10 levels in the medium after co-incubation (n=5). Data are presented\nas mean ± SD, statistical significance was analyzed by One-way ANOVA with Tukey’s\npost hoc test.\n[Methods section:\nBridging effect of CII-RM@aCD4Tn on CD4+ T cells and DCs\nTo assess the contribution of different APC subsets to iCD4 induction, CD11c+ DCs,\nCD11b⁺ macrophages, Ly6G⁺ neutrophils and CD19⁺ B cells derived from male DBA/1\nmice were co-cultured with CD4+ T cells (1 × 106) at a number ratio of 1:1 in the presence\nof CII-RM@aCD4Tn for 3 days. IL-10 levels in culture medium were quantified by ELISA.\nResults section:\nTo further identify the in vivo cellular targets, Cy5-Cll-RM@aCD4Tn-associated cells\nwere analyzed in iLN. Among Cy5⁺ CD45⁺ leukocytes, NVs were predominantly targeted\nto CD4+ T cells (44.8%) and CD11c⁺ DCs (31.3%), followed by CD19⁺ B cells (11.7%),\nwhereas uptake by CD11b⁺ macrophages (7.9%) and Ly6G⁺ neutrophils (3.2%) was\ncomparatively low (Supplementary Fig. 12a). When CD4+ T cells were co-cultured with\npurified APC subsets in the presence of Cll-RM@aCD4Tn, CD11c⁺ DCs elicited the highest\nIL-10 levels in the medium (Supplementary Fig. 12b,c), implying that DCs are the dominant\nAPC subset engaging the NVs to drive tolerogenic responses.]\nIntroduction (line 44) to more accurately reflect this point. The text now reads:\n[Introduction section:\nTo overcome these limitations, nanotechnology-based strategies have been explored\nto deliver antigens and immunosuppressive agents specifically to antigen presenting cells\n(APCs), particularly specialized dendritic cells (DCs), thereby boosting the generation of\nantigen-specific T in situ4, 6, 12, 25, 26.\nregs\nReferences\n4. Liu, Y. et al. Generation of tolerogenic antigen-presenting cells in vivo via the\ndelivery of mRNA encoding PDL1 within lipid nanoparticles. Nat. Biomed. Eng.\n(2025).\n6. Sohn, H.S. et al. Tolerogenic nanoparticles induce type II collagen-specific\nregulatory T cells and ameliorate osteoarthritis. Sci. Adv. 8, eabo5284 (2022).\n12. Scotland, B.L. et al. Cell and biomaterial delivery strategies to induce immune\ntolerance. Adv. Drug Deliv. Rev. 203, 115141 (2023).\n25. Cifuentes-Rius, A., Desai, A., Yuen, D., Johnston, A.P.R. & Voelcker, N.H. Inducing\nimmune tolerance with dendritic cell-targeting nanomedicines. Nat. Nanotechnol.\n16, 37-46 (2021).\n26. Krienke, C. et al. A noninflammatory mRNA vaccine for treatment of experimental\nautoimmune encephalomyelitis. Science 371, 145-153 (2021).]\nQ3: The iCD4 nomenclature is not widely used and would benefit from being\nintroduced and discussed in more detail. Is this a reflection of the T differentiation not\nreg\nincorporating a purification step? What is the frequency of T generated? Are the batch-\nregs\nto-batch variation when performing the NP preparation?\nA3: We thank the reviewer for raising this point. We agree that the iCD4 nomenclature\nis not yet widely standardized. In the revised manuscript, we therefore introduce iCD4\nexplicitly at first mention in the Results and clarify that iCD4 is used here as an abbreviation\nfor “immunosuppressive CD4⁺ T cells”, defined by an immunosuppressive\nphenotype/function in our system rather than implying a formally recognized lineage.\nSpecifically, iCD4 encompasses both Foxp3⁺ regulatory T cells (T ) and Foxp3⁻\nregs\n91\nsuppressive CD4⁺ subsets. This usage is consistent with high-impact literature in which\n“immunosuppressive CD4⁺ T cells” is used as a functional umbrella term. For example, a\nrecent Nature study reported that CNS-derived regulatory self-peptides evoked\n“immunosuppressive CD4⁺ T cells” that controlled CNS autoimmunity in a CTLA4- and\nTGFβ-dependent manner and highlighted an unconventional FOXP3⁻ suppressor CD4⁺ T-\ncell population19. Likewise, the Cell study of Memory of Inflammation in Regulatory T Cells\nintroduces T as “a specialized subset of immunosuppressive CD4 T cells” defined by\nregs\nFoxp3 expression20.\nThe revised text read as follows:\n[Results section:\nImmunosuppressive CD4+ T cells (iCD4) including Foxp3⁺ T and Foxp3⁻\nregs\nsuppressive CD4⁺ subsets, are essential for immune regulation and autoimmunity\nprevention, holding therapeutic promise for a range of conditions12, 18, 32, 33.\nReferences\n12. Scotland, B.L. et al. Cell and biomaterial delivery strategies to induce immune\ntolerance. Adv. Drug Deliv. Rev. 203, 115141 (2023).\n18. Boardman, D.A. et al. Expression of a chimeric antigen receptor specific for donor\nHLA class I enhances the potency of human regulatory T cells in preventing human\nskin transplant rejection. Am. J. Transplant. 17, 931-943 (2017).\n32. Kim, M.W. et al. Endogenous self-peptides guard immune privilege of the central\nnervous system. Nature 637, 176-183 (2025).\n33. van der Veeken, J. et al. Memory of Inflammation in Regulatory T Cells. Cell 166,\n977-990 (2016).]\nIn addition, we agree that we not purify the induced cells and the percentage of Foxp3⁺\nT obtained under our in vitro induction setting is not high. During method optimization,\nregs\nwe observed that CD4⁺ T cells upregulated CD25 upon activation, and increasing the\nconcentration of TGF-β indeed led to a gradual increase in Foxp3 expression together with\nhigher levels of CTLA4 (Supplementary Fig. 1a-c). However, high TGF-β levels also\nresulted in markedly reduced cell yields (Supplementary Fig. 1d). Our aim in this study was\nnot to generate a highly purified Foxp3⁺ T population in vitro, but rather to obtain CD4⁺\nreg\nT cells with sufficiently high CTLA4 expression to effectively engage CD80/86 on dendritic\ncells in the bispecific NV design. We therefore selected 10 ng/mL TGF-β as a condition\n92\nthat provided an appropriate balance between CTLA4 upregulation and cell recovery, and\nthis condition was used for all subsequent experiments. To more accurately reflect their\nphenotype, we designated these induced cells as immunosuppressive CD4⁺ T cells (iCD4).\nRegarding batch-to-batch variation, repeated inductions under the same conditions\nconsistently showed stable upregulation of CTLA4 on iCD4 cells (Supplementary Fig. 1e).\nTo fully clarify this point, we have now included the complete data in Supplementary Fig.\n1.\nThe revised figures and accompanying text read as follows:\nSupplementary Fig. 1. Ex vivo induction of immunosuppressive CD4⁺ T cells (iCD4).\na, Representative FC contour plots illustrating CD25+ Foxp3+ T in CD4+ cells after\nregs\ninduction with varying concentrations of TGF-β. b, Percentage of CD25+ Foxp3+ T in\nregs\nCD4+ cells (n=3). c, MFI of CTLA4 after induction in CD4+ T cells (n=3). d, Cell expansion\nfold of iCD4 (n=3). e, MFI of CTLA4 in iCD4 across different batch at 10 ng mL-1 TGF-β\n(n=3). f, Representative FC histograms and quantification of PD-1 and LAG-3 after iCD4\ninduction (n=3). Data are presented as mean ± SD, statistical significance was analyzed\nby One-way ANOVA with Tukey’s post hoc test (b-e) or unpaired two-tailed t-test (f).\n93\n[Methods section:\nInduction of CD4+ T cells into immunosuppressive CD4⁺ T cells (iCD4) in vitro\nFor iCD4 differentiation, TGF-β (2-20 ng ml-1) and RM (50 nM) were added59, 60.\nCells were cultured for 4 days, and iCD4 induction was verified by FC (Aria III, BD) to\ndetect CD25, Foxp3, CTLA4, LAG-3, and PD-1.\nReferences:\n59. Chen, W. et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+\n13\nregulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med.\n198, 1875-1886 (2003).\n60. Battaglia, M., Stabilini, A. & Roncarolo, M.G. Rapamycin selectively expands\nCD4+CD25+FoxP3+ regulatory T cells. Blood 105, 4743-4748 (2005).\nResults section:\niCD4 with stabled high CTLA4 express were successfully induced from CD4+ T cells\nisolated from spleen and LNs, showing approximately threefold expansion at 10 ng mL-1\nTGF-β (Supplementary Fig. 1a-e). Additionally, the key immunomodulatory markers PD-1\n(P<0.0001) and LAG-3 (P=0.0012) were significantly upregulated following stimulation\n(Supplementary Fig. 1f).]\nQ4: Chapter 7: the authors should be careful when to use the phrase ‘therapeutic’, the\ndata presented in figure 5 is ‘preventing’ CIA not ‘treating’. This figure (5) could even be\nmoved into the supplementary data set. In contrast the data in extending figure 7a which\nis ‘therapeutic’ could be moved in as a main figure.\nA4: We thank the reviewer for this thoughtful comment and for highlighting the\nimportance of distinguishing preventive and therapeutic settings in CIA. In our DBA/1 CIA\nmodel, we routinely observe that clinical signs of arthritis (paw swelling and redness) begin\nto appear shortly after the second immunization on day 21. By day 22, mice began to\nexhibit mild clinical symptoms, and by day 27 the paws show overt swelling and erythema.\nTherefore, both regimens in which CII-RM@aCD4Tn was administered on days 22, 25,\nand 28 (Fig. 4) and on days 27, 30, and 33 (Supplementary Fig. 27) represent therapeutic\ninterventions initiated after disease onset, with the former corresponding to an early\ntherapeutic window and the latter to a late therapeutic setting in more established disease.\nIn contrast, the truly preventive protocol in our study is the regimen in which CII-\nRM@aCD4Tn was administered three and two weeks before CIA induction\n94\n(Supplementary Fig. 28), that is, before any clinical signs of arthritis were present. To avoid\nconfusion, we have revised the manuscript to explicitly refer to these three regimens as\n“Preventive” (pre-immunization), “Early therapeutic” (starting on day 22 after immunization),\nand “Late therapeutic” (starting on day 27 after immunization) throughout the text and\nfigure legends.\nWe would also like to note that in multiple CIA studies, interventions starting at day 22\nafter the booster immunization at day 21 are commonly described as “treat” rather than\n“preventive”21-23. For example, in a Sci Rep study of MASM, treatment beginning on day\n22 is defined as “treat”21. We have added appropriate references to place our timing in the\ncontext of these established CIA protocols.\nWe appreciate the reviewer’s suggestion regarding figure placement. Because the\nearly therapeutic regimen (day 22 start) provides the most comprehensive evaluation of\nclinical scores, joint pathology, and functional outcomes, we feel it is helpful to retain this\ndataset as a main figure to clearly present the overall therapeutic profile. However, we are\nhappy to move this figure to the Supplementary Information and promote the later\ntherapeutic dataset to the main figures if the reviewer consider this arrangement more\nappropriate.\nQ5: Figure 6a is not showing any triple positive cells!? This would be important to show.\nA5: We thank the reviewer for this important comment. We agree that visualizing CD4⁺\nCD25⁺ Foxp3⁺ triple-positive cells in the synovium are critical to directly illustrate T\nreg\nlocalization.\nIn the revised manuscript, we have re-analyzed the sections and updated Fig. 6a (now\nrepresented as Fig. 5a) to better address this point. First, we increased the magnification\nand provided higher-resolution images, including separated channels and merged overlays,\nso that co-localization of CD4, CD25, and Foxp3 can be clearly discerned. Second, for\nquantification, we now present the immunofluorescence data as Foxp3⁺ area normalized\nto CD4⁺ area (Foxp3⁺ area/CD4⁺ area), which more accurately reflects the proportion of\nFoxp3⁺ T cells within the CD4⁺ compartment.\nThe revised figures and accompanying text read as follows:\nregs\nsynovium. Scale bars, 60 μm. Blue, DAPI; Red, CD4; Green, CD25; Yellow, Foxp3. b,\nFoxp3+ area in synovium (n=6). c, Foxp3+ area/CD4+ area in synovium (n=6).\n[Results section:\nImmunofluorescence analysis revealed a marked increase in the proportion of Foxp3+\nT in the synovium after CII-RM@aCD4Tn treatment (Fig. 5a-c).]\nregs\nQ6: Extended figure 8 is identical to main figure 7. Probably a mix-up, please check!\nA6: We thank the reviewer for pointing out this mistake. Main Figure 7 was\ninadvertently duplicated from the Extended Data Figure 8 (now presented as\nSupplementary Fig. 28) in the original submission. We have carefully checked all figures\nand corrected this mix-up in the revised manuscript.\nQ7: Information (methods) and data on the cell transfer experiment (main figure 8) is\nmissing!\nA7: We thank the reviewer for pointing this out and apologize for the confusion. In the\noriginal submission, the adoptive cell transfer experiments were presented in main Figure\n7, which was identical to Extended figure 8 (now presented as Supplementary Fig. 28).\nThese data are now correctly shown in main Figure 7 in the revised manuscript.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\n1. McCright, J., Naiknavare, R., Yarmovsky, J. & Maisel, K. Targeting lymphatics for\nnanoparticle drug delivery. Front. Pharmacol. 13, 887402 (2022).\n2. He, P. et al. Advances in nanomedicines for lymphatic imaging and therapy. J.\nNanobiotechnology 21, 292 (2023).\n3. Lee, J. et al. Nanoparticles for lymph node-directed delivery. Pharmaceutics 15\n(2023).\n4. Boada, C. et al. Rapamycin-loaded biomimetic nanoparticles reverse vascular\ninflammation. Circ. Res. 126, 25-37 (2020).\n5. Lin, G. et al. Enhancing retention and permeation of rapamycin for osteoarthritis\ntherapy using a two-stage drug delivery system. Mater. Today Bio. 29, 101279\n(2024).\n6. Miao, Z.L. et al. Preparation of a liposomal delivery system and its in vitro release\nof rapamycin. Exp. Ther. Med. 9, 941-946 (2015).\n7. Raffin, C., Vo, L.T. & Bluestone, J.A. T(reg) cell-based therapies: challenges and\nperspectives. Nat. Rev. Immunol. 20, 158-172 (2020).\n8. Ferreira, L.M.R., Muller, Y.D., Bluestone, J.A. & Tang, Q. Next-generation\nregulatory T cell therapy. Nat. Rev. Drug Discov. 18, 749-769 (2019).\n9. Mosanya, C.H. & Isaacs, J.D. Tolerising cellular therapies: what is their promise for\nautoimmune disease? Ann. Rheum. Dis. 78, 297-310 (2019).\n10. Hunter, Z. et al. A biodegradable nanoparticle platform for the induction of\nantigen-specific immune tolerance for treatment of autoimmune disease. ACS\nNano 8, 2148-2160 (2014).\n11. Kelly, C.P. et al. TAK-101 Nanoparticles Induce Gluten-Specific Tolerance in Celiac\nDisease: A Randomized, Double-Blind, Placebo-Controlled Study.\nGastroenterology 161, 66-80.e68 (2021).\n12. Saunders, M.N. et al. Allergen-encapsulating nanoparticles reprogram\npathogenic allergen-specific Th2 cells to suppress food allergy. Adv. Healthc.\nMater. 14, e2400237 (2025).\n13. Sohn, H.S. et al. Tolerogenic nanoparticles induce type II collagen-specific\nregulatory T cells and ameliorate osteoarthritis. Sci. Adv. 8, eabo5284 (2022).\n14. Fu, L. et al. PRDM16-dependent antigen-presenting cells induce tolerance to gut\nantigens. Nature 642, 756-765 (2025).\n15. Campos Canesso, M.C. et al. Identification of antigen-presenting cell-T cell\ninteractions driving immune responses to food. Science 387, eado5088 (2025).\n16. Akkaya, B. et al. Regulatory T cells mediate specific suppression by depleting\npeptide-MHC class II from dendritic cells. Nat. Immunol. 20, 218-231 (2019).\n17. Chen, W. et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+\nregulatory T cells by TGF-beta induction of transcription factor Foxp3. J. Exp. Med.\n198, 1875-1886 (2003).\n18. Battaglia, M., Stabilini, A. & Roncarolo, M.G. Rapamycin selectively expands\nCD4+CD25+FoxP3+ regulatory T cells. Blood 105, 4743-4748 (2005).\n98\n19. Kim, M.W. et al. Endogenous self-peptides guard immune privilege of the central\nnervous system. Nature 637, 176-183 (2025).\n20. van der Veeken, J. et al. Memory of Inflammation in Regulatory T Cells. Cell 166,\n977-990 (2016).\n21. Zou, Y. et al. Therapeutic effects of matrine derivate MASM in mice with collagen-\ninduced arthritis and on fibroblast-like synoviocytes. Sci. Rep. 7, 2454 (2017).\n22. Chen, S.Y. et al. Interleukin-23 mediates osteoclastogenesis in collagen-induced\narthritis by modulating microRNA-223. Int. J. Mol. Sci. 23 (2022).\n23. Jia, Q. et al. Astragalin suppresses inflammatory responses and bone destruction\nin mice with collagen-induced arthritis and in human fibroblast-like synoviocytes.\nFront. Pharmacol. 10, 94 (2019).\n99"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The Reviewer appreciates the efforts to address the prior comments. However, there are several critical contextual edits and full discussion of the data is still required.\n1. The Authors should to revise the following sentence from the abstract. “However, current approaches either require prolonged ex vivo expansion of rare Tregs or generate insufficient antigen-specific Tregs response in vivo.”\nThere are several Ag-specific therapies presently in clinical trials, phase II and beyond, which have shown good safety profiles, efficacy, and either decreases in effector T cells or increases in Tregs. The Authors do address this briefly in the discussion section, however, as the sentence is presently written, the suggestion is that no progress in the Ag-specific tolerance space has been made.\n2. In the introduction, (Line 30), the Authors state the following: “Being distinct from conventional immunosuppression that broadly dampens immunity, restoring immune tolerance offers significant advantages in terms of antigen-specificity (targeting only pathogenic immune responses), long-term efficacy (reprogramming immune memory), and minimal systemic toxicity (preserving host defense against infections). Therefore, immune tolerance is an attractive therapeutic strategy for a wide range of immune-mediated conditions.”\nThis Reviewer is in full agreement with the Authors, and the precision of an Ag-specific therapy is the ultimate goal. To this end, the Authors have now included the non-specific Ag control treatment in Figure 6B. The data show that in the absence of the full formulation, treatment is not as efficacious. However, these data should also be interpreted for what the data do show. The data show that even in the absence of the disease-specific Ag, the RM@aCD4Tn (no Ag; disease score ~4), CII-RM@aCD4Th (lacking CTLA4; disease score ~4), OVA-RM@aCD4Tn (non-specific Ag; disease score ~4), and CII + RM@aCD4Tn (soluble Ag not directly with the therapeutic; disease score ~4) all have a highly significant therapeutic benefit. This is further illustrated with the rechallenge study data presented in Figure 6I. This then runs counter to the Authors stated goal and conclusions. These data definitively show that whilst their drug does have benefit with the disease-associated Ag, the data also show that the drug is broadly immunosuppressive.\nThe Authors must address this issue. The statistical analysis for these four groups (Figure 6B) are compared to CII-RM@aCD4Th (the main line drug; disease score ~1), however the Authors do not show the statistics for these four drugs compared to PBS treated disease control group (disease score ~12). Based on the Day 42 disease scores, the other component parts of the full formulation minus the disease-specific Ag decreased disease from a score of ~12 to a score of ~4, and the specific Ag only decreased the disease score to ~1. The non-specific portion of the treatment is further presented in Figure 3H, where OVA-RM@aCD4Tn significantly decreased the level of IFN-gamma secreted by the DBA/1 mouse cells activated in the presence of CII ex vivo. Taken in combination, these data show that the drug in general is highly immunosuppressive in an Ag-non-specific manner. This must be clearly stated and acknowledged in both the associated results section and in the discussion.\n3. The question of inducing Ag-specific Treg, whilst true that the full formulation induces the greatest percentage increase (Figure 3B), the non-Ag containing formulations also increase the percentage of Tregs to an extent. This is putatively in correlation with Comment #2. As such, the Authors must acknowledge this outcome.", "reviewer_2": "The authors have adequately addressed all issues raised by the reviewer. Now I recommend acceptance of this manuscript.", "reviewer_3": "The revised manuscript is substantially improved. My only remaining comment is as follows:\n1) Given that Te yield was too low for their use to be of practical use, this limitation could be noted and Te data could be further minimized in the manuscript (i.e. presented only in the supplementary methods and not included in the main text).", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_6": "The authors addressed all concerns of the reviewers.", "reviewer_7": "I would like to thank the authors for their extensive revision which has adequately addressed my concerns"}, "author_response": "Reply to comments from reviewers\nDC-CD4 bispecific tolerogenic nanovesicles induce antigen-specific regulatory T cells\nand ameliorate collagen-induced arthritis in mice\n(Nature Communications, NCOMMS-25-47323A)\nWe thank the reviewers for the additional round of peer review and for their constructive\ncomments on our revised submission. We have carefully considered each suggestion,\nimplemented the requested revisions, and thoroughly rechecked the manuscript for\nconsistency. Here, we describe the changes that we made:\nText in italic: comments from the reviewers.\nBlue letters: revised and newly added text in the revised version.\nREVIEWER COMMENTS\nReviewer #1\nThe Reviewer appreciates the efforts to address the prior comments. However, there\nare several critical contextual edits and full discussion of the data is still required.\n1. The Authors should to revise the following sentence from the abstract. “However,\ncurrent approaches either require prolonged ex vivo expansion of rare T or generate\nregs\ninsufficient antigen-specific T response in vivo.”\nregs\nThere are several Ag-specific therapies presently in clinical trials, phase II and beyond,\nwhich have shown good safety profiles, efficacy, and either decreases in effector T cells or\nincreases in T . The Authors do address this briefly in the discussion section, however,\nregs\nas the sentence is presently written, the suggestion is that no progress in the Ag-specific\ntolerance space has been made.\n2. In the introduction, (Line 30), the Authors state the following: “Being distinct from\nconventional immunosuppression that broadly dampens immunity, restoring immune\ntolerance offers significant advantages in terms of antigen-specificity (targeting only\npathogenic immune responses), long-term efficacy (reprogramming immune memory), and\nminimal systemic toxicity (preserving host defense against infections). Therefore, immune\ntolerance is an attractive therapeutic strategy for a wide range of immune-mediated\nconditions.”\nThis Reviewer is in full agreement with the Authors, and the precision of an Ag-specific\ntherapy is the ultimate goal. To this end, the Authors have now included the non-specific\nAg control treatment in Figure 6B. The data show that in the absence of the full formulation,\ntreatment is not as efficacious. However, these data should also be interpreted for what\nthe data do show. The data show that even in the absence of the disease-specific Ag, the\nRM@aCD4Tn (no Ag; disease score ~4), CII-RM@aCD4Th (lacking CTLA4; disease\nscore ~4), OVA-RM@aCD4Tn (non-specific Ag; disease score ~4), and CII +\nRM@aCD4Tn (soluble Ag not directly with the therapeutic; disease score ~4) all have a\nhighly significant therapeutic benefit. This is further illustrated with the rechallenge study\ndata presented in Figure 6I. This then runs counter to the Authors stated goal and\nconclusions. These data definitively show that whilst their drug does have benefit with the\ndisease-associated Ag, the data also show that the drug is broadly immunosuppressive.\nThe Authors must address this issue. The statistical analysis for these four groups\n(Figure 6B) are compared to CII-RM@aCD4Th (the main line drug; disease score ~1),\nhowever the Authors do not show the statistics for these four drugs compared to PBS\ntreated disease control group (disease score ~12). Based on the Day 42 disease scores,\nthe other component parts of the full formulation minus the disease-specific Ag decreased\ndisease from a score of ~12 to a score of ~4, and the specific Ag only decreased the\ndisease score to ~1. The non-specific portion of the treatment is further presented in Figure\n3H, where OVA-RM@aCD4Tn significantly decreased the level of IFN-gamma secreted\nby the DBA/1 mouse cells activated in the presence of CII ex vivo. Taken in combination,\nthese data show that the drug in general is highly immunosuppressive in an Ag-non-\nspecific manner. This must be clearly stated and acknowledged in both the associated\nresults section and in the discussion.\n3. The question of inducing Ag-specific T , whilst true that the full formulation induces\nreg\nthe greatest percentage increase (Figure 3B), the non-Ag containing formulations also\nincrease the percentage of Tregs to an extent. This is putatively in correlation with\nComment #2. As such, the Authors must acknowledge this outcome.\nReviewer #2\nThe authors have adequately addressed all issues raised by the reviewer. Now I\nrecommend acceptance of this manuscript.\nReviewer #3\nThe revised manuscript is substantially improved. My only remaining comment is as\nfollows:\n1. Given that Te yield was too low for their use to be of practical use, this limitation\ncould be noted and Te data could be further minimized in the manuscript (i.e. presented\nonly in the supplementary methods and not included in the main text).\nReviewer #4\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #5\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #6\nThe authors addressed all concerns of the reviewers.\nReviewer #7\nI would like to thank the authors for their extensive revision which has adequately\naddressed my concerns.\nResponse to reviewer’s comments:\nReviewer #1\nThe Reviewer appreciates the efforts to address the prior comments. However, there\nare several critical contextual edits and full discussion of the data is still required.\nQ1: The Authors should to revise the following sentence from the abstract. “However,\ncurrent approaches either require prolonged ex vivo expansion of rare T or generate\nregs\ninsufficient antigen-specific T response in vivo.”\nregs\nThere are several Ag-specific therapies presently in clinical trials, phase II and beyond,\nwhich have shown good safety profiles, efficacy, and either decreases in effector T cells or\nincreases in T . The Authors do address this briefly in the discussion section, however,\nregs\nas the sentence is presently written, the suggestion is that no progress in the Ag-specific\ntolerance space has been made.\nA1: We thank the reviewer for this insightful and constructive comment. We agree that\nthe original wording in the Abstract may have unintentionally underrepresented the\nprogress made in antigen-specific tolerance strategies, including approaches currently in\nclinical development. To avoid potential overgeneralization in the Abstract and to improve\nconcision in line with Nature Communications abstract length expectations, we have\nremoved this sentence. This revision provides a more balanced and precise description.\nQ2: In the introduction, (Line 30), the Authors state the following: “Being distinct from\nconventional immunosuppression that broadly dampens immunity, restoring immune\ntolerance offers significant advantages in terms of antigen-specificity (targeting only\npathogenic immune responses), long-term efficacy (reprogramming immune memory), and\nminimal systemic toxicity (preserving host defense against infections). Therefore, immune\ntolerance is an attractive therapeutic strategy for a wide range of immune-mediated\nconditions.”\nThis Reviewer is in full agreement with the Authors, and the precision of an Ag-specific\ntherapy is the ultimate goal. To this end, the Authors have now included the non-specific\nAg control treatment in Figure 6B. The data show that in the absence of the full formulation,\ntreatment is not as efficacious. However, these data should also be interpreted for what\nthe data do show. The data show that even in the absence of the disease-specific Ag, the\nRM@aCD4Tn (no Ag; disease score ~4), CII-RM@aCD4Th (lacking CTLA4; disease\nscore ~4), OVA-RM@aCD4Tn (non-specific Ag; disease score ~4), and CII +\nRM@aCD4Tn (soluble Ag not directly with the therapeutic; disease score ~4) all have a\nhighly significant therapeutic benefit. This is further illustrated with the rechallenge study\ndata presented in Figure 6I. This then runs counter to the Authors stated goal and\nconclusions. These data definitively show that whilst their drug does have benefit with the\ndisease-associated Ag, the data also show that the drug is broadly immunosuppressive.\nThe Authors must address this issue. The statistical analysis for these four groups\n(Figure 6B) are compared to CII-RM@aCD4Th (the main line drug; disease score ~1),\nhowever the Authors do not show the statistics for these four drugs compared to PBS\ntreated disease control group (disease score ~12). Based on the Day 42 disease scores,\nthe other component parts of the full formulation minus the disease-specific Ag decreased\ndisease from a score of ~12 to a score of ~4, and the specific Ag only decreased the\ndisease score to ~1. The non-specific portion of the treatment is further presented in Figure\n3H, where OVA-RM@aCD4Tn significantly decreased the level of IFN-gamma secreted\nby the DBA/1 mouse cells activated in the presence of CII ex vivo. Taken in combination,\nthese data show that the drug in general is highly immunosuppressive in an Ag-non-\nspecific manner. This must be clearly stated and acknowledged in both the associated\nresults section and in the discussion.\nA2: We thank the reviewer for this insightful and important comment. We appreciate\nthe careful interpretation of the data and agree that the results indicate the presence of an\nantigen-independent immunomodulatory component within our system.\nAs correctly noted, in the CIA model, RM@aCD4Tn (G5), CII-RM@aCD4Th lacking\nCTLA4 (G6), OVA-RM@aCD4Tn (G7), and CII + RM@aCD4Tn (G8) all showed significant\ntherapeutic benefit compared to PBS group. These findings indicate that the NV platform\nitself exerts baseline immunosuppressive effects independent of disease-associated\nantigen incorporation. This observation is consistent with the intrinsic properties of the NV,\nwhich are derived from immunosuppressive CD4 T cells and express immunoinhibitory\nmolecules such as CTLA4, PD-1, and LAG-3 (Supplementary Fig. 1). Accordingly,\n⁺\nadministration of Tn alone (G4) or aCD4-modified NVs without disease-specific antigen\n(G5) produced measurable immunosuppressive effects (Fig. 4). Therefore, while\nincorporation of the disease-associated antigen further enhances therapeutic efficacy,\ncomplete antigen-restricted immune modulation is not fully achieved in the current design.\nIn response to the reviewer’s comment, we have revised the manuscript as follows:\n(1) Statistical comparisons between these treatment groups and the PBS control\ngroup have been added in Fig. 6B;\n(2) The Results section has been clarified to explicitly state that the NV platform\nconfers baseline immunosuppressive activity;\n(3) The Discussion has been expanded to acknowledge that the therapeutic effect\ncomprises both a baseline immunosuppressive component and an antigen-specific\ntolerogenic component.\nThe revised figure and text read as follows:\nFig. 6| a, Experimental scheme of CIA mice treatment with various formulations. b,\nArthritis score curve (n=6). c, Paw thickness on day 42 (n=6).\n[Resultssection:\nsystem\nHowever, CII-RM@aCD4Tn also demonstrated a significant reduction in IFN-γ\nproduction compared to CII alone in OT-II mice (G3 vs G4, P=0.0001) (Fig. 3e,f),\nwhich is likely attributable to the intrinsic baseline immunosuppressive properties of the iCD4-\nderived NVs.\n7. Mechanistic validation and durable efficacy of CII-RM@aCD4Tn\nIndeed, RM@aCD4Tn (G5), CII-RM@aCD4Tn lacking CTLA4 (G6), OVA-\nRM@aCD4Tn (G7), and CII + RM@aCD4Tn (G8) all exhibited significant therapeutic\nbenefit compared to PBS (Fig. 6a-f and Supplementary Fig. 29).\nDiscussionsection:\nMeasurable immunosuppressive effects were also observed in the absence of\ncognate antigen loading (Fig. 3a-d, G4 and G5; Fig. 3e,f, G4; Fig. 3g,h, G2; Fig. 4b,\nG4 and G5; Fig. 6b, G5 and G7). This baseline activity is likely attributable to the\nintrinsic immunomodulatory properties of the iCD4-derived NVs, which express high\nlevels of inhibitory molecules such as CTLA4, PD-1, and LAG-3 (Supplementary Fig.\n1). Therefore, while the NV platform itself confers a degree of antigen-independent\nimmune suppression, incorporation of the cognate antigen substantially enhances T\nreg\ncell expansion and functional suppression. In this framework, antigen loading provides\nan additional layer of specificity and potency rather than absolute exclusivity.]\nQ3: The question of inducing Ag-specific T , whilst true that the full formulation induces\nreg\nthe greatest percentage increase (Figure 3B), the non-Ag containing formulations also\nincrease the percentage of T to an extent. This is putatively in correlation with\nregs\nComment #2. As such, the Authors must acknowledge this outcome.\nA3: We thank the reviewer for this important comment. We agree that, although the\nfull antigen-containing formulation induced the greatest increase in T frequency, the non-\nreg\nAg containing formulations also resulted in a moderate elevation of T percentages\nreg\ncompared to PBS. In response, we have revised the Results section to explicitly\nacknowledge this observation and provide a clearer description of the relative magnitude\nof these effects. The updated text now states that the non-Ag containing formulations (G4\nand G5) modestly increased T frequencies.\nreg\nThe revised text read as follows:\n[Result section:\nAlthough the non-Ag containing formulations (G4 and G5) modestly increased the\npercentage of T cells compared to PBS, OVA-RM@aCD4Tn induced the highest\nreg\nproportions and absolute numbers of CD25+ Foxp3+ T cells in the LNs, with 9.7-fold\nreg\nand 1.3-fold higher percentages than PBS and OVA-RM@Tn, respectively (Fig. 3b,c\nand Supplementary Fig. 17).]"}]

Decision letter