Antibody-conjugated mesenchymal stromal cell drug delivery system for the treatment of autoimmune diseases in mice

Domain: chemistry
Published: 2026-01-22
Editorial outcome: minor_revision

Abstract
Autoimmune diseases (AIDs), such as psoriasis and rheumatoid arthritis, are driven by immune dysfunction, leading to chronic inflammation and tissue damage. Mesenchymal stromal cells (MSCs) possess immunomodulatory and tissue repair properties; however, the therapeutic applicability of MSCs faces limitations due to the low homing efficiency upon systemic infusion. Inspired by antibody-drug conjugates (ADC), here we develop an antibody-conjugated MSC-based drug delivery system (AcM-DDS) that combines CD4 monoclonal antibody-conjugated MSC (CD4-mBMSC) with liposome-encapsulated Cedirogant, a RORγt inverse agonist. AcM-DDS targets CD4⁺ T cells, key drivers of AID, and precisely delivers RORγt antagonists to suppress Th17-mediated inflammation. In mouse models of imiquimod-induced psoriasis and collagen-induced arthritis, AcM-DDS enhances MSC homing to inflamed tissues, reduces Th17 activity, lowers pro-inflammatory cytokine production, and preserves cartilage and bone integrity. Thus, our results indicate the promising applicability of our engineered platform for targeted immune modulation and provide support for the suitability of this therapeutic approach for the treatment of AIDs.

Introduction
Mesenchymal stromal cells (MSC) have shown promising potential in the treatment of autoimmune diseases (AID) such as psoriasis (PO) 1–3, systemic lupus erythematosus (SLE) 4–6, multiple sclerosis (MS) 7, inflammatory bowel disease (IBD) 8–10, and rheumatoid arthritis (RA) 11–13. Their ability to modulate immune responses and promote tissue repair makes them a valuable therapeutic option for these conditions. MSCs exert therapeutic effects through multiple mechanisms: secreting anti-inflammatory factors and exosomes that suppress aberrant T and B cell activation 14,15, promoting the expansion of regulatory T cells (Treg) to restore immune homeostasis 16, and migrating to damaged tissues to facilitate tissue regeneration 17,18. A key advantage of MSC is their availability from multiple sources, such as bone marrow, adipose tissue, and umbilical cords 19. Their low expression of major histocompatibility complex molecules reduces the risk of immune rejection in allogeneic transplantation 20, and their genetic stability and multipotency during in vitro expansion further enhance their clinical utility 21,22. Clinical studies have demonstrated that MSC transplantation effectively reduces psoriatic lesions and alleviates pruritus 1–3, while also showing safety and efficacy in treating RA 11–13, SLE 4–6, MS 7, and IBD 8–10. These findings highlight the immense therapeutic potential and clinical value of MSCs in managing AIDs.

However, the low homing efficiency of MSCs remains a critical limitation in their clinical application for AID 23. Following systemic administration, a large proportion of MSCs become trapped in non-target organs, failing to migrate effectively to sites of inflammation or tissue damage, thereby reducing therapeutic efficacy 24. MSC homing is mediated by chemokine gradients, adhesion molecules, and their corresponding receptors, which guide MSCs to inflamed or damaged tissues 25. However, under pathological conditions, the dysregulated expression of chemokines and adhesion molecules, along with a hostile inflammatory microenvironment, often impairs these mechanisms, leading to inefficient MSC migration and retention at target sites 26. Consequently, higher doses of MSCs are required to achieve the desired therapeutic effects, which not only increase treatment costs but also raises the risk of adverse effects 27. To enhance the homing efficiency of BMSCs, genetically modified BMSCs overexpressing receptors such as CXCR6 and CCR2 have been developed, enabling improved targeting to inflamed sites via increased responsiveness to inflammatory signals 28,29. However, the limitations of genetic modification, including safety concerns, hinder their widespread application 30. Therefore, enhancing MSC homing efficiency is essential to improving their clinical efficacy and expanding their therapeutic applications in AIDs (Fig. 1a).

Antibody-drug conjugates (ADC), which combine monoclonal antibodies (Ab) with cytotoxic agents 31, offer a potential solution. By exploiting antibody specificity, ADCs ensure precise drug localization at pathological sites, minimizing off-target effects, and maximizing therapeutic efficacy 32. Inspired by this concept, Antibody-conjugated MSCs (Ac-MSC) have been developed to improve MSC homing (Fig. 1b). Conjugating MSCs with disease-specific antibodies enables these cells to selectively target inflamed or damaged tissues by binding to overexpressed pathological markers. This approach enhances MSC accumulation at disease sites, amplifying their immunomodulatory and regenerative potential.

Additionally, Ac-MSCs can function as “live cell drug carriers”, internalizing therapeutic agents and releasing them within the inflammatory microenvironment. Unlike ADCs, which require antigens (Ag) to undergo receptor-mediated endocytosis for efficient payload delivery 33, Ac-MSCs transport their internalized payload to inflammation sites, where it is extracellularly released and diffuses into adjacent target cells (Fig. 1b). This mechanism eliminates the need for antigen internalization, allowing Ac-MSCs to target a wide range of surface proteins and achieve site-specific drug accumulation rather than cell-restricted delivery. By integrating targeted drug delivery with the inherent therapeutic functions of MSCs, this strategy reduces required cell doses, lowering costs and minimizing systemic side effects. By combining ADC targeting precision with MSC multifunctionality, the Antibody-conjugated MSC Drug Delivery System (AcM-DDS) represents a promising strategy to enhance the efficacy of MSC-based therapies and expand their clinical applications in AID.

Lesion sites in AIDs are characterized by an accumulation of aberrantly activated CD4+ T cells, which drive immune dysregulation and inflammation 34. Among CD4+ T cells, Th17 cells play a pivotal role in autoimmune pathogenesis by secreting pro-inflammatory cytokines such as IL-17A 35–37. However, BMSCs do not significantly inhibit Th17 cells in some AID patients, which partly explains their uncertain clinical efficacy 38. The differentiation and function of Th17 cells are regulated by the transcription factor RAR-related orphan receptor gamma t (RORγt), whose hyperactivation in AID drives excessive IL-17A production, exacerbating inflammation and disrupting immune homeostasis 39,40. Although targeting RORγt with inverse agonists such as Cedirogant (ABBV-157) stabilizes the protein in its inactive conformation and represents a promising therapeutic approach for modulating Th17 activity 41, the clinical development of these compounds has been hindered by off-target toxicities 42.

To overcome these limitations, we conjugated CD4 monoclonal antibodies (CD4mAb) to mouse bone marrow-derived MSC (mBMSC) using bioorthogonal chemistry, generating CD4mAb-BMSCs that selectively targeted CD4+ T cells and localized to lesion sites (Fig. 1c). We proposed that augmenting BMSCs with a small-molecule drug capable of directly suppressing Th17 cells would improve their therapeutic efficacy against AIDs. Accordingly, CD4mAb-BMSCs were loaded with liposomes encapsulating Cedirogant, leveraging the high encapsulation efficiency and biocompatibility of liposomes for controlled delivery. This AcM-DDS framework therefore integrates targeted recognition of pathogenic T cells with localized pharmacologic inhibition of RORγt, establishing a coherent strategy for restoring immune balance in AID.

Here we present the application of this AcM-DDS platform for the treatment of AIDs. The system effectively suppresses Th17 differentiation and IL-17A overexpression in two Th17-driven disease models: the imiquimod (IMQ)-induced PO model and the collagen-induced arthritis (CIA) model. In PO mice, AcM-DDS significantly inhibits Th17 differentiation and IL-17A overexpression, reduces pro-inflammatory cytokine release, alleviates inflammation and improves skin lesions (Fig. 1d). In CIA mice, it downregulates joint Th17 activity and IL-17A levels, thereby mitigating cartilage and bone destruction (Fig. 1e). These results demonstrate the therapeutic potential of AcM-DDS in controlling Th17-mediated pathology and highlight its broader applicability to other inflammatory disorders characterized by dysregulated T-cell responses.

Results
Enhanced homing efficiency of CD4 antibody-conjugated mBMSC to inflammatory lesions in PO mice
Bioorthogonal technology enables cell membrane modification while preserving biological integrity 43. Ac₄ManNAz metabolically labels cells by introducing azide groups into membrane glycoproteins and glycolipids via the sialic acid biosynthesis pathway 44. Simultaneously, lysine residues on antibodies react with DBCO-NHS esters to yield DBCO-modified antibodies 43, which rapidly conjugate with azide-functionalized MSC membranes through catalyst-free click chemistry at room temperature, generating antibody-conjugated MSC (Fig. 2a).

Th17 cells are central to the pathogenesis of psoriasiform inflammation. Consistent with this, histological analysis revealed that most CD4⁺ T cells in psoriasiform lesions are Th17 cells (Supplementary Fig. 1). Additionally, the CD4 antibody Ibalizumab has been approved by the FDA, indicating a favorable safety profile 45. More importantly, CD4 here is not a therapeutic target but serves to direct BMSC toward inflamed regions. Based on this evidence, CD4mAb were conjugated onto metabolically azidated mBMSC to generate CD4-mBMSC with selective affinity for CD4⁺ T cells. Confocal microscopy confirmed successful azide incorporation on the mBMSC membrane after Ac₄ManNAz treatment, as evidenced by robust DBCO-FAM (a green fluorescent dye) fluorescence (Fig. 2b). Subsequently, CD4mAb were conjugated with DBCO-PEG4-NHS ester through amide coupling and purified by ultrafiltration, and then co-incubated with N₃-mBMSC via bioorthogonal click chemistry to obtain CD4-mBMSC. Flow cytometry analysis revealed a concentration-dependent increase in fluorescence intensity upon incubation of N₃-mBMSC with DBCO-CD4mAb-FITC. Quantitative fluorescence analysis indicated that when 25 µg of DBCO-CD4mAb-FITC was conjugated with 1 × 10⁶ N₃-mBMSC, approximately 2500 antibody molecules anchored per cell on average (Fig. 2c). Confocal microscopy further confirmed successful surface conjugation of CD4mAb (Fig. 2d).

To validate the binding capability of CD4-mBMSC, CD4⁺ T cells were isolated from the spleens of C57BL/6J mice using magnetic bead sorting (Supplementary Fig. 2a). N₃-mBMSC and CD4-mBMSC pre-labeled with Dil (a red fluorescent membrane dye) and co-cultured with CD4⁺ T cells. Confocal imaging revealed that, compared to N₃-mBMSC, CD4-mBMSC exhibited significantly enhanced binding to multiple CD4⁺ T cells, confirming their targeted binding capacity toward CD4⁺ T cells in vitro (Fig. 2e, f, Supplementary Fig. 2b, c). Furthermore, the in vivo targeting efficiency of CD4-mBMSC was evaluated in IMQ-induced PO mice. DiD (a near-infrared lipophilic fluorescent dye)-labeled mBMSC and CD4-mBMSC were intravenously administered, followed by in vivo imaging using IVIS. CD4-mBMSC showed faster accumulation and longer retention in the spleen and inflamed dorsal skin regions than unmodified mBMSC, indicating superior in vivo targeting efficiency (Fig. 2g, h). Consistently, flow cytometric analysis of dorsal skin tissues 72 h post-injection revealed a significantly higher proportion of DiD⁺ cells in the CD4-mBMSC group, further confirming enhanced accumulation of CD4-modified mBMSC in inflamed skin (Supplementary Fig. 2d).

Uptake Cedirogant-loaded liposome (CdgLipo) to construct AcM-DDS
RORγt inverse agonists suppress IL-17A for AID treatment; however, challenges related to selectivity and off-target effects have raised safety concerns, resulting in the discontinuation of clinical trials 46. To overcome these limitations, we aimed to load RORγt inverse agonists into CD4-mBMSC to enable targeted drug delivery at the disease site. For this purpose, we encapsulated Cedirogant (Cdg)—a Phase II RORγt inverse agonist previously discontinued due to safety issues 47—into liposomes (CdgLipo) composed of DPPC, DSPE-PEG2000, and cholesterol (Fig. 3a). HPLC analysis revealed that the liposomes had a drug loading capacity of approximately 8.48% (w/w, drug-to-lipid ratio) and an encapsulation efficiency of 92.8% (percentage of total Cdg encapsulated in liposomes). Characterization of CdgLipo, using transmission electron microscopy (TEM) (Supplementary Fig. 3a), Malvern particle size analysis (Supplementary Fig. 3b) and nano-flow cytometry (Supplementary Fig. 3c), revealed an average particle size of 76.3 ± 14.8 nm, with a particle concentration of 9.55 × 10¹⁰ particles/mL (Supplementary Fig. 3d). No significant changes in particle size were observed over 7 days, but a marked increase in size was noted over a longer period (Supplementary Fig. 3e). The zeta potential remained stable, with no significant fluctuations over 27 days (Supplementary Fig. 3f). After 12 hours of incubation with mBMSC, Calcein-AM/PI staining showed no signs of cytotoxicity (Supplementary Fig. 3g).

Then, CD4-mBMSC were incubated with CdgLipo for one hour, and intracellular localization of liposomes was observed predominantly around mitochondria by Bio-TEM (Fig. 3b). To further investigate cellular uptake, Cy5-labeled CdgLipo (Cy5-CdgLipo) was compared to free Cy5, showing superior internalization by mBMSC (Supplementary movies 1 and 2). Mitochondrial and lysosomal staining showed a strong colocalization of Cy5-CdgLipo fluorescence with mitochondria, indicating effective lysosomal escape (Fig. 3c). Furthermore, quantitative fluorescence analysis based on Cy5 intensity allowed estimation of nanoparticle uptake per cell, indicating that each CD4-mBMSC internalized approximately 1.15 × 10⁴ CdgLipo particles on average.

To evaluate drug release kinetics, we assessed Cdg release from CdgLipo alone and from CD4-mBMSC after internalization. While free CdgLipo exhibited nearly complete Cdg release within 48 hours, intracellular CdgLipo displayed a more controlled release profile, with approximately 76.31% of Cdg released over the same period (Fig. 3d). Interestingly, in a separate experiment, we observed that both N₃-mBMSC and mBMSC internalized Cy5Lipo with comparable efficiency (Fig. 3e). But N₃-mBMSC released Cy5 significantly more slowly (Fig. 3f, Supplementary Fig. 4a). This suggests that glycoprotein azidation on the cell membrane may impact small molecule efflux. Further investigation using the P-glycoprotein inhibitor verapamil revealed that N₃-mBMSC demonstrated an efflux capacity comparable to verapamil-treated mBMSC (Supplementary Fig. 4b). These findings indicate that azidation impairs P-glycoprotein-mediated efflux in mBMSC, enhancing their potential for drug delivery applications.

Furthermore, we analyzed osteogenic differentiation in four cell groups: mBMSC, mBMSC incubated with CdgLipo, CD4-mBMSC, and AcM-DDS. ALP staining (early osteogenesis) and Alizarin Red (AR) staining (late osteogenesis) showed no significant differences in differentiation capacity among these groups (Fig. 3g). Chondrogenic differentiation, assessed by Alcian blue (AB) staining of cell-derived spheroids (Fig. 3h) and adipogenic differentiation, evaluated by Oil Red O (ORO) staining (Fig. 3i), also revealed no appreciable differences, indicating comparable chondrogenic and adipogenic potentials.

In addition, we further performed bulk RNA-Seq analysis and found that both antibody-conjugated BMSC and antibody-conjugated, drug-loaded BMSC affect less than 0.2% of the total transcriptome (log₂FC > 2 or < −2, p < 0.05). Moreover, the highly variable genes were not involved in signaling pathways related to adipogenic, osteogenic, or chondrogenic differentiation of BMSC (Supplementary Fig. 5a-e). Notably, the expression levels of Fgf2 and Ptgs2—both known to promote BMSC proliferation 48,49—were markedly increased, which is consistent with our observation that AcM-DDS exhibited enhanced proliferation compared to mBMSC (Supplementary Figs. 5f, 3j). Meanwhile, live/dead staining showed no notable differences in viability among the groups (Fig. 3k). All of these results suggest that the AcM-DDS system, constructed using CdgLipo and CD4-mBMSC, preserves MSC stemness and proliferation capacity while demonstrating superior drug delivery potential.

AcM-DDS effectively treats IMQ-induced PO in an acute inflammation mouse model
The IMQ-induced PO model is an acute inflammatory mouse model induced by topical IMQ, closely mimicking the pathological and immune features of human PO, and widely utilized in the preclinical development of PO therapies 50. In this study, dorsal hair was removed from male Balb/c mice, followed by topical Vaseline or IMQ application. IMQ-treated mice were randomly assigned to five groups: Model, mBMSC, CD4-mBMSC, CdgLipo, and AcM-DDS. For MSC-based treatments, 1 × 10⁶ cells per mouse were administered via tail vein injection, which is a common used dosage and route in preclinical MSC therapy studies for autoimmune diseases 51, ensuring reliable efficacy and safety. For Cdg, the administered dose (4.5 mg/kg) corresponded to the maximal amount of drug internalized by 1 × 10⁶ mBMSC. IMQ and Vaseline were applied daily for five days, and all mice were sacrificed on day 7 for analysis (Fig. 4a).

Following IMQ application, all IMQ-treated groups exhibit a slight reduction in body weight compared to the Sham group (Supplementary Fig. 6a). Dorsal skin lesions developed by day 4, with scaling appearing on day 5 in all groups except AcM-DDS (Supplementary Fig. 6b). By day 7, the AcM-DDS group remained largely free of scaling, while the mBMSC, CD4-mBMSC, and CdgLipo groups showed marked improvement over the Model group. Notably, CD4-mBMSC group exhibited superior therapeutic effects compared to mBMSC, indicating that antibody conjugation enhances MSC-based therapy (Fig. 4b). Histological analysis supported these findings. The hematoxylin and eosin (H&E) staining revealed severe epidermal thickening and pronounced wrinkling in the Model group, which were significantly alleviated in all treatment groups, with the most pronounced improvement in AcM-DDS (Fig. 4c, d). Additionally, Masson staining demonstrated that AcM-DDS markedly reduced fiber deposition in the dorsal skin (Fig. 4e, f).

For comparison, additional groups were treated with the positive control methotrexate 52 (MTX, daily oral gavage), free Cdg (single intravenous injection), or repeated doses of CdgLipo (two intravenous injections during the 5-day IMQ treatment period). Based on the daily assessment of skin scaling, along with H&E and Masson staining results, both MTX and free Cdg induced moderate improvements, while repeated CdgLipo dosing produced slightly better outcomes. However, none of these treatments achieved the therapeutic efficacy of AcM-DDS, as evidenced by more limited lesion resolution and histopathological recovery. Consistently, the AcM-DDS group exhibited the most pronounced reductions in spleen enlargement, epidermal thickness, and dermal fibrosis (Supplementary Fig. 7a–i).

To investigate whether ACM-DDS regulates Th17-cell-mediated inflammation, immunofluorescence (IF) staining for CD4, IL-17A, and RORγt was performed on frozen skin sections. IL-17A and RORγt were highly expressed at the epidermal-dermal junction in the Model group but significantly reduced in the mBMSC and CdgLipo groups, slightly higher in CD4-mBMSC, and nearly absent in AcM-DDS (Fig. 4g–i). CD4 expression was moderately reduced in all treatment groups compared with the Model group, with no significant differences among them (Supplementary Fig. 8). Collectively, these results demonstrate that AcM-DDS achieves superior therapeutic efficacy primarily through potent inhibition of Th17-cell-mediated inflammation in the skin.

AcM-DDS attenuates Th17-driven cytokine expression and systemic inflammation in IMQ-induced PO mice
To further elucidate the immunomodulatory effects of AcM-DDS, we first evaluate local cytokine expression in the dorsal skin of IMQ-induced PO mice. IF staining revealed marked upregulation of IL-21 and IL-22 in the Model group, both of which are critical downstream mediators of Th17-driven inflammation. In contrast, AcM-DDS treatment led to a pronounced suppression of IL-21 and IL-22 expression compared with other treatment groups (Fig. 5a–d), suggesting potent inhibition of Th17-associated inflammatory pathways.

Luminex multiplex assay was performed to assess the expression of 22 inflammatory cytokines in total protein extracts from dorsal skin tissues (Fig. 5e). Compared to the Sham group, the Model group exhibited a significantly elevation in multiple pro-inflammatory cytokines, including Eotaxin, G-CSF, GM-CSF, IL-12p40, IL-12p70, IL-17A, IL-1α, IL-1β, IL-4, KC, MIP-1α, and MIP-1β (Supplementary Fig. 9). Treatment with AcM-DDS, as well as mBMSC, CD4-mBMSC, and CdgLipo, led to an overall downward trend in these cytokines, indicating effective attenuation of inflammation. Notably, although reductions in IL-17A and GM-CSF were comparable among the cell-based and liposomal treatments (Fig. 5f, g), IF analysis revealed a more pronounced local suppression of IL-17A, IL-21 and IL-22 by AcM-DDS, suggesting that its superior modulation may primarily occur at the tissue microenvironment level rather than in total tissue cytokine content. Interestingly, CD4-mBMSC showed stronger suppression of G-CSF and IL-12p40 than mBMSC, suggesting that even in the absence of Cdg loading, enhanced localization and prolonged residence of MSC at inflamed sites contributes significantly to their anti-inflammatory activity. Although IL-17A and RORγt expression appeared only partially reduced in the CD4-mBMSC group by IF, this likely reflects localized measurement limits, as the Luminex assay—analyzing whole-skin lysates from multiple cellular sources—revealed a pronounced overall suppression of inflammatory cytokines.

Given that systemic inflammation in the IMQ-induced PO model often manifests as splenomegaly and elevated IL-17A levels 50, we next examined the spleen to assess potential systemic effects of AcM-DDS. Consistent with prior observations, significant splenic enlargement was observed in all IMQ-treated groups compared to Sham (Supplementary Fig. 10). Histological examination revealed substantial red pulp expansion and tissue edema in the Model group, which persisted in the mBMSC, CD4-mBMSC, and CdgLipo groups but was substantially alleviated by AcM-DDS treatment (Fig. 5h-i). Additionally, H&E staining of heart, liver, lungs, and kidneys showed no histopathological abnormalities across all groups, indicating good biosafety (Supplementary Fig. 11). Immunohistochemistry (IHC) analysis further demonstrated a significant reduction in IL-17A expression across treatment groups, with the AcM-DDS group exhibiting the most pronounced suppression (Fig. 5j-k). All of these results indicate that AcM-DDS effectively attenuates systemic inflammation and mitigates splenic immune activation in the IMQ-induced PO model.

AcM-DDS ameliorates joint inflammation and bone erosion in a CIA mouse model of RA
Given the diverse types of AID and the limitations of the IMQ-induced PO model as an acute inflammation model, evaluating the efficacy of the AcM-DDS system in chronic autoimmune settings remains challenging. The CIA mouse model, a well-established model for RA research, induces an autoimmune response against type II collagen, leading to chronic joint inflammation, progressive cartilage degradation, and bone erosion 53. Here, we used the CIA-induced mouse RA model to investigate the application of the AcM-DDS in the treatment of chronic autoimmune diseases. To establish this model, male DBA/1J mice were immunized twice via tail injection of a collagen-Freund’s adjuvant emulsion, with a two-week interval between immunizations. One week after the initial immunization, mice were assigned to the same treatment groups and dosing regimen as in the IMQ model, receiving retro-orbital venous injections every 10 days for one month (Fig. 6a). IVIS imaging revealed that, compared with DiD-mBMSC, retro-orbital injection of DiD-CD4-mBMSC resulted in preferential accumulation in the limbs and inflammatory regions of CIA mice within 24 h (Supplementary Fig. 12). Throughout the study, all groups exhibited stable weight gain (Supplementary Fig. 13a). By day 18 post-immunization, the Model group developed limb swelling, with 5 out of 6 mice displaying pronounced joint inflammation at the study endpoint. The mBMSC group exhibited early-stage swelling with lower severity, resulting in a final incidence of 3/6. In the CdgLipo group, inflammation onset was delayed until day 28; however, by the endpoint, 4/6 mice exhibited severe joint swelling, aligning with the limited clinical efficacy of IL-17A monoclonal antibodies in RA treatment. The CD4-mBMSC group displayed a milder disease phenotype, with arthritis incidence limited to 2/6 mice. Notably, AcM-DDS treatment significantly attenuated disease progression, with only 1/6 mice exhibiting mild inflammation at the endpoint (Fig. 6b).

Micro-CT analysis of hind limb joints (knee, ankle, and foot) revealed severe bone erosion and structural degradation in the Model group (Fig. 6c). The CdgLipo group exhibited notable but less severe bone loss, whereas the mBMSC groups showed minimal knee cartilage destruction but substantial foot bone erosion. The CD4-mBMSC group demonstrated improved joint integrity compared to mBMSC, while the AcM-DDS group exhibited near-intact bone structures, comparable to the Sham group. Three-dimensional quantitative analysis of the subchondral bone in the knee joint revealed significant improvements in bone volume fraction (BV/TV), structural model index (SMI), and trabecular number (Tb.N) parameters in all mBMSC-based treatment groups, while the CdgLipo group showed no apparent recovery (Fig. 6d, e, Supplementary Fig. 13b, c). Histological analysis of decalcified knee (Supplementary Fig. 13d) and ankle (Supplementary Fig. 13e) joints sections via H&E staining further supported these findings. Both the Model and CdgLipo groups displayed evident synovial inflammation, cartilage degradation and subchondral bone damage. Toluidine blue staining of knee joints (Fig. 6f, g) and safranin O/fast green staining of ankle joints (Fig. 6h, i) revealed varying degrees of cartilage degeneration among mBMSC-based treatment groups. Noticeable loss of acidic glycosaminoglycans was observed in the mBMSC group, indicating evident cartilage degradation. The CD4-mBMSC group also showed signs of cartilage degeneration, but to a lesser extent compared with the mBMSC group, suggesting partial chondroprotection. In contrast, the AcM-DDS group exhibited well-preserved cartilage structure and proteoglycan content, showing the most pronounced protective effect among all treatments.

AcM-DDS reduces inflammatory marker expression in knee joints and spleens of CIA mice
RA differs from PO in pathogenesis. Whereas PO is primarily driven by Th17 cell-mediated inflammation, RA involves a more complex immune network 54,55. In addition to IL-17A, TNF and IL-6 drives synovial inflammation and joint destruction 55. Moreover, MMP3 accelerates cartilage degradation by breaking down the extracellular matrix, exacerbating joint damage 56. To further elucidate the differential therapeutic effects among treatment groups, we performed IHC analysis of CD4 (Fig. 7a), IL-17A (Supplementary Fig. 14a), RORγt (Supplementary Fig. 14b), TNF (Fig. 7b), IL-6 (Fig. 7c), and MMP3 (Fig. 7d) in knee joint paraffin sections of CIA model mice. Compared with the Model, CD4 expression was significantly reduced in the mBMSC, CD4-mBMSC, and CdgLipo groups, with no notable differences among them. In contrast, AcM-DDS treatment nearly abolished their expression (Fig. 7e). Similarly, IL-17A and RORγt levels were significantly lower in all four treatment groups compared to the Model group, though no significant differences were observed among them, indicating robust suppression of Th17 cell differentiation (supplementary Fig. 14c, d). In contrast, TNF and IL-6 exhibited distinct expression patterns. While CdgLipo failed to suppress TNF, CD4-mBMSC showed greater inhibitory effects than mBMSC, comparable to AcM-DDS (Fig. 7f). Similarly, IL-6 expression persisted to some extent in the mBMSC, CD4-mBMSC, and CdgLipo groups but was nearly undetectable in the AcM-DDS group (Fig. 7g). For MMP3, CdgLipo had minimal impact on MMP3 expression, whereas mBMSC, CD4-mBMSC, and AcM-DDS effectively suppressed MMP3 expression (Fig. 7h). These findings suggest that variations in inflammatory cytokine regulation among treatment groups may underlie differences in therapeutic efficacy.

Furthermore, we assessed the proportion of Th17 (CD4⁺IL-17A⁺) cells in splenic tissues using flow cytometry. Compared to the Model group (24.9%), the proportion of Th17 cells was significantly reduced in the mBMSC (8.5%), CD4-mBMSC (11.5%), CdgLipo (11.4%), and AcM-DDS (7.37%) groups, with levels comparable to the Sham group (5.50%) (Fig. 7i, supplementary Fig. 14e). In situ IF staining for CD4, RORγt, and IL-17A in frozen spleen sections further corroborated the flow cytometry findings (Fig. 7j, supplementary Fig. 14f). Considering the potential safety concerns associated with long-term administration, serum biochemical analyses were performed to evaluate hepatic and renal function (ALT, AST, γ-GT, UREA, CREA, and UA). All parameters remained within normal ranges across groups (supplementary Fig. 15a). Moreover, H&E staining of paraffin-embedded sections of the heart, liver, lung, and kidney (supplementary Fig. 15b), as well as the jejunum and colon (supplementary Fig. 16), revealed no apparent pathological alterations, indicating that these treatments did not induce organ toxicity. These results collectively demonstrate that AcM-DDS effectively suppresses systemic Th17 differentiation, thereby mitigating inflammation both in the spleen and at the joint level, ultimately contributing to the attenuation of RA pathogenesis.

Methods
Cell culture
C57BL/6 mouse bone marrow MSC (mBMSC) were purchased from Oricell (Oricellbio, cat# MUBMX-01001, CN). The mBMSC were cultured in αMEM medium (Gibco, cat# C12571500, USA) supplemented with 10% fetal bovine serum (Hyclone, cat# SV30208.02, USA), 1% Penicillin-Streptomycin solution (Gibco, cat# 15140122, USA), 1 ng/mL murine FGF-basic protein (Peprotech, cat# 450-33, USA), and 1% GlutaMAX (Gibco, cat# 07946, USA).

Preparation of CD4 monoclonal antibody conjugated mBMSC (CD4-mBMSC)
To prepare the DBCO-modified antibody, 15 μg of CD4 monoclonal antibody (Invitrogen, cat# 14-0041-85, USA) was reacted with 69 μg of DBCO-PEG4-NHS ester (Confluore, cat# BCD-7, CN) in 1 mL PBS under gentle stirring (200 rpm) at 4°C overnight. The following day, the reaction mixture was centrifuged using a 100 kDa ultrafiltration device (Millipore, cat# UFC5100096, GER) to remove any unreacted DBCO-PEG4-NHS ester, yielding DBCO-CD4mAb. Similarly, DBCO-PEG4-NHS ester was conjugated with FITC-labeled CD4 monoclonal antibody (eBioscience, cat# 11-0041-82, USA) under the same conditions to obtain DBCO-FITC-CD4mAb.

For cell surface modification, 1 × 10⁶ mBMSC were incubated overnight with 6 mL of 10 μM N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) (MCE, cat# HY-W728531, USA). The next day, cells were treated with 1 mL of 100 nM DBCO-FAM (Confluore, cat# BF-11, CN), DBCO-CD4mAb, or DBCO-FITC-CD4mAb and incubated at 37°C for 30 min. Afterward, stain the nuclei with Hoechst 33342 (Beyotime, cat# C1027, CN) and observe the fluorescence on the cell membranes using a confocal fluorescence microscope (ZEISS, LSM900, Germany). The number of antibodies anchored on each cell was further quantified by flow cytometry using AccuCheck™ ERF reference beads (Invitrogen, cat# A55950, USA), revealing that approximately 2,500 antibody molecules were conjugated per cell on average.

Formulation and characterization of Cedirogant-loaded liposome (CdgLipo)
Dissolve 1,2-Dipalmitoyl-rac-glycero-3-phosphocholine (Aladdin, cat# D353387, CN), cholesterol (Aladdin, cat# C104028, CN), DSPE-PEG2000 (Melopeg, cat# 070701, CN), and Cedirogant (Hifuture, cat# 2055496-11-0, CN) in dichloromethane in a mass ratio of 6:2:2:1. Remove the dichloromethane by rotary evaporation under reduced pressure to form a thin film. Hydrate the film by adding PBS and sonicating at 42°C for 30 minutes to obtain CdgLipo. To prepare Cy5-CdgLipo, add 0.15 mass ratio of lipophilic Cy5 (Aladdin, C266403#, CN) to the formulation and follow the same procedure.

To assess drug loading and encapsulation efficiency, mix 100 μL of Cy5-CdgLipo with 900 μL of acetonitrile, then centrifuge and analyze the supernatant using HPLC (SHIMADZU, LC-20ADxr, JP). Determine the particle size and zeta potential of the liposomes using a Malvern particle size analyzer (Malvern, ZETASIZER NANO ZS, UK). Evaluate the size and particle number of the liposomes using Nano-Flow Cytometry (NanoFCM, cat# N30E, CN). Capture the morphology of the liposomes with transmission electron microscopy (TEM, G2 Spirit Biotwin, CN) imaging.

Construction of AcM-DDS
To construct the AcM-DDS, 50 μL of CdgLipo suspension (2 mg/mL Cdg, approximately 9.55E+10 particles) was added to 1 mL of α-MEM complete medium and co-incubated with CD4-mBMSC for 1 h. After incubation, the cells were washed three times with PBS and counted to obtain the AcM-DDS construct. The Cdg uptake efficiency was quantified by collecting the culture supernatant and lysing the cells with acetonitrile, followed by HPLC analysis, which indicated that approximately 90 μg of Cdg was internalized per 1 × 10⁶ cells. Furthermore, based on the particle number obtained from Nano-Flow analysis, a linear correlation between the fluorescence intensity of Cy5-labeled CdgLipo and Cdg content was established, allowing the estimation that each CD4-mBMSC internalized approximately 1.15 × 10⁴ CdgLipo particles on average.

Biodistribution of CD4-mBMSC
Label 1 × 10⁶ mBMSC or CD4-mBMSC with 10 μM DiD cell membrane dye (Invitrogen, cat# V22887, USA). The labeled cells were injected into IMQ-induced PO mice via the tail vein or into CIA mice via retro-orbital venous injection. Whole-body fluorescence images were acquired at different time points post-injection using an in vivo imaging system (IVIS Spectrum, PerkinElmer, USA) to monitor the biodistribution of the transplanted cells.

Animals
Eight-week-old, male BALB/c mice (n = 80, cat# BSTSC-013) were purchased from BesTest Bio-Tech (Zhuhai, CN). Six to seven-weeks-old, male DBA/1J mice (n = 80, cat# N000219) were purchased from GemPharmatech (Foshan, CN). All mice were kept in a specific pathogen-free (SPF) animal facility at Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. Mice were accommodated in group housing under conditions of 20–24°C temperature and 40–60% humidity, following a 12-hour light/12-hour dark cycle. Mice were fed a standard chow diet (Keao, cat# 12231, CN) with ad libitum access to food and water. Experimental and control mice were housed in separate cages but within the same rack and environmental conditions, without cross-contact between groups. Euthanasia was performed by CO2 inhalation followed by cervical dislocation.

IMQ-induced PO mouse model
Male BALB/c mice were used to establish the imiquimod (IMQ)-induced psoriasis-like (PO) model. Under isoflurane anesthesia, a 2.5 × 2.5 cm area of dorsal fur was removed using depilatory cream and thoroughly cleaned. Beginning the following day, IMQ cream (MEDSHINE, CN) was applied daily to the depilated area for seven consecutive days to induce psoriatic inflammation, while the Sham group received Vaseline.

Two independent experiments were conducted. In the first experiment, mice were randomly assigned to six groups (n = 5 per group): Sham, Model, mBMSC, CD4-mBMSC, CdgLipo, and AcM-DDS. Starting on Day 2, treatments were administered intravenously as follows: mBMSC or CD4-mBMSC (1 × 10⁶ cells in 200 μL suspension), CdgLipo (4.5 mg/kg Cdg), or AcM-DDS (1 × 10⁶ CD4-mBMSC preloaded with 4.5 mg/kg Cdg liposomes). Sham and Model groups received equivalent volumes of saline. On Day 7, mice were euthanized, and their affected skin and internal organs were collected for subsequent analysis.

In the second experiment, a separate cohort of IMQ-induced mice was used to further evaluate the pharmacological efficacy and dosing schedule of free and liposomal Cdg formulations. Mice were divided into five groups: Sham (n = 3), Model (n = 3), MTX (positive control, n = 5), Cdg (n = 5), and CdgLipo (n = 5). MTX was administered daily via oral gavage at 1 mg/kg in a carboxymethyl cellulose sodium (CMC-Na) vehicle. Cdg was delivered as a single intravenous injection on Day 2 (4.5 mg/kg), whereas CdgLipo was administered intravenously on Days 2 and 5 (4.5 mg/kg per injection). Sham and Model groups received saline or Vaseline, respectively. All other experimental conditions, including IMQ application, euthanasia, and tissue collection, were identical to those in the first experiment.

Mouse model of collagen-induced arthritis
The CIA mouse model was established following the method previously reported 63. Thirty-six male DBA/1J mice were randomly divided into six groups, with six mice in each group, according to the grouping scheme used for psoriasis mice. For the initial immunization, bovine type II collagen (Chondrex, cat# 20022, USA) emulsified with complete Freund’s adjuvant (Chondrex, cat# 7001, USA) was subcutaneously injected into the base of the tail at a dose of 100 μL per mouse, while the Sham group received an equal volume of saline. Fourteen days later, a booster immunization was administered using type II collagen emulsified with incomplete Freund’s adjuvant (Chondrex, cat# 7002, USA). Treatment was initiated one week after the initial immunization, following the same dosing regimen as the psoriasis mouse model. Subsequent immunizations were administered every ten days, for a total of three immunizations. At the end of the experiment, blood, spleen, lymph nodes, and knee joints were collected from the mice.

Binding ability of CD4-mBMSC with CD4+ T cells
A single 8-week-old male BALB/c mouse was euthanized, and its spleen was harvested. The spleen was mechanically dissociated to obtain splenocytes, which were subsequently subjected to magnetic bead-based sorting for CD4⁺ T cell isolation (STEMCELL, cat# 19852A, CAN) according to the manufacturer’s protocol. To assess sorting efficiency, 100,000 cells were stained with FITC-CD4 monoclonal antibody and analyzed by flow cytometry (Easycell, WELLGROW, CN) to determine the proportion of CD4⁺ T cells. For co-culture experiments, CD4⁺ T cells were suspended and incubated with pre-seeded, Dil-labeled CD4-mBMSC for 3 hours. Following incubation, all culture media were removed, and the cells were gently washed with PBS. Nuclei were then counterstained with Hoechst 33342 (Beyotime, cat# C1022, CN), and cell binding interactions were visualized using confocal microscopy.

Luminex cytokines assay
After euthanizing the mice, the inflamed skin area was collected in a sterile environment. The samples were washed with PBS and rapidly frozen in liquid nitrogen. These samples were then sent to Shanghai Huaying Biomedical Technology Co., Ltd. for protein extraction and cytokines concentration test. Protein quantification was performed using the BCA assay. The Luminex 2000 system (Luminex Corporation, Austin, USA) was employed to measure the expression levels of Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-12p40, IL-12p70, IL-13, IL-17A, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF in the samples.

Osteogenic, adipogenic, and chondrogenic Differentiation of mBMSC-based group
Four groups of cells were used for differentiation assays: mBMSC, CdgLipo-mBMSC (mBMSC pre-loaded with CdgLipo), CD4-mBMSC, and AcM-DDS.

For osteogenic induction, an osteogenic induction medium consisting of 100 nM dexamethasone (Sigma-Aldrich, cat# D4902, USA), 10 mM β-glycerophosphate disodium salt hydrate (Sigma-Aldrich, cat# G9422, USA), and 50 μg/mL vitamin C (Sigma-Aldrich, cat# A8960, USA), all formulated in αMEM complete medium and filtered for sterilization. Seed mBMSC at a density of 1 × 10^5 cells per well in a 6-well plate (Corning, cat# 3516, USA) with 2 mL of normal complete medium per well. When cells reach 90% confluence, switch to osteogenic induction medium, refreshing it with new induction medium every two days. Continue the induction for 14 to 21 days. Fix cells using 4% paraformaldehyde solution (Beyotime, cat# P0099, CN) and proceed with staining using 1% Alizarin Red (Aladdin, cat# A100195, CN) or ALP staining (Beyotime, cat# P0321, CN).

For adipogenic differentiation, an adipogenic induction medium (OriCell, cat# MUXMX-90031, CN) was used according to the manufacturer’s instructions. After 28 days of induction, cells were fixed and stained with Oil Red O (Sigma, cat# O0625, GER) to visualize intracellular lipid droplets.

For chondrogenic differentiation, 5 × 10⁴ cells from each group were seeded into ultra-low attachment 96-well plates and cultured in chondrogenic induction medium (OriCell, cat# MUXMX-90041, CN) for 28 days. The resulting cartilage spheroids were fixed, cryoprotected with sucrose, embedded, and sectioned for Alcian Blue staining to evaluate glycosaminoglycan deposition.

Transcriptome sequencing
The mBMSC, CdgLipo-mBMSC, CD4-mBMSC, and AcM-DDS cells samples were collected and lysed using TRIzol reagent (Invitrogen, cat# 15-596-018, USA). Total RNA was extracted and subjected to bulk mRNA sequencing by BGI Genomics (Shenzhen, CN). Reads were quality-checked, filtered, and aligned to the mouse genome (mm10). Gene expression was quantified as FPKM, and differential expression between groups was analyzed using DESeq2 (FDR < 0.05). Functional enrichment analyses (GO and KEGG) were performed to interpret relevant biological processes.

CdgLipo uptake efficiency
Seed 4 × 10^5 mBMSC in a confocal culture dish. After Ac4ManNAz treatment and antibody coupling, stain the cells with Lysotracker Deep Red (Invitrogen, cat# L12492, USA), Mito-tracker Red (Invitrogen, cat# M22425, USA), and Hoechst 33342, and observe the staining using a confocal microscope. Immediately after, add Cy5-CdgLipo, and capture images after 2 minutes of uptake.

After incubating CD4-mBMSC with CdgLipo for one hour, the culture supernatant was collected, and the cells were trypsinized, counted, and lysed. The intracellular and supernatant Cdg concentrations were quantified using HPLC. The results indicated that the intracellular Cdg content reached 0.15 mg per million cells. Furthermore, based on the particle number obtained from Nano-Flow analysis, a linear correlation between the fluorescence intensity of Cy5-labeled CdgLipo and Cdg content was established, allowing the estimation that each CD4-mBMSC internalized approximately 1.15 × 10⁴ CdgLipo particles on average.

Cdg release efficiency from CDGLipo
To assess the release efficiency of Cdg from CdgLipo, 1 mg/mL CdgLipo was placed in a 3 kDa ultrafiltration membrane bag (Millipore, GER) and submerged in PBS under continuous stirring at 300 rpm. At designated time points (0, 1, 3, 5, 16, 24, 36, and 48 h), aliquots from the external PBS solution were collected, and the Cdg concentration was quantified using HPLC.

To evaluate the intracellular release kinetics of Cdg following cellular uptake, CD4-mBMSC were incubated with 1 mg/mL Cy5-CdgLipo for one hour, after which the medium was replaced with fresh culture medium. At 0, 1, 3, 5, 16, 24, 36, and 48 h, the culture supernatants were collected, and fluorescence intensity was measured using a microplate reader to quantify the extracellular release of Cy5-labeled Cdg.

Bio-TEM
Seed 1 × 10^6 mBMSC in a T25 flask. Following Ac4ManNAz treatment and antibody coupling, incubate the cells with CdgLipo for 1 hour. Detach with a cell scraper, centrifuge at 1100 xg for 5-10 minutes, and collect into a 1.5 mL microcentrifuge tube. Fix with 2.5% glutaraldehyde at 4°C overnight. Discard the fixative, and wash three times with 0.1 M phosphate buffer (pH 7.0) for 15 minutes each. Subsequently, fix with 1% osmium tetroxide for 1-2 hours, then wash three times again with phosphate buffer. Dehydrate using a series of ethanol concentrations (30%, 50%, 70%, 80%, 90%, 95%), each for 15 minutes, followed by 100% ethanol for 20 minutes. Transition to pure acetone for 20 minutes. Infiltrate with a resin-acetone mixture (1:1) for 1 hour, a mixture (3:1) for 3 hours, then pure resin overnight. Cure the embedded samples at 70°C overnight. Section the samples using a LEICA EM UC7 ultramicrotome into slices of 70-90 nm. Stain with lead citrate and saturated uranyl acetate in 50% ethanol for 5-10 minutes each. The sections are now ready for transmission electron microscopy observation and imaging.

Cell viability assay
To assess the viability of modified mBMSC, 5000 or 10000 mBMSC were seeded in 6-well plates and subjected to experimental treatments to generate CD4-mBMSC or AcM-DDS. Additionally, another group of mBMSC was incubated with CdgLipo for 1 hour, followed by drug removal. After 72 hours, cell viability was evaluated using the CCK-8 assay (GOONIE, cat# 100-106, CN). Furthermore, to determine cell viability and apoptosis, cells were harvested, stained with Calcein-AM/7-AAD (Beyotime, cat# C2015M, CN), and analyzed by flow cytometry to distinguish live and dead cell populations.

Differential uptake and release of free Cy5 and Cy5-CdgLipo by N3-mBMSC
To investigate the differences in uptake and release between N3-mBMSC and mBMSC, both cell types were incubated with either free Cy5 (0.15 mg/mL) or an equivalent amount of Cy5 encapsulated in Cy5-CdgLipo for 1 hour. Following incubation, cells were digested and subjected to flow cytometry analysis to quantify intracellular Cy5 fluorescence intensity, reflecting the uptake efficiency.

For release kinetics, after a 1-hour uptake, cells were washed and replaced with fresh culture medium. At designated time points (0 h, 6 h, 24 h, and 48 h), cells were digested and analyzed via flow cytometry to measure Cy5 fluorescence intensity, providing a quantitative assessment of Cy5 release efficiency over time. Additionally, at 0 h, 24 h, and 48 h, confocal microscopy was used to capture in situ fluorescence images, providing a spatial visualization of Cy5 retention and release.

Efflux efficiency of Calcein in mBMSC and N3-mBMSC after Verapamil treatment
To assess the efflux efficiency of calcein in mBMSC and N3-mBMSC, cells were pretreated with verapamil (20 μM) for 4 hours. Following treatment, cells were stained with calcein (5 μM) and Hoechst 33342 for 30 minutes. After staining, fluorescence intensity was monitored using confocal microscopy at 1 h, 6 h, and 12 h post-staining to evaluate the efflux dynamics of calcein. This experiment aims to determine the impact of verapamil on calcein retention and efflux efficiency in both cell types.

Micro-CT analysis of joint in CIA mice
Knee joints and ankle joints from CIA model mice in each treatment group were collected and fixed in neutral formalin for 24 hours, followed by ethanol fixation. Micro-CT scanning was performed using the Bruker μCT system (Skyscan 1276 Micro-CT, Kontich, Belgium) under the following parameters: voxel size of 6.5 μm (medium resolution), 70 kV, 200 mA, 0.25 mm Al filter, and an integration time of 350 ms. Three-dimensional reconstructions were generated from contoured two-dimensional images using a distance transformation-based algorithm within the CTvox software (version 3.3.0).

Histological staining
Skin and internal organs from IMQ mice, as well as knee and ankle joints and internal organs from CIA mice, were fixed in formalin for 24 hours, dehydrated, and embedded in paraffin. Sections of 6 μm thickness were prepared, dried, dewaxed with xylene, rehydrated through graded alcohols, and stained with hematoxylin (Servicebio, cat# G1004, CN) and eosin (Beyotime, cat# C0109, CN). Following dehydration and mounting with neutral resin, slides were scanned for H&E assessment.

Masson’s trichrome staining (Solarbio, cat# G1346, CN) was performed on mouse back skin sections according to the manufacturer’s instructions, using the same section preparation procedure. Toluidine blue staining (Servicebio, cat# G2543, CN) was applied to CIA mouse knee joint sections, while Safranin O/Fast Green staining (Sigma-Aldrich, cat# F7258-25G, USA) (MCE, Cat# HY-D0215, USA) was performed on CIA ankle joint sections to evaluate cartilage integrity.

IHC staining
For the spleen sections of the IMQ mouse model, after rehydration, they were blocked with 10% goat serum (Beyotime, CN) and incubated overnight at 4°C with IL-17A monoclonal antibody (Invitrogen, cat# 14-7179-82, USA). For the knee joint sections of the CIA model, they were incubated with IL-17A, RORγt (Abcam, cat# ab207082, USA), TNF (Abcam, cat# ab307164, USA), IL-6 (Abcam, cat# ab290735, USA), and MMP3 (Abcam, cat# ab52915, USA) monoclonal antibody. The next day, sections were treated with a goat anti-rabbit secondary antibody (Abcam, cat# ab6721, USA) or goat anti-mouse secondary antibody (Abcam, cat# ab6789, USA) for 1 hour, developed with DAB (Abcam, cat# ab64238, USA), mounted, and scanned for IHC results.

IF staining
Affected skin of the IMQ mouse model and spleen from the CIA mouse model were flash-frozen, embedded in OCT, and cryo-sectioned at 6 μm. After warming to room temperature, sections were fixed with 4% paraformaldehyde, blocked with 10% goat serum, and incubated overnight at 4°C with IL-17A monoclonal antibody, Anti-RORgamma, or CD4 monoclonal antibody. In addition, frozen skin sections were also stained with IL-21 monoclonal antibody (eBioscience, cat# 16-7211-82, USA) and IL-22 monoclonal antibody (eBioscience, cat# 16-7222-82, USA). The next day, they were treated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (Abcam, cat# ab150225, USA) or Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Abcam, cat# ab150077, USA). Finally, sections were mounted using a DAPI medium and imaged with a confocal microscope (ZEISS, SUPRA 55#, Germany).

Flow cytometry analysis of Th17 cells in the spleen of CIA mice
Fresh spleens were harvested from CIA model mice in each experimental group. The tissues were mechanically dissociated using a mesh filter, passed through a 100 μm cell strainer, subjected to red blood cell lysis, and resuspended in PBS for cell counting. To block Fc receptor binding, 1 × 10⁶ cells were pre-incubated with Ms CD16/CD32 Pure 2.4G2 (BD, cat# BD553141, USA). Surface staining for CD4 was performed using FITC-conjugated anti-CD4 monoclonal antibody. Subsequently, cells were fixed and permeabilized using the Cytofix/Cytoperm Soln Kit (BD, cat# BD554714, USA) before intracellular staining with Alexa 647-conjugated anti-IL-17A monoclonal antibody (BD, cat# BD560184, USA). Following staining and washing, cells were analyzed via flow cytometry to quantify the proportion of Th17 (CD4⁺IL-17A⁺) cells in the spleen. All antibodies used for IHC and IF staining, including their detailed information and working concentrations, are listed in Supplementary Table 1.

Statistical & reproducibility
Data are expressed as mean ± SD, with sample sizes detailed in the figure legends. Differences between groups were evaluated using unpaired Student’s t-tests or one-way/two-way ANOVA, followed by Tukey’s post hoc tests, as specified. In vivo experiments involved 5 to 10 mice per condition. A p-value of ≤ 0.05 was considered statistically significant, and values less than 0.0001 are reported as p < 0.0001. All statistical analyses were performed using GraphPad Prism 8 software.

Ethic statement
All animal handling and use was approved by the Institutional Animal Care and Use Committee (IACUC) of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. The study involving CIA mice was approved under registration number SIAT-IACUC-210901-YGS-WY-A2044 on September 17, 2021, and the study involving IMQ-induced mice was approved under registration number SIAT-IACUC-240409-YGS-WY-A2588 on April 18, 2024. All procedures were conducted in accordance with the ARRIVE guidelines for reporting animal research. All mice used in the study were male to ensure model consistency and minimize variability; sex was not considered a biological variable, as CIA is more susceptible in males, and IMQ-induced PO inflammation does not show strong sex bias.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents a novel and well-constructed strategy for the targeted treatment of AIDs by engineering CD4mAb-BMSCs combined with a liposomal delivery system carrying RORγt inverse agonists. The study is conceptually innovative and supported by comprehensive in vitro and in vivo data, including two disease models (PO and RA). The dual approach—integrating targeted cell therapy and controlled drug release—offers strong potential for translational application. However, several key issues require clarification and additional discussion before the manuscript can be considered for publication. These include mechanistic insights into selective targeting, the rationale for specific experimental choices, and aspects related to clinical safety and translatability. I provide specific comments below to help the authors further improve the clarity, completeness, and impact of their work.\nIntroduction\n1. It would be helpful for readers if the authors could elaborate on the underlying mechanism by which antibody conjugation to the cell surface prevents non-specific cellular internalization. Clarifying this point in the manuscript would greatly aid in understanding how selective delivery to Th17 cells is achieved.\n2. Have the authors considered potential off-target effects when CD4mAb-BMSCs bind to naïve CD4⁺ T cells rather than Th17 cells? Any possible immunological side effects arising from non-specific targeting should be discussed.\n3. Could the authors explain why liposomes were chosen as the delivery vehicle for the RORγt inverse agonists? It would be helpful to understand whether liposomes offer any particular advantages over other nanoparticle systems in this context.\nResults\n1. Figure 3f, Supplementary Figure 4a: The authors suggest that glycoprotein azidation of the cell membrane may influence small molecule efflux. Please provide a more detailed mechanistic explanation and supporting evidence to substantiate this claim.\n2. Figure 3h, Could the authors clarify why osteogenic differentiation was evaluated rather than adipogenic or chondrogenic differentiation? For applications in arthritis models, chondrogenic potential would appear more directly relevant. Additionally, the increased proliferation observed in AcM-DDS compared to mBMSCs warrants further explanation.\n3. Figure 6f, The use of Safranin O staining might provide clearer visualization of cartilage morphology and thickness compared to Toluidine Blue. The authors may consider discussing this alternative method.\n4. Figure 7a–c, Supplementary Figure 9: Articular cartilage consists solely of chondrocytes. Therefore, the DAB signals observed within the cartilage layers are more likely to represent gene expression in chondrocytes rather than infiltrating immune cells. Since immune cells such as T cells typically reside in the synovium or synovial fluid, analysis of these regions may be more appropriate for confirming T cell localization.\nDiscussion\n1. The Discussion section largely reiterates content from the Introduction and Results. It would be more impactful if this section focused on interpretation and implications of the findings. For instance, potential mechanisms that enable selective drug delivery to Th17 cells without unintended release could be discussed.\n2. The authors suggest that cell-mediated delivery could help overcome the clinical safety issues previously associated with Cedirogant. If so, it is necessary to clearly explain how AcM-DDS addresses these safety concerns. Moreover, supporting data regarding the safety of this delivery system should be included to substantiate this claim.\n3. Systemically administered mBMSCs may accumulate in major organs such as the liver, lungs, and spleen, raising potential safety concerns including tumorigenicity. In addition, when translating to human applications, issues such as the immunogenicity of CD4 antibodies and Cedirogant, dosing, and scalability should be considered. Discussion of these factors would enhance the clinical relevance and translational potential of the study.\nAdditional Comments\n1. The manuscript would benefit from a discussion on the in vivo retention time of AcM-DDS and the duration of its anti-inflammatory effects. If relevant experiments have been conducted, presenting this data would strengthen the translational significance of the work.\n2. The immunomodulatory effects of BMSCs and the pharmacological effects of Cedirogant are not clearly delineated. Without appropriate experimental controls (e.g., CD4mAb-BMSCs + empty liposomes, or repeated dosing of CdgLipo alone), it is difficult to assess their relative contributions. Additional discussion or clarification of this limitation would improve the mechanistic understanding of the system.\n3. While AcM-DDS is reported to suppress Th17 cells primarily through IL-17A inhibition, Th17 cells also secrete other proinflammatory cytokines such as IL-21, IL-22, and GM-CSF. Inclusion or discussion of these additional markers would help define the extent of Th17 modulation more comprehensively.", "reviewer_2": "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_3": "This study demonstrated the construction of a “live drug carrier”, proposed to be CD-4 tagged BM-MSCs, loaded with Cdg encapsulating liposomes for application in autoimmune diseases like psoriasis and arthritis. However the current study has certain gaps which need to be addressed before it can be considered suitable for publication.\n1. As per the authors, MSCs act as live drug carriers and will release the drug in inflammatory milieu without the need for internalization. However, the authors may clarify that if MSCs burst to release the drug in the inflammatory milieu, it will also be released in the circulation, what effect that may have? Authors may evaluate the same in the mice blood as well.\n2. The specificity of the graphical abstract with respect to the article maybe improved. Currently, the abstract does not represent the article, but seems like a generalized poster. Specifically, there is no specific mention of the RORγT antagonist, and it is not evident from the graphical abstract that the study was performed in psoriasis and arthritis models. The layout and content need to be improved.\n3. In Fig. 2 (d), the authors could have captured different frames and presented the quantification of the results, apart from only the representative images to demonstrate the CD-4+ T cells and CD4-BM-MSCs\n4. The authors may quantitate the percentage of MSCs that reached the skin upon intra venous administration and check the localization by quantifying the presence of CD-4 labelled or unlabelled cells at site by ex vivo methods\n5. In Fig. 2 (f), the authors suggest a significant difference in the skin and the spleen, however the authors may re-check whether there is a significant difference in the liver group at 24h as well.\n6. As per the statement of the authors, Cedirogant was discontinued due to off-targeting and safety issues. However, this does not seem in line with available literature and data about the drug, since as per the reports available, cedirogant was well tolerated and no safety concerns were reported (NCT05044234). Additionally, as per other data, cedirogant was discontinued by the manufacturer due to some new unfavourable toxicology findings in preclinial studied, Therefore, the authors may explain the plausibility of their hypothesis, strategizing to load cedirogant for mitigating off-targeting.\n7. The authors may elaborate further about the statement in Line 169- “HPLC analysis determined that the drug loading capacity of Cdg in liposomes was approximately 8.48%, with an encapsulation efficiency of 92.8%” It is unclear whether 8,48% of Cdg was loaded into liposomes and 92.8% lipososomes were able to take up the drug, or does it mean something else.\n8. The authors may quantify how many CD-4 receptors are present per MSCS, and how many drug loaded lipososmes are internalized per MSC, as this will be relevant to determine the clinical applicability.\n9. The authors could have included an only drug group (without liposomes encapsulation) to demonstrate the off-targeting effect suggested by them. Additionally, a positive control, e.g. a commercial treatment option could have been included.\n10. It is unclear how the authors determined the dose of the treatment groups in animals. The authors may test variable doses and present the data for the chosen dose.\n11. Time point based skin localization study in animal could have been included to determine the highest localisation of drug at a given time point. This could have also given insights on whether liposome is accelerating the time of delivery, as early relief might also be important clinically.\n12. The data in Fig 6 (d-f) may be quantified and evaluated for statistical significance\n13. The authors may opine about the fate of modified MSCs if kept in culture, and the liposomal load if they passage or divide, and what could be the shelf life of this modified MSC construct. Additionally, the authors may evaluate whether liposome internalization is affecting surface labelling?\n14. Since the MSCs are heavily modified and being proposed as a drug carrier, the authors may be very precise and exact about the reproducibility of the protocols, with respect to number of receptors and liposomes loaded.\n15. The authors mention in Line 407-409 that “However, such approaches, coupled with the challenges of MSC production standardization, elevate treatment costs and increase clinical complexity”. However, they may elaborate on how such heavy modifications of MSCs will not increase the cost of production and therapy.\n16. The author may reference that CD-4 antibodies do not raise any safety concerns, as mentioned in Line 423-428.\n17. The authors may elaborate more about the limitations of the study.\n18. In line 474-375, the authors suggest that their system shows a broad translational potential for immune disorders, however as also demonstrated in their own study, it seems that the applicability of this system primarily depends on the pathophysiology of the disease.", "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": "This manuscript presents a state-of-the-art drug delivery strategy that combines antibody-conjugated mesenchymal stem cells with drug-loaded liposomes to achieve targeted drug release at inflammatory sites. The therapeutic potential is evaluated in both acute (psoriasis) and chronic (rheumatoid arthritis) mouse models of autoimmune diseases.\nThe Antibody-Conjugated MSCs Drug Delivery System (AcM-DDS) is highly innovative and conjugates several therapeutic advantages specifically associated with the trophic and immunomodulatory effects of BMSC (confirmed in the in vivo experiments by the administration of BMSC alone) with the possibility to 1) conjugate antigen-specific antibodies (in this case for the CD4 antigen expressed on T cells) onto the BMSC surface to enhance their homing efficiency, 2) transform antibody-conjugated BMSCs (CD4-mBMSC) into cargo cells (AcM-DDS ) for small-molecule drugs (in this study, Cdg, a RORγt inverse agonist) enabling targeted delivery to sites of inflammation.\nThe study is well written and results are robust, built on carefully conducted and technologically solid experiments supporting this novel strategy for drug delivery in the treatment of autoimmune diseases. A few aspects of the study require clarification and revision to strengthen the manuscript.\nGeneral comment:\nBecause AcM-DDS can target any membrane protein, enabling more flexible and precise drug delivery, we wonder why authors used the broadly expressed CD4 antigen to target T cells, involved in AID pathogenesis. Although the delivery of Cdg, a RORγt inverse agonist, mainly restricts the drug effect on Th17 cells, the use of CD4 to guide AcM-DDS toward the sites of inflammation in IMQ-induced PO mice and in the CIA mouse model of RA poses, in a larger perspective, some concerns about the risk of targeting CD4+ T cells, a T cell subsets playing major roles in physiological immune responses. While the lack of abnormalities, observed in major organs (heart, liver, lungs and kidneys), may reflect the safety of AcM-DDS in these mouse models, assessed in a pathogen free environment, a different outcome may be observed upon an infectious trigger and in humans. Authors should comment on this issue in the discussion.\nMajor points:\n1. Why did authors change the administration protocol for the PO and RA mouse models experiments (i.v. in the tail vein for the IMQ study vs retro-orbital venous injection for the CIA mouse model)? Importantly, no data are provided to evaluate the biodistribution of MSCs following retroorbital administration. Authors should assess whether this alternative route leads to differences in organ accumulation\n2. In general, authors use citations quite unscrupulously particularly in the introduction section. A few examples: In lines 30-32, the statement “Mesenchymal stem cells (MSCs) have shown promising potential in the treatment of autoimmune diseases (AIDs) such as psoriasis (PO)1, systemic lupus erythematosus (SLE)2, multiple sclerosis (MS)3, inflammatory bowel disease (IBD)4, and rheumatoid arthritis (RA)5” is not directly supported by the references. Ref 6 (Pittenger et al, 1999) does not cover the anti-inflammatory properties of MSC. Ref 8 refers to MSC and B cells (and not Tregs) and Ref 9 uses a mice model defective of Tregs and therefore is not appropriate to support the effect of MSC on Tregs. More references are not totally appropriate along the text. Overall, authors are invited to carefully go through the text to ensure that citations properly support what they are claiming.\nMinor comments\n1. Authors use the term “Mesenchymal Stem Cells”, rather than “Mesenchymal Stromal Cells” which is now preferred. As the manuscript does not address any issue related to their regenerative potential and they only demonstrated that modified BMSC can differentiate into the osteogenic lineage, we would suggest using the term “Mesenchymal Stromal Cells” in the title and along the whole manuscript.\n2. In the legend for Figure 1, the authors should include a brief description of the design framework and experimental workflow.\n3. In lines 141-143, the authors claim “CD4-mBMSCs exhibited significantly enhanced binding to multiple CD4⁺ T cells, confirming their targeted binding capacity toward CD4⁺ T cells at inflammatory sites”. These data refer to confocal imaging, and quantitative analyses are needed to support the data.\n4. Why distribution of CD4-mBMSC did drop at 72 hours following i.v. administration (compared to high levels at 24 and 96h)?\n5. Although the manuscript describes the preparation of the AcM-DDS system, it does not provide quantitative data on the percentage of cells successfully conjugated or loaded.\n6. In Figure 5d/e and Supplementary Figure 7, the authors present heat maps and cytokine quantifications to support the anti-inflammatory effect of AcM-DDS. In lines 277–279, they claim that “Importantly, AcM-DDS treatment led to a substantial downregulation of these cytokines, suggesting a potent anti-inflammatory effect mediated by suppression of Th17-driven immune responses”. However, based on the quantification of IL-17A, the reduction appears comparable across the different treatment groups and does not clearly indicate a distinct effect from AcM-DDS. Authors should comment in the text.\n7. Why was IL-17A and RORγt expression only partially reduced in the CD4-mBMSCs mice group, considering that in the Luminex Multiplex Assay the effect on inflammatory cytokines was quite significant (Fig 5)?\n8. In lines 131 and 140, the fluorophores FAM and DiI are mentioned. These should be identified as fluorescent dyes when first introduced.\n9. In lines 142-143, the authors state that “confirming their targeted binding capacity toward CD4⁺ T cells at inflammatory sites”. However, the data comes from in vitro co-culture experiments. Therefore, the statement “at inflammatory sites” is misleading.\n10. In the legend of Figure 2e, the authors should add the imaging technique used to acquire the data.\n11. The sections describing the generation of CD4-antibody-conjugated MSCs lack clarity, and the methodology is difficult to follow. The authors should improve the readability of the text.\n12. For the supplementary movies, the authors should add a figure legend.", "reviewer_6": "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": "Response Letter to Reviewers\nDear Reviewers,\nFirst, we would like to express our sincere gratitude for your thorough review of our\nmanuscript and for the positive feedback you provided. We also greatly appreciate the\nvaluable comments and suggestions that you have offered. We recognize that these inputs are\ncrucial for improving the quality of our work. Over the past three months, we have conducted\nadditional animal experiments and updated the related data in response to the concerns raised\nby the reviewers.\nIn response to the reviewers’ comments, we have conducted additional animal\nexperiments, revised several sections of the manuscript, and updated the corresponding\nfigures and data. Specifically, we optimized the experimental design of the IMQ-induced\npsoriasis model, included new data on AcM-DDS biodistribution and therapeutic efficacy,\nimproved the mechanistic analyses, and refined the statistical and methodological\ndescriptions throughout the paper. These revisions have substantially strengthened the\nreliability and overall impact of our study.\nWe hope that our revisions and additions address the concerns raised and enhance the\nscientific value of our work. We sincerely thank you again for your detailed review, and we\nlook forward to receiving your feedback and, hopefully, acceptance of our revised\nmanuscript.\nSincerely,\nYan WANG\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript presents a novel and well-constructed strategy for the targeted treatment of\nAIDs by engineering CD4mAb-BMSCs combined with a liposomal delivery system carrying\nRORγt inverse agonists. The study is conceptually innovative and supported by\ncomprehensive in vitro and in vivo data, including two disease models (PO and RA). The dual\napproach—integrating targeted cell therapy and controlled drug release—offers strong\npotential for translational application. However, several key issues require clarification and\nadditional discussion before the manuscript can be considered for publication. These include\nmechanistic insights into selective targeting, the rationale for specific experimental choices,\nand aspects related to clinical safety and translatability. I provide specific comments below to\nhelp the authors further improve the clarity, completeness, and impact of their work.\nIntroduction\n1. It would be helpful for readers if the authors could elaborate on the underlying mechanism\nby which antibody conjugation to the cell surface prevents non-specific cellular\ninternalization. Clarifying this point in the manuscript would greatly aid in understanding how\nselective delivery to Th17 cells is achieved.\nResponse:\nThank you for your valuable suggestion. Unlike ADCs, which require target antigens (Ag) to\nundergo receptor-mediated endocytosis for efficient payload delivery (Fig. 1a), Ac-MSCs\ntransport their internalized payload to inflammation sites, where it is extracellularly released\nand diffuses into adjacent target cells (Fig. 1b). Therefore, our system does not ensure that\nCdg enters only Th17 cells but rather promotes the local enrichment of Cdg around CD4⁺ T\ncells at inflamed sites. Since Th17 cells predominate among CD4⁺ T cells in inflamed tissues,\nincreasing the local concentration of Cdg in their vicinity facilitates its action on Th17 cells.\nThis mechanism eliminates the need for antigen internalization, allowing Ac-MSCs to target a\nwide range of surface proteins and achieve site-specific drug accumulation rather than cell-\nrestricted delivery.\nWe have also incorporated this explanation into the revised introduction (Page 3, line 73-78).\n2. Have the authors considered potential off-target effects when CD4mAb-BMSCs bind to\nnaïve CD4⁺ T cells rather than Th17 cells? Any possible immunological side effects arising\nfrom non-specific targeting should be discussed.\nResponse:\nThank you for your comment. Although CD4 is broadly expressed among helper T-cell\nsubsets, our in vivo data indicates that AcM-DDS preferentially accumulates in inflamed\ntissues enriched with activated CD4⁺ T cells (Fig. 2g and 2h), where Th17 cells predominate\n(Supplementary Fig. 1). As naïve CD4⁺ T cells are relatively limited in inflamed sites, their\npotential engagement by AcM-DDS is expected to be limited. Consistent with this, no\napparent immunological side effects or abnormal immune activation were observed in either\nthe RA or PO models following repeated administration. Moreover, since MSCs are\ninherently immunomodulatory and non-cytotoxic, transient interactions with non-activated\nCD4⁺ T cells are unlikely to cause adverse immune responses.\nWe have also incorporated this explanation into the revised discussion (Page 25, line 478-\n485).\n3. Could the authors explain why liposomes were chosen as the delivery vehicle for the\nRORγt inverse agonists? It would be helpful to understand whether liposomes offer any\nparticular advantages over other nanoparticle systems in this context.\nResponse:\nWe thank the reviewer for this valuable comment. Liposomes were chosen as the delivery\nvehicle for the RORγt inverse agonists mainly due to their favorable structural features, high\ndrug-loading capacity, and well-established clinical safety profile.\nStructurally, liposomes possess a phospholipid bilayer architecture that closely resembles\nbiological membranes, which not only enhances biocompatibility but also facilitates efficient\ncellular uptake and intracellular drug release. In addition, their amphiphilic nature enables the\nefficient encapsulation of both hydrophilic and hydrophobic drugs; in this study, the\nhydrophobic RORγt inverse agonists were effectively incorporated into the lipid bilayer,\nachieving a high drug-loading efficiency and good stability.\nImportantly, liposomes are one of the few nanoscale delivery systems that have been\nextensively validated in clinical settings, with several FDA-approved formulations (e.g.,\nDoxil, AmBisome), demonstrating excellent safety and translational potential. These\nadvantages collectively make liposomes a rational and reliable choice for constructing the\nAcM-DDS platform in our study.\nWe have also incorporated this explanation into the revised introduction (Page 4, line 99-102).\nResults\n1. Figure 3f, Supplementary Figure 4a: The authors suggest that glycoprotein azidation of the\ncell membrane may influence small molecule efflux. Please provide a more detailed\nmechanistic explanation and supporting evidence to substantiate this claim.\nResponse:\nWe sincerely thank the reviewer for this insightful comment and apologize for any lack of\nclarity in our original manuscript that may have led to this misunderstanding. In fact, we had\nperformed additional experiments to further investigate whether metabolic glycan labeling\nand subsequent azidation affect small-molecule efflux. As shown in Supplementary Fig. 4b,\nfurther investigation using the P-glycoprotein (P-gp) inhibitor verapamil revealed that N3-\nmBMSCs exhibited an efflux capacity comparable to that of verapamil-treated mBMSCs,\nsuggesting that glycoprotein azidation does not markedly impair membrane transporter\nfunction.\nWe acknowledge, however, that this validation remains preliminary. Due to the peripheral\nnature of this observation relative to the central focus of our study, we were unable to pursue\nmore extensive mechanistic experiments at this stage. Nevertheless, we are currently\nperforming transcriptomic analyses of mBMSCs, CD4-mBMSCs, CdgLipo-mBMSCs, and\nAcM-DDS cells, which may provide additional insight into potential changes in membrane\ntransport–related pathways (NEW supplemented Fig. 5a-5f). We plan to further explore this\nmechanism in future studies.\nWe have also incorporated this modification into the revised Results (Page 10, line 224-231).\n2. Figure 3h, Could the authors clarify why osteogenic differentiation was evaluated rather\nthan adipogenic or chondrogenic differentiation? For applications in arthritis models,\nchondrogenic potential would appear more directly relevant. Additionally, the increased\nproliferation observed in AcM-DDS compared to mBMSCs warrants further explanation.\nResponse:\nWe sincerely thank the reviewer for this important comment and apologize for the oversight\nin our initial submission. In the revised version, we have now added these results:\nchondrogenic differentiation was evaluated by Alcian Blue staining (NEW Fig. 3h) and\nadipogenic differentiation by Oil Red O staining (NEW Fig. 3i), showing comparable\ndifferentiation potential among the different groups.\nRegarding the increased proliferation of AcM-DDS compared to mBMSCs, this is consistent\nwith our transcriptomic analysis, which revealed upregulated expression of proliferation-\nrelated genes such as Fgf2 and Ptgs2 (NEW Supplementary Fig. 5f).\nWe have also incorporated this modification into the revised Results (Page 10, line 220-223).\n3. Figure 6f, The use of Safranin O staining might provide clearer visualization of cartilage\nmorphology and thickness compared to Toluidine Blue. The authors may consider discussing\nthis alternative method.\nResponse:\nWe sincerely thank the reviewer for this valuable suggestion. In the initial submission, we had\nrepeatedly attempted Safranin O/Fast Green staining on paraffin-embedded knee joint\nsections; however, the staining results were unstable and unsatisfactory, likely due to pH\nvalues in the decalcifying solution, as reported in the literature (Hayer et al., 2021). Because\nthe available knee sections were exhausted during these attempts, we adopted toluidine\nblue—a more stable alternative under our conditions—for the initial histological assessment.\nDuring the revision stage, we carefully re-collected the preserved ankle joints, initiated a new\ndecalcification process, and successfully performed Safranin O/Fast Green staining. The new\nresults are presented in Figure 6h-i. Moreover, H&E staining of the corresponding ankle\njoints has been added and integrated with the knee joint histology (previously shown in Fig.\n6e) in the NEW Supplementary Figure 13e.\nWe have also incorporated this modification into the revised Results (Page 21, line 411-418).\n4. Figure 7a–c, Supplementary Figure 9: Articular cartilage consists solely of chondrocytes.\nTherefore, the DAB signals observed within the cartilage layers are more likely to represent\ngene expression in chondrocytes rather than infiltrating immune cells. Since immune cells\nsuch as T cells typically reside in the synovium or synovial fluid, analysis of these regions\nmay be more appropriate for confirming T cell localization.\nResponse:\nWe sincerely thank the reviewer for this valuable comment. Since RA is primarily driven by\nsynovial inflammation, we agree that the synovium is the more relevant tissue for analyzing T\ncell localization. In the revised manuscript, while maintaining the original data, we have re-\nfocused the magnified images to specifically highlight the synovium, meniscus, and cartilage\nregions. These modifications can be found in Figure 7a-d and Supplementary Figure 14a-b,\nallowing for a more accurate representation of T cell distribution within the relevant tissues.\nWe have also incorporated this modification into the revised Results (Page 22, line 425-433).\nDiscussion\n1. The Discussion section largely reiterates content from the Introduction and Results. It\nwould be more impactful if this section focused on interpretation and implications of the\nfindings. For instance, potential mechanisms that enable selective drug delivery to Th17 cells\nwithout unintended release could be discussed.\nResponse:\nThank you for your suggestion. The AcM-DDS system was designed to enrich the local\nconcentration of Cedirogant around CD4⁺ T cells rather than to achieve cell-internal release as\nin ADC therapeutics. In vitro release assays showed that approximately 25% of the payload\nwas released within 24 h, indicating that limited diffusion occurs before complete tissue\nhoming. Therefore, we acknowledge that unintended partial release cannot be entirely\nexcluded. A major direction for future optimization is to ensure that nanoparticles are released\nfrom MSC carriers only after tissue homing. To reduce premature drug release from\nintracellularly loaded MSCs and to ensure release only after homing, several complementary\nstrategies are feasible. The possible strategies include inducible CRISPRi/siRNA to reversibly\ndownregulate ABC transporters, or engineering MSCs to undergo inflammation-triggered\nexocytosis.\nWe have also incorporated this explanation into the revised Discussion (Page 25, line 514-\n526).\n2. The authors suggest that cell-mediated delivery could help overcome the clinical safety\nissues previously associated with Cedirogant. If so, it is necessary to clearly explain how\nAcM-DDS addresses these safety concerns. Moreover, supporting data regarding the safety of\nthis delivery system should be included to substantiate this claim.\nResponse:\nThank you for valuable comment. Cedirogant previously showed unfavorable systemic\nexposure and metabolic instability in preclinical studies, including body weight loss and\ngastrointestinal toxicity observed in a 39-week dog study (Tyring et al., 2024). In our\nexperiments, the treatment durations were considerably shorter (1 week for the PO model and\n5 weeks for the RA model). Histological analysis of the small intestine and colon revealed no\napparent injury in either the Cdg-alone or AcM-DDS groups. These findings suggest that, in\nour models, AcM-DDS primarily enhances local Cedirogant efficacy at inflammatory sites\nrather than reducing gastrointestinal side effects.\nWe have also incorporated this explanation into the revised Discussion (Page 25, line 501-\n506).\nIn response to the reviewer’s concern regarding the systemic safety of our AcM-DDS system,\nwe have now included additional biochemical analyses of hepatic and renal function in RA\nmice (Supplementary Fig. 15a). The results showed that serum levels of ALT, AST, BUN, and\ncreatinine remained within the normal physiological range across all treatment groups, with\nno significant differences compared to the control group. These findings further confirm that\nrepeated administration of AcM-DDS does not induce hepatotoxicity or nephrotoxicity,\nthereby strengthening the overall safety profile of our delivery system.\nWe have also incorporated this modification into the revised Results (Page 22, line 448-451).\n3. Systemically administered mBMSCs may accumulate in major organs such as the liver,\nlungs, and spleen, raising potential safety concerns including tumorigenicity. In addition,\nwhen translating to human applications, issues such as the immunogenicity of CD4 antibodies\nand Cedirogant, dosing, and scalability should be considered. Discussion of these factors\nwould enhance the clinical relevance and translational potential of the study.\nResponse:\nAs mentioned in our previous response, we have supplemented the manuscript with\nSupplementary Fig. 15a, which presents additional biochemical analyses of hepatic and renal\nfunction in RA mice. The serum levels of ALT, AST, BUN, and creatinine in all treatment\ngroups remained within normal physiological ranges, showing no significant differences\ncompared with the control group. These data further substantiate the systemic safety of our\nAcM-DDS platform and demonstrate that repeated administration does not cause detectable\nhepatic or renal toxicity. (Page 22, line 448-451)\nThe overall safety profile of AcM-DDS depends primarily on two components: the\nbiodistribution of the carrier BMSCs and the localization of the encapsulated small-molecule\ndrug. AcM-DDS functions by promoting BMSC accumulation at inflammatory sites and\nconsequently enriching the local concentration of Cdg in these regions. Therefore, at an\nequivalent systemic dose, the potential systemic toxicity of AcM-DDS should not exceed that\nof either BMSCs or Cdg administered alone. Notably, the localized enrichment at inflamed\ntissues may produce higher local drug concentrations, which could enhance efficacy but also\nrequires dose optimization to avoid local adverse effects. In our current dosing regimen, no\ntissue damage or inflammatory exacerbation was observed at the injection or inflammatory\nsites.\nBinding of the CD4 antibody to the CD4 receptor on T cells represents another potential\nsource of immunological concern, as it could interfere with endogenous ligand interactions\nsuch as CXCL12 binding. However, it is worth noting that CD4-targeting monoclonal\nantibodies have already been approved for clinical use and exhibit favorable safety profiles.\nThe CD4 antibody selected in this study, therefore, offers a relatively safer targeting option\ncompared to unvalidated antigens. Together, these findings support the safety and\ntranslational feasibility of AcM-DDS, although further optimization of dosing and long-term\nevaluation will be required for future clinical application.\nWe have also incorporated this explanation into the revised Discussion (Page 26, line 539-\n543).\nAdditional Comments\n1. The manuscript would benefit from a discussion on the in vivo retention time of AcM-DDS\nand the duration of its anti-inflammatory effects. If relevant experiments have been\nconducted, presenting this data would strengthen the translational significance of the work.\nResponse:\nThank you for your careful review and insightful comments. Bioluminescence tracking\nrevealed that AcM-DDS persisted at inflammatory sites for up to 96 hours post-injection (Fig\n2g), correlating with sustained IL-17A suppression. The extended retention underscores its\npotential for enhanced anti-inflammatory efficacy. Although real-time expression of IL-17A\nor other inflammatory cytokines were not continuously monitored, endpoint analyses\ndemonstrated that AcM-DDS treatment achieved the greatest reduction in IL-17A, IL-21, IL-\n22, and other proinflammatory cytokines compared with all control groups. As shown in Fig.\n2g and 2h, CD4-mBMSCs exhibited longer retention at inflamed tissues than unmodified\nBMSCs. Furthermore, in the cytokine profiling assays, CD4-mBMSCs showed stronger\nsuppression of G-CSF and IL-12p40 than mBMSCs (Fig 5e), suggesting that even in the\nabsence of CDG loading, enhanced localization and prolonged residence of MSCs at inflamed\nsites contribute significantly to their anti-inflammatory activity.\nWe have also incorporated this modification into the revised Results (Page 16, line 331-334).\n2. The immunomodulatory effects of BMSCs and the pharmacological effects of Cedirogant\nare not clearly delineated. Without appropriate experimental controls (e.g., CD4mAb-BMSCs\n+ empty liposomes, or repeated dosing of CdgLipo alone), it is difficult to assess their relative\ncontributions. Additional discussion or clarification of this limitation would improve the\nmechanistic understanding of the system.\nResponse:\nThank you for your comments. The immunomodulatory functions of BMSCs are well\nestablished and include suppression of T-cell activation, promotion of Treg differentiation,\ninhibition of proinflammatory cytokine release, and secretion of trophic factors that support\ntissue repair. These properties contribute to the therapeutic effects observed in both the PO\nand RA models, where BMSCs alone attenuated inflammation and improved tissue\nmorphology.\nCedirogant, a potent RORγt inverse agonist, acts primarily by downregulating Th17 activity\nand IL-17A–driven cytokine signaling. In both PO and RA models, Th17-derived cytokines\n(IL-17A, IL-21, and IL-22) are important mediators of immune-driven pathology. However,\nin the RA model, CdgLipo alone provided limited bone-protective benefit, as shown in Fig.\n6b–i, despite comparable suppression of IL-17A⁺CD4⁺ T cells (11.4% in the Cdg group vs.\n11.5% in the CD4-mBMSC group, Fig. 7i).\nWhen combined within the AcM-DDS system, the dual actions of BMSC-mediated\nimmunomodulation and Cedirogant-mediated Th17 inhibition produced a clear synergistic\neffect. AcM-DDS treatment exhibited superior anti-inflammatory and bone-protective\noutcomes compared with either BMSC or Cedirogant monotherapy, including CD4-mBMSC.\nThese findings suggest that Th17 suppression contributes to therapeutic efficacy of BMSCs to\npromote tissue protection and repair. The detailed mechanisms underlying this synergy\nwarrant further investigation.\n3. While AcM-DDS is reported to suppress Th17 cells primarily through IL-17A inhibition,\nTh17 cells also secrete other proinflammatory cytokines such as IL-21, IL-22, and GM-CSF.\nInclusion or discussion of these additional markers would help define the extent of Th17\nmodulation more comprehensively.\nResponse:\nWe thank the reviewer for this insightful comment. We completely agree that Th17 cells\nsecrete multiple proinflammatory cytokines beyond IL-17A, including IL-21, IL-22, and GM-\nCSF, which together contribute to Th17-driven immune responses. In the revised manuscript,\nwe have expanded our analysis to include these cytokines. Specifically, immunofluorescence\nstaining demonstrated that AcM-DDS markedly reduced IL-21 (NEW Fig. 5a-b) and IL-22\n(NEW Fig. 5c-d) expression in dorsal skin tissues, while Luminex multiplex assay confirmed\na significant suppression of GM-CSF (NEW Fig. 5g). These results further substantiate that\nAcM-DDS exerts a broad inhibitory effect on Th17-associated cytokine production rather\nthan acting solely through IL-17A suppression.\nWe have also incorporated this modification into the revised Results (Page 16, line 315-320).\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nThis study demonstrated the construction of a “live drug carrier”, proposed to be CD-4 tagged\nBM-MSCs, loaded with Cdg encapsulating liposomes for application in autoimmune diseases\nlike psoriasis and arthritis. However the current study has certain gaps which need to be\naddressed before it can be considered suitable for publication.\n1. As per the authors, MSCs act as live drug carriers and will release the drug in inflammatory\nmilieu without the need for internalization. However, the authors may clarify that if MSCs\nburst to release the drug in the inflammatory milieu, it will also be released in the circulation,\nwhat effect that may have? Authors may evaluate the same in the mice blood as well.\nResponse:\nThank you for raising this important point. We fully acknowledge the concern regarding the\npotential release of the drug into circulation if MSCs were to burst in the inflammatory\nmilieu.\nIn our study, we did consider evaluating the effects of drug release in circulation. However,\ndue to the low drug loading in the MSCs, we encountered significant technical challenges in\nquantifying the drug concentration in the blood and local tissues of mice. Despite our efforts,\nincluding attempts to use LC-MS for drug concentration measurements, the sensitivity and\ndetection limits of the current analytical techniques were not sufficient to reliably measure the\ndrug levels in circulation or tissues at these low concentrations.\nWe have discussed these technical limitations in the manuscript and acknowledge that more\nsensitive methods could help address this issue in future studies (Page 26, line 517-523).\n2. The specificity of the graphical abstract with respect to the article maybe improved.\nCurrently, the abstract does not represent the article, but seems like a generalized poster.\nSpecifically, there is no specific mention of the RORγT antagonist, and it is not evident from\nthe graphical abstract that the study was performed in psoriasis and arthritis models. The\nlayout and content need to be improved.\nResponse:\nThank you for the constructive feedback. We agree that the original graphical abstract lacked\nspecificity, so we have revised Figure 1 accordingly. Without changing the overall structure,\nwe now explicitly highlight the use of RORγt inverse agonists and clearly indicate the disease\ncontexts (psoriasis and arthritis) examined in this study. In addition, we added new panels d-e\nas application examples: CD4mAb-conjugated BMSCs loaded with RORγt inverse agonists\ndemonstrate therapeutic efficacy in the IMQ-induced psoriasis-like (PO) model (panel d) and\nthe collagen-induced arthritis (CIA) model (panel e). We also refined the layout to improve\nclarity and narrative flow. We believe these changes address the concerns regarding\nspecificity, disease models, and layout, and better align the graphical abstract with the\nmanuscript. We greatly appreciate your suggestion.\n3. In Fig. 2 (d), the authors could have captured different frames and presented the\nquantification of the results, apart from only the representative images to demonstrate the CD-\n4+ T cells and CD4-BM-MSCs\nResponse:\nWe sincerely thank the reviewer for this constructive suggestion. In response, we have\nperformed quantification of the binding shown in the original Fig. 2d (now Fig.2e), as\nrequested. Specifically, N₃-mBMSCs and CD4-mBMSCs were co-cultured with CD4⁺ T cells,\nand the ratio of CD4⁺ T cells bound per MSC was calculated across 10 randomly selected\nfields from three independent experiments (NEW Fig. 2f). We also repeated the experiments,\nand the results were consistent with those previously observed: the duration of co-culture did\nnot affect the binding. Additionally, we included different T cell-to-MSC ratios and found that\nhigher numbers of T cells led to increased binding to CD4-mBMSCs (NEW Supplementary\nFig. 2b-c). These quantitative analyses confirm and complement the representative images in\nFig. 2e, validating the targeted binding capability of CD4-mBMSCs toward CD4⁺ T cells.\nWe have also incorporated this modification into the revised Results (Page 7, line 148-158).\n4. The authors may quantitate the percentage of MSCs that reached the skin upon intra venous\nadministration and check the localization by quantifying the presence of CD-4 labelled or\nunlabelled cells at site by ex vivo methods\nResponse:\nWe sincerely thank the reviewer for this valuable suggestion. In response, we quantified the\naccumulation of MSCs in inflamed skin following intravenous administration. DiD-labeled\nmBMSCs and CD4-mBMSCs were injected into IMQ-induced PO mice, and dorsal skin was\nharvested 72 h post-injection for flow cytometric analysis. Consistent with the in vivo\nfluorescence imaging (Fig. 2g-h), a significantly higher proportion of DiD⁺ cells was detected\nin the CD4-mBMSC group compared with unmodified mBMSCs, indicating enhanced\nhoming and retention of CD4-modified MSCs at the inflammatory site (NEW Supplementary\nFig. 2d). This ex vivo data quantitatively confirm the superior targeting efficiency of CD4-\nmBMSCs.\nWe have also incorporated this modification into the revised Results (Page 7, line 159-161).\n5. In Fig. 2 (f), the authors suggest a significant difference in the skin and the spleen, however\nthe authors may re-check whether there is a significant difference in the liver group at 24h as\nwell.\nResponse:\nThank you for raising this important point. We carefully re-examined the raw data for the\nliver at 24 h and performed repeated statistical analyses. Both an unpaired two-tailed t test\nand a Tukey’s post hoc test (following two-way ANOVA) consistently indicated no\nstatistically significant difference for the liver at 24 h (p = 0.0589). These results are fully\ndocumented and can be verified in the original source excel data we have provided.\n6. As per the statement of the authors, Cedirogant was discontinued due to off-targeting and\nsafety issues. However, this does not seem in line with available literature and data about the\ndrug, since as per the reports available, cedirogant was well tolerated and no safety concerns\nwere reported (NCT05044234). Additionally, as per other data, cedirogant was discontinued\nby the manufacturer due to some new unfavourable toxicology findings in preclinial studied,\nTherefore, the authors may explain the plausibility of their hypothesis, strategizing to load\ncedirogant for mitigating off-targeting.\nResponse:\nWe sincerely thank the reviewer for this constructive comment. Cedirogant (ABBV-157) was\ndiscontinued due to preclinical toxicology findings related to GI track. In the 39-week dog\ntoxicity study, body weight loss and gastrointestinal toxicity were observed (Tyring et al.,\n2024). The chronic suppression of Th17 cells could disrupt the delicate balance between Th17\ncells and Tregs in the gut. Th17 cells are critical for maintaining intestinal homeostasis by\nproducing cytokines such as IL-17 and IL-22, which support epithelial barrier integrity,\nantimicrobial peptide production, and mucosal defense (Ohara et al., 2024). This disruption\nmay impair mucosal immunity, reduce epithelial regeneration, and compromise barrier\nfunction, leading to intestinal toxicity. In our short-term models (1-5 weeks), histological\nanalyses revealed no intestinal damage in either the CdgLipo or AcM-DDS groups (NEW\nsupplementary Fig 16). These findings suggest that, in our models, AcM-DDS primarily\nenhances local Cdg efficacy at inflammatory sites rather than reducing gastrointestinal side\neffects.\nWe have also incorporated this explanation into the revised Discussion (Page 25, line 501-\n508).\n7. The authors may elaborate further about the statement in Line 169- “HPLC analysis\ndetermined that the drug loading capacity of Cdg in liposomes was approximately 8.48%,\nwith an encapsulation efficiency of 92.8%” It is unclear whether 8,48% of Cdg was loaded\ninto liposomes and 92.8% lipososomes were able to take up the drug, or does it mean\nsomething else.\nResponse:\nWe sincerely thank you for this valuable comment. We have revised the sentence to clarify the\nmeaning of both parameters. Specifically, the drug loading capacity (8.48%) refers to the\ndrug-to-lipid weight ratio, while the encapsulation efficiency (92.8%) represents the\nproportion of total Cdg encapsulated within the liposomes. The revised text in the manuscript\nnow reads:\n“HPLC analysis revealed that the liposomes had a drug loading capacity of approximately\n8.48% (w/w, drug-to-lipid ratio) and an encapsulation efficiency of 92.8% (percentage of\ntotal Cdg encapsulated in liposomes).” (Page 9, Line 185-187)\n8. The authors may quantify how many CD-4 receptors are present per MSCS, and how many\ndrug loaded lipososmes are internalized per MSC, as this will be relevant to determine the\nclinical applicability.\nResponse:\nWe sincerely thank the reviewer for this insightful and instructive comment, which helped us\nrecognize the importance of quantitatively characterizing the surface modification and\ncellular uptake efficiency of our system. In the revised manuscript, we have now provided\ndetailed quantitative data addressing both aspects.\n“Quantitative fluorescence analysis indicated that when 25 µg of DBCO-CD4mAb-FITC\nwas conjugated with 1 × 10⁶ N₃-mBMSCs, approximately 2,500 antibody molecules were\nanchored per cell on average.” (Page 6, Line 144-146)\n“Furthermore, quantitative fluorescence analysis based on Cy5 intensity allowed\nestimation of nanoparticle uptake per cell, indicating that each CD4-mBMSC internalized\napproximately 1.15 × 10⁴ CdgLipo particles on average.” (Page 9, Line 201-204)\nThe corresponding experimental procedures for these quantifications have also been added to\nthe Methods section for clarity and reproducibility.\n9. The authors could have included an only drug group (without liposomes encapsulation) to\ndemonstrate the off-targeting effect suggested by them. Additionally, a positive control, e.g.\na commercial treatment option could have been included.\nResponse:\nWe sincerely thank the reviewer for this valuable suggestion. In the revised manuscript, we\nhave now added animal experiments in IMQ-induced PO mice to address this point (NEW\nSupplementary Fig. 7a-i). Specifically, we included a free Cedirogant (Cdg) treatment group\nto evaluate the effect of the drug without liposome encapsulation, as well as a positive control\ngroup using methotrexate (MTX), which is widely used as a positive control in the IMQ-\ninduced PO model (Xie et al., 2018). Our results indicate that both free Cdg and MTX\nproduced modest therapeutic improvements, whereas repeated dosing of CdgLipo showed\nslightly enhanced efficacy. Importantly, all these treatments were still less effective than\nAcM-DDS, demonstrating the superior targeting and therapeutic advantage of our antibody-\nguided, liposome-loaded MSC system. We greatly appreciate the reviewer’s suggestion,\nwhich helped us strengthen the experimental validation and provide a clearer comparison with\nconventional treatments.\nWe have also incorporated this modification into the revised Results (Page 14, line 280-288).\n10. It is unclear how the authors determined the dose of the treatment groups in animals. The\nauthors may test variable doses and present the data for the chosen dose.\nResponse:\nWe appreciate the reviewer’s comment regarding the dosing details in our animal\nexperiments. For the MSC-based treatment groups, we administered 1 × 106 cells per mouse,\nas this is the most commonly used dosage in preclinical studies of MSCs for autoimmune\ndiseases, providing a widely accepted reference for efficacy and safety (González et al.,\n2009).\nFor Cedirogant (Cdg), each mouse received 4.5 mg/kg, which corresponds to the maximum\namount of drug that can be internalized by 1 × 106 MSCs. Importantly, there are currently no\npublished preclinical animal studies for Cdg, so we referred to human clinical trial data.\nIn Phase 1 studies (Maier et al., 2024), oral doses of Cdg ranged from 20–750 mg as a single\ndose and 75–375 mg/day for multiple doses. Using the FDA’s Meeh-Rubner conversion\nfactor (Reagan-Shaw et al., 2008), this roughly corresponds to 15.4–77.1 mg/kg/day oral\ndosing in mice. Considering the oral bioavailability in humans (Maier et al., 2024), the\nequivalent intravenous dose in mice would be approximately 6 mg/kg. Our chosen dose of\n4.5 mg/kg per 1 × 106 MSCs is therefore a conservative estimate, lower than the clinical-\nequivalent dose.\nSince this study is primarily a proof-of-concept, we did not explore a full range of high,\nmedium, and low doses. Future studies may optimize dosing to evaluate dose-dependency and\nfurther refine therapeutic efficacy.\nWe have also incorporated this modification into the revised Results (Page 13, line 262-266).\n11. Time point based skin localization study in animal could have been included to determine\nthe highest localisation of drug at a given time point. This could have also given insights on\nwhether liposome is accelerating the time of delivery, as early relief might also be important\nclinically.\nResponse:\nThank you for your valuable suggestion regarding the time-point based skin localization\nstudy. In our current study, we performed in vivo imaging (IVIS) to monitor the distribution\nof BMSCs and observed that the fluorescence signal peaked in the skin between 72- and 96-\nhours post-injection, indicating the highest localization of BMSCs in the inflamed tissue (Fig.\n2g). However, we understand that evaluating the drug concentration at these time points is\ncrucial for further insights.\nWe attempted to quantify the drug levels in skin tissue using LC-MS, but the results were not\nsuccessful. The primary challenge was that the amount of drug loaded into the MSCs was\nrelatively low, and consequently, the drug concentration in the tissues was below the detection\nthreshold of the LC-MS method. To overcome this limitation, we would need to increase the\ndrug load in the MSCs or use a larger animal model (e.g., rats), where the drug quantity in\ntissues would be more detectable. We appreciate the reviewer’s suggestion and will consider\nthese strategies in future studies to enable more accurate quantification of drug delivery over\ntime.\nWe have also incorporated this explanation into the revised Discussion (Page 26, line 517-523).\n12. The data in Fig 6 (d-f) may be quantified and evaluated for statistical significance\nResponse:\nWe thank the reviewer for the insightful suggestion. In the revised manuscript, we have added\nquantitative analysis for the data shown in originally Fig 6d-f. Specifically, for micro-CT\nanalysis (now Fig. 6c), we quantified the subchondral bone parameters, including BV/TV,\nSMI, and Tb. N, and observed significant improvements in the mBMSCs-based treatment\ngroups (Fig. 6d-e, Supplementary Fig. 13b-c). Quantification of histological data (Fig. 6g, 6i,\nSupplementary Fig. 13d-e) was also included, with statistical significance now evaluated for\nall groups.\nWe have also incorporated this modification into the revised Results (Page 21, line 403-409).\n13. The authors may opine about the fate of modified MSCs if kept in culture, and the\nliposomal load if they passage or divide, and what could be the shelf life of this modified\nMSC construct. Additionally, the authors may evaluate whether liposome internalization is\naffecting surface labelling?\nResponse:\nThank you for your question. CD4 antibody-conjugated mBMSCs and the full AcM-DSS\nsystem retained normal osteogenic, chondrogenic, and adipogenic differentiation (Fig 3g-i).\nDue to chemical conjugation and intracellular small-molecule loading, the system is\nunsuitable for extended passaging. Surface-conjugated antibodies remain stable after\ncryopreservation, but small-molecule payloads may partially release after ~24 h; thus, loaded\ncompounds should be freshly prepared.\nWe have also incorporated this explanation into the revised Discussion (Page 27, line 572-\n576).\n14. Since the MSCs are heavily modified and being proposed as a drug carrier, the authors\nmay be very precise and exact about the reproducibility of the protocols, with respect to\nnumber of receptors and liposomes loaded.\nResponse:\nWe appreciate the reviewer’s valuable comment. In the revised manuscript, we have provided\ndetailed quantitative descriptions of all procedures involved in the construction of the\nAcM-DDS system in the Methods section, including receptor conjugation and liposome\nloading steps. Each protocol has been independently repeated multiple times to ensure high\nreproducibility and consistency of results.\nWe have also incorporated this modification into the revised Methods (Page 29, line 630-\n640).\n15. The authors mention in Line 407-409 that “However, such approaches, coupled with the\nchallenges of MSC production standardization, elevate treatment costs and increase clinical\ncomplexity”. However, they may elaborate on how such heavy modifications of MSCs will\nnot increase the cost of production and therapy.\nResponse:\nThank you for your valuable question. We acknowledge that the AcM-DDS platform involves\nadditional processing steps compared with conventional BMSC therapy. The conjugation of\nantibodies to MSCs is technically straightforward, and the main added cost arises from the\nantibody itself. Likewise, the incorporation of small-molecule payloads primarily contributes\nto the expense of the active compound rather than the cell-handling process. In current\nBMSC-based therapies, such as Ryoncil (remestemcel-L), the major cost lies in cell\nproduction—approximately USD 194,000 per intravenous infusion in the U.S. Notably, in our\nPO model, the number of BMSCs that accumulated at inflammatory sites was about six times\nhigher for AcM-DDS than for unmodified BMSCs. Therefore, equivalent or superior\ntherapeutic efficacy could be achieved with one-sixth the number of BMSCs, suggesting that\nthe overall treatment cost of AcM-DDS, even after accounting for the antibody and small-\nmolecule components, would remain substantially lower than that of conventional BMSC\ntherapy.\n16. The author may reference that CD-4 antibodies do not raise any safety concerns, as\nmentioned in Line 423-428.\nResponse:\nThank you for the suggestion. As CD4 antibody Ibalizumab has been approved by the FDA,\nindicating a favorable safety profile. We have added a reference in Line 133-134 of the\nrevised manuscript.\n17. The authors may elaborate more about the limitations of the study.\nResponse:\nWe thank the reviewer for the insightful suggestion. Limitations of this study include the use\nof murine MSCs and disease models that may not fully capture the complexity of human\nimmune responses. In future studies, antibodies with higher cellular specificity could be\nselected to direct payloads toward more defined immune subsets, such as Th17, Th1, or Th2\ncells, thereby enhancing targeting precision. In addition, all manufacturing steps were\nconducted at the laboratory scale, and the scalability of the current AcM-DDS preparation\nprocess to pilot or GMP-level production remains to be validated.\nWe have also incorporated this explanation into the revised discussion (Page 27, line 575-\n581).\n18. In line 474-375, the authors suggest that their system shows a broad translational potential\nfor immune disorders, however as also demonstrated in their own study, it seems that the\napplicability of this system primarily depends on the pathophysiology of the disease.\nResponse:\nThanks for your careful review. The AcM-DDS platform offers broad translational potential\nthrough two distinct avenues. First, antibody-conjugated BMSC therapy alone represents a\ntechnically feasible and clinically relevant approach for autoimmune diseases. Similar\nantibody-conjugated immune cell therapies—such as antibody-tagged NK cells and γδT\ncells—are already in clinical development (Acea Therapeutics, clinical pipeline). If our\nproduct progresses toward clinical translation, the use of a clinically approved CD4 antibody\n(Ibalizumab, FDA-approved) provides an additional safety and regulatory advantage.\nTherefore, from both a manufacturing and safety perspective, CD4mAb-BMSC–based\ntherapy has a realistic potential for clinical application. Of course, the pathological features of\ndiseased tissues can provide specific target receptors for selecting appropriate antibodies in\nour delivery system. Therefore, elucidating the pathological characteristics of the lesion—\nparticularly the disease-specific receptors—is essential for the effective application of our\nplatform.\nSecond, the full AcM-DDS system, which combines antibody-conjugated BMSCs with drug-\nloaded liposomes, requires freshly prepared and additional validation of payload safety. Since\nCedirogant has not yet completed all clinical evaluations, clinical translation of the AcM-DDS\nsystem would likely involve substituting Cedirogant with an FDA-approved small-molecule\npayload. These considerations together support the flexibility and adaptability of the AcM-\nDDS platform for future therapeutic development.\nWe have also incorporated this explanation into the revised discussion (Page 27, line 564-\n575).\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nThis manuscript presents a state-of-the-art drug delivery strategy that combines antibody-\nconjugated mesenchymal stem cells with drug-loaded liposomes to achieve targeted drug\nrelease at inflammatory sites. The therapeutic potential is evaluated in both acute (psoriasis)\nand chronic (rheumatoid arthritis) mouse models of autoimmune diseases.\nThe Antibody-Conjugated MSCs Drug Delivery System (AcM-DDS) is highly innovative and\nconjugates several therapeutic advantages specifically associated with the trophic and\nimmunomodulatory effects of BMSC (confirmed in the in vivo experiments by the\nadministration of BMSC alone) with the possibility to 1) conjugate antigen-specific\nantibodies (in this case for the CD4 antigen expressed on T cells) onto the BMSC surface to\nenhance their homing efficiency, 2) transform antibody-conjugated BMSCs (CD4-mBMSC)\ninto cargo cells (AcM-DDS ) for small-molecule drugs (in this study, Cdg, a RORγt inverse\nagonist) enabling targeted delivery to sites of inflammation.\nThe study is well written and results are robust, built on carefully conducted and\ntechnologically solid experiments supporting this novel strategy for drug delivery in the\ntreatment of autoimmune diseases. A few aspects of the study require clarification and\nrevision to strengthen the manuscript.\nGeneral comment:\nBecause AcM-DDS can target any membrane protein, enabling more flexible and precise drug\ndelivery, we wonder why authors used the broadly expressed CD4 antigen to target T cells,\ninvolved in AID pathogenesis. Although the delivery of Cdg, a RORγt inverse agonist, mainly\nrestricts the drug effect on Th17 cells, the use of CD4 to guide AcM-DDS toward the sites of\ninflammation in IMQ-induced PO mice and in the CIA mouse model of RA poses, in a larger\nperspective, some concerns about the risk of targeting CD4+ T cells, a T cell subsets playing\nmajor roles in physiological immune responses. While the lack of abnormalities, observed in\nmajor organs (heart, liver, lungs and kidneys), may reflect the safety of AcM-DDS in these\nmouse models, assessed in a pathogen free environment, a different outcome may be observed\nupon an infectious trigger and in humans. Authors should comment on this issue in the\ndiscussion.\nResponse:\nWe sincerely appreciate your recognition of our study and your thoughtful review. CD4 was\nchosen as the targeting antigen during the proof-of-concept stage of the AcM-DDS platform.\nThe selection criteria included (i) high expression of CD4 in inflamed tissues enriched with\nactivated T cells, (ii) presence of CD4 on Th17 cells, which are the intended recipients of\nCedirogant delivery, and (iii) the availability of an FDA-approved CD4 antibody\n(Ibalizumab), which provides a favorable safety precedent. Importantly, CD4 itself is not a\ntherapeutic target in this context but serves to direct BMSCs and their encapsulated small\nmolecules toward inflamed regions. The successful proof-of-concept using CD4 supports the\nfeasibility of AcM-DDS, and future iterations can employ disease-specific antigens (e.g., IL-\n23R or CCR6) for enhanced targeting precision.\nWe have also incorporated this explanation into the revised Results (Page 6, line 131-135).\nMajor points:\n1. Why did authors change the administration protocol for the PO and RA mouse models\nexperiments (i.v. in the tail vein for the IMQ study vs retro-orbital venous injection for the\nCIA mouse model)? Importantly, no data are provided to evaluate the biodistribution of MSCs\nfollowing retroorbital administration. Authors should assess whether this alternative route\nleads to differences in organ accumulation\nResponse:\nThank you for your thoughtful question. We chose retro-orbital venous injection for the CIA\nmouse model due to significant challenges with tail vein injection in this model. Specifically,\ncollagen and Freund’s adjuvant emulsions are injected subcutaneously near the tail base twice\nduring the model setup, which always leads to severe local inflammation, causing persistent\nlesions or even tail loss in some cases. This made tail vein injection unfeasible.\nGiven this issue, we opted for retro-orbital venous injection, which has turned out to be\ntechnically easier to perform. In our preliminary experiments, we used IVIS imaging to assess\nthe biodistribution of DiD-labeled mBMSCs and CD4-mBMSCs. The results showed that\nCD4-mBMSCs preferentially accumulated in the limbs and at the tail base, where the\ninflammation from the CIA model was most pronounced. We have included these data in\nSupplementary Fig. 12 to clarify any concerns about biodistribution. We hope this explanation\naddresses your concern and clarifies the rationale for our approach.\nWe have also incorporated this explanation into the revised Results (Page 19, line 374-377).\n2. In general, authors use citations quite unscrupulously particularly in the introduction\nsection. A few examples: In lines 30-32, the statement “Mesenchymal stem cells (MSCs) have\nshown promising potential in the treatment of autoimmune diseases (AIDs) such as psoriasis\n(PO)1, systemic lupus erythematosus (SLE)2, multiple sclerosis (MS)3, inflammatory bowel\ndisease (IBD)4, and rheumatoid arthritis (RA)5” is not directly supported by the references.\nRef 6 (Pittenger et al, 1999) does not cover the anti-inflammatory properties of MSC. Ref 8\nrefers to MSC and B cells (and not Tregs) and Ref 9 uses a mice model defective of Tregs and\ntherefore is not appropriate to support the effect of MSC on Tregs. More references are not\ntotally appropriate along the text. Overall, authors are invited to carefully go through the text\nto ensure that citations properly support what they are claiming.\nResponse:\nWe sincerely thank the reviewer for pointing out this important issue. We apologize for the\ninappropriate or imprecise use of some references in the initial submission. In the revised\nmanuscript, we have thoroughly re-examined and carefully cross-checked all citations\nthroughout the text to ensure that each reference accurately supports the corresponding\nstatement.\nMinor comments\n1. Authors use the term “Mesenchymal Stem Cells”, rather than “Mesenchymal Stromal\nCells” which is now preferred. As the manuscript does not address any issue related to their\nregenerative potential and they only demonstrated that modified BMSC can differentiate into\nthe osteogenic lineage, we would suggest using the term “Mesenchymal Stromal Cells” in the\ntitle and along the whole manuscript.\nResponse:\nWe sincerely thank the reviewer for this insightful and constructive suggestion. We fully agree\nwith the point raised regarding terminology. Indeed, “Mesenchymal Stromal Cells (MSCs)” is\nthe currently preferred nomenclature, particularly in contexts where regenerative potential is\nnot the primary focus. In light of this valuable feedback, we have carefully revised the\nmanuscript, including the title and all relevant sections, to consistently use “Mesenchymal\nStromal Cells” throughout. We greatly appreciate your guidance in helping us improve the\nclarity and accuracy of our manuscript.\n2. In the legend for Figure 1, the authors should include a brief description of the design\nframework and experimental workflow.\nResponse:\nWe sincerely appreciate this insightful suggestion. We have revised the Figure 1 legend to\ninclude a concise overview of the study design and experimental workflow. In addition, we\nadded panels d–e to depict the therapeutic application of CD4mAb-conjugated BMSCs loaded\nwith RORγt inverse agonists in the IMQ-induced PO and CIA models. We believe these\nupdates enhance the clarity and informativeness of Figure 1.\n3. In lines 141-143, the authors claim “CD4-mBMSCs exhibited significantly enhanced\nbinding to multiple CD4⁺ T cells, confirming their targeted binding capacity toward CD4⁺ T\ncells at inflammatory sites”. These data refer to confocal imaging, and quantitative analyses\nare needed to support the data.\nResponse:\nWe sincerely thank the reviewer for raising this important point, which was also noted by\nanother reviewer. We acknowledge that the original manuscript did not present explicit\nquantification of CD4⁺ T cell binding to CD4-mBMSCs, and we apologize for this oversight.\nIn response, we have now performed quantification of the binding shown in the original Fig.\n2d (now Fig. 2e). Specifically, N₃-mBMSCs and CD4-mBMSCs were co-cultured with CD4⁺\nT cells, and the ratio of CD4⁺ T cells bound per MSC was calculated across 10 randomly\nselected fields from three independent experiments (NEW Fig. 2f). Repeated experiments\nyielded consistent results, confirming that the duration of co-culture did not influence\nbinding. Furthermore, by examining different T cell-to-MSC ratios, we observed that higher T\ncell numbers led to increased binding to CD4-mBMSCs (NEW Supplementary Fig. 2b-c).\nWe have also incorporated this explanation into the revised Results (Page 7, line 148-159).\n4. Why distribution of CD4-mBMSC did drop at 72 hours following i.v. administration\n(compared to high levels at 24 and 96h)?\nResponse:\nThank you for this question, but in our experiments, we did not observe a decrease in\nCD4‑mBMSC distribution at 72 h (Fig. 2g). We apologize if our phrasing led to any\nmisunderstanding.\n5. Although the manuscript describes the preparation of the AcM-DDS system, it does not\nprovide quantitative data on the percentage of cells successfully conjugated or loaded.\nResponse:\nWe sincerely thank you for raising this insightful and important point regarding the\nquantitative characterization of our system. A similar concern was also raised by another\nreviewer, and we have taken it very seriously. In the revised manuscript, we now provide\ndetailed quantification of antibody conjugation: when 25 µg of DBCO-CD4mAb-FITC was\nincubated with 1 × 10⁶ N₃-mBMSCs, approximately 2,500 antibody molecules were anchored\nper cell on average (Page 6, Line 144-146).\nIn addition, while addressing this request, we also performed quantitative analysis of\nnanoparticle uptake. Based on Cy5 fluorescence intensity, each CD4-mBMSC internalized\napproximately 1.15 × 10⁴ CdgLipo particles per cell on average (Page 9, Line 201-204). The\nexperimental procedures for these quantifications have been detailed in the Methods section\nfor clarity and reproducibility.\n6. In Figure 5d/e and Supplementary Figure 7, the authors present heat maps and cytokine\nquantifications to support the anti-inflammatory effect of AcM-DDS. In lines 277–279, they\nclaim that “Importantly, AcM-DDS treatment led to a substantial downregulation of these\ncytokines, suggesting a potent anti-inflammatory effect mediated by suppression of Th17-\ndriven immune responses”. However, based on the quantification of IL-17A, the reduction\nappears comparable across the different treatment groups and does not clearly indicate a\ndistinct effect from AcM-DDS. Authors should comment in the text.\nResponse:\nWe sincerely thank you for this important comment and apologize for the previous wording,\nwhich was not sufficiently precise. We have now revised the manuscript to clarify that,\naccording to the Luminex results, the reduction of IL-17A in the mBMSCs, CD4-mBMSCs,\nCdgLipo, and AcM-DDS groups was generally comparable. Nevertheless,\nimmunofluorescence analysis of local dorsal skin tissue indicated that AcM-DDS produced a\nmore pronounced suppression of Th17-related cytokines such as IL17A, IL-21 and IL-22,\nsuggesting that its superior anti-inflammatory effect is primarily exerted at the local tissue\nlevel.\n“Treatment with AcM-DDS, as well as mBMSCs, CD4-mBMSCs, and CdgLipo, led to an\noverall downward trend in these cytokines, indicating effective attenuation of\ninflammation. Notably, although reductions in IL-17A and GM-CSF were comparable\namong the cell-based and liposomal treatments (Fig. 5f-g), immunofluorescence analysis\nrevealed a more pronounced local suppression of IL17A, IL-21 and IL-22 by AcM-DDS,\nsuggesting that its superior modulation may primarily occur at the tissue\nmicroenvironment level rather than in total tissue cytokine content.” (Page 6, Line 325-\n331)\n7. Why was IL-17A and RORγt expression only partially reduced in the CD4-mBMSCs mice\ngroup, considering that in the Luminex Multiplex Assay the effect on inflammatory cytokines\nwas quite significant (Fig 5)?\nResponse:\nWe thank the reviewer for this insightful comment. The apparent discrepancy reflects\ndifferences in assay scope and tissue kinetics. The Luminex assay analyzes whole‑skin lysates\nand integrates signals from multiple cellular sources, hence the pronounced global decrease in\ninflammatory cytokines with CD4‑mBMSCs. By contrast, immunofluorescence captures a\nlocalized, section‑level snapshot. At this compartment, IL‑17A/RORγt can be partially\nsustained by non‑CD4 sources (e.g., dermal γδ T cells, ILC3s) and residual upstream\nIL‑23/IL‑1β–chemokine signaling, with additional temporal lag between upstream\nsuppression and terminal epidermal readouts. Consequently, IL‑17A/RORγt appear only\npartially reduced by IF at the sampling time point, while whole‑tissue Luminex reveals a\nrobust overall anti‑inflammatory effect. These readouts are complementary rather than\ncontradictory.\nWe have also incorporated this explanation into the revised Results (Page 17, line 334-338).\n8. In lines 131 and 140, the fluorophores FAM and DiI are mentioned. These should be\nidentified as fluorescent dyes when first introduced.\nResponse:\nThank you for the careful review and helpful suggestion. We agree and have revised the\nmanuscript to explicitly identify FAM and DiI as fluorescent dyes at their first mention. We\nhave also applied this convention consistently to any other fluorescent dyes upon first\nintroduction throughout the text.\n9. In lines 142-143, the authors state that “confirming their targeted binding capacity toward\nCD4⁺ T cells at inflammatory sites”. However, the data comes from in vitro co-culture\nexperiments. Therefore, the statement “at inflammatory sites” is misleading.\nResponse:\nWe sincerely thank you for this careful and insightful comment. We fully agree that the phrase\n“at inflammatory sites” could be misleading, as the current data are derived from in vitro co-\nculture experiments rather than in vivo inflammatory models. Accordingly, we have revised\nthe sentence to read:\n“Confocal imaging revealed that, compared to N₃-mBMSCs, CD4-mBMSCs exhibited\nsignificantly enhanced binding to multiple CD4⁺ T cells, confirming their targeted binding\ncapacity toward CD4⁺ T cells in vitro.” (Page 7, line 151-153)\n10. In the legend of Figure 2e, the authors should add the imaging technique used to acquire\nthe data.\nResponse:\nThank you for your careful review and suggestion. We have revised the original legend of Fig\n2e (now Fig 2g) to explicitly state the imaging technique.\n“g, In vivo fluorescence imaging acquired using an IVIS imaging system of DiD-stained\nmBMSCs or CD4-mBMSCs in IMQ-induced PO mice at 24 h, 72 h, and 96 h after\nintravenous injection.”\n11. The sections describing the generation of CD4-antibody-conjugated MSCs lack clarity,\nand the methodology is difficult to follow. The authors should improve the readability of the\ntext.\nResponse:\nWe thank the reviewer for this valuable comment. We apologize for the lack of clarity in the\noriginal description. In the revised manuscript, we have substantially revised and expanded\nthis section to provide a clearer, step-by-step description of the process, including detailed\ninformation on reagent concentrations, reaction conditions, incubation times, and purification\nsteps.\nWe have also incorporated this modification into the revised Methods (Page 29, line 630-\n640).\n12. For the supplementary movies, the authors should add a figure legend.\nResponse:\nWe appreciate the reviewer’s suggestion. Legends have been added to the supplementary\nmovies as follows:\n“Supplementary Movie a: Real‑time confocal imaging of mBMSCs taking up\nCy5‑CdgLipo in situ.\nSupplementary Movie b: Real‑time confocal imaging of mBMSCs taking up free Cy5 in\nsitu.”\nReviewer #6 (Remarks to the Author):\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\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReference\nGonzález, M. A., Gonzalez-Rey, E., Rico, L., Büscher, D., & Delgado, M. (2009). Treatment\nof Experimental Arthritis by Inducing Immune Tolerance With Human Adipose-\nDerived Mesenchymal Stem Cells. Arthritis and Rheumatism, 60(4), 1006-1019.\nhttps://doi.org/10.1002/art.24405\nHayer, S., Vervoordeldonk, M. J., Denis, M. C., Armaka, M., Hoffmann, M., Bäcklund, J.,\nNandakumar, K. S., Niederreiter, B., Geka, C., Fischer, A., Woodworth, N., Blüml, S.,\nKollias, G., Holmdahl, R., Apparailly, F., & Koenders, M. I. (2021). 'SMASH'\nrecommendations for standardised microscopic arthritis scoring of histological\nsections from inflammatory arthritis animal models. Ann Rheum Dis, 80(6), 714-726.\nhttps://doi.org/10.1136/annrheumdis-2020-219247\nMaier, C. S., Eckert, D., Laroux, F. S., Hew, K. M., Suleiman, A. A., Liu, W., & Mohamed,\nM. F. (2024). Cedirogant Population Pharmacokinetics and Pharmacodynamic\nAnalyses of Interleukin-17A Inhibition in Two Phase 1 Studies in Healthy\nParticipants and Participants with Moderate to Severe Psoriasis. Clin Pharmacol\nDrug Dev, 13(5), 474-484. https://doi.org/10.1002/cpdd.1377\nOhara, D., Takeuchi, Y., & Hirota, K. (2024). Type 17 immunity: novel insights into intestinal\nhomeostasis and autoimmune pathogenesis driven by gut-primed T cells Cellular &\nMolecular Immunology, 21(11), 1183-1200. https://doi.org/10.1038/s41423-024-\n01218-x\nReagan-Shaw, S., Nihal, M., & Ahmad, N. (2008). Dose translation from animal to human\nstudies revisited. Faseb Journal, 22(3), 659-661. https://doi.org/10.1096/fj.07-\n9574LSF\nTyring, S., Moore, A., Morita, A., Hong, H. C. H., Song, I. H., Eccleston, J., Levy, G.,\nMohamed, M. E. F., Qian, Y. L., Wu, T. S., Pan, A. Q., Hew, K., & Papp, K. A.\n(2024). Cedirogant in adults with psoriasis: a phase II, randomized, placebo-\ncontrolled clinical trial. Clinical and Experimental Dermatology, 49(11), 1347-1355.\nhttps://doi.org/10.1093/ced/llae152\nXie, X.-j., Di, T.-t., Wang, Y., Wang, M.-x., Meng, Y.-j., Lin, Y., Xu, X.-l., Li, P., & Zhao, J.-x.\n(2018). Indirubin ameliorates imiquimod-induced psoriasis-like skin lesions in mice\nby inhibiting inflammatory responses mediated by IL-17A-producing γδ T cells.\nMolecular Immunology, 101, 386-395.\nhttps://doi.org/https://doi.org/10.1016/j.molimm.2018.07.011\nResponse Letter to Reviewers\nWe sincerely appreciate the reviewers’ time and constructive evaluations. Below, we\nprovide a point-to-point response to all comments.\nReviewer #1\nComment:\nThe authors have responded sincerely and thoroughly to all review comments. The\nadditional experiments and analyses substantially strengthen the robustness, clarity,\nand translational relevance of the study. I recommend that this manuscript is\nacceptable for publication in Nature Communications.\nResponse:\nWe thank the reviewer for the positive evaluation and supportive recommendation.\nWe are grateful that the additional experiments and clarifications strengthened the\nmanuscript.\nReviewer #3\nComment:\nThank you for your detailed responses to the comments. The improvements in\nmethodology, data interpretation, and discussion have strengthened the manuscript.\nThe manuscript has been recommended for publication.\nResponse:\nWe appreciate the reviewer’s supportive recommendation. We are glad that the\nrevisions improved the manuscript and thank the reviewer for recommending\nacceptance.\nReviewer #4\nComment:\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse:\nWe thank the reviewer and the co-reviewer for contributing to the evaluation of our\nwork.\nReviewer #5\nComment:\nI thank the authors for their careful attention to my comments. The manuscript has\nimproved in clarity and organization, and the additional data strengthen the study. My\nprevious concerns have been satisfactorily addressed, and I recommend the\nmanuscript for acceptance.\nResponse:\nWe sincerely thank the reviewer for the constructive comments during the previous\nround and for recognizing the improvements made. We are glad that our revisions and\nadditional data addressed all concerns.\nReviewer #6\nComment:\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse:\nWe appreciate the reviewer and the co-reviewer for their contribution and supportive\nengagement in this review process."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have responded sincerely and thoroughly to all review comments. The additional experiments and analyses significantly substantially strengthen the robustness, clarity, and translational relevance of the study. I recommend that this manuscript is acceptable for publication in Nature Communications.", "reviewer_3": "Thank you for your detailed responses to the comments. The improvements in methodology, data interpretation, and discussion have strengthened the manuscript. The manuscript has been recommended for publication.", "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 thank the authors for their careful attention to my comments. The manuscript has improved in clarity and organization, and the additional data strengthen the study. My previous concerns have been satisfactorily addressed, and I recommend the manuscript for acceptance.", "reviewer_6": "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": ""}]

Decision letter