Antigen-specific immunotherapy with a CD4^+ T cell neoepitope restrains CD8^+ T cell differentiation in murine pancreatic islet grafts

Domain: biology
Published: 2026-03-24
Editorial outcome: accept

Abstract
Pancreatic islet transplantation offers a potential cure for autoimmune type 1 diabetes (T1D) but requires immunosuppression to prevent recurrent, T cell-mediated autoimmunity. Antigen-specific immunotherapy is a targeted alternative that induces peripheral tolerance in autoreactive T cells without broad immunosuppression. Tolerance induction with antigen-specific therapies may induce dominant tolerance, extending to T cell clones other than those targeted by the therapy. However, the mechanism of this suppression remains unclear. Using pancreatic islet transplantation in NOD mice, we show that induction of tolerance to a CD4+ T cell hybrid insulin peptide neoepitope reprograms the fate of antigen-specific CD8+ T cells within the grafts. Induction of tolerance to a single hybrid insulin peptide transiently protected islet grafts from autoimmune destruction, and graft survival could be extended with continued therapeutic dosing. Treatment limited a cytolytic/interferon-stimulated differentiation program in antigen-specific CD8+ T cells and reduced TCR avidity within grafts. This was mediated in part by IL-10-producing regulatory CD4+ T cells that suppressed dendritic cell activation. IL-10 blockade reversed these effects, restoring CD8+ T cell differentiation, as well as the licensing of dendritic cells. These findings reveal an IL-10-dependent mechanism by which CD4+ T cell-targeted immunotherapy restrains pathogenic CD8+ T cell fates through the suppression of dendritic cells in islet grafts.

Introduction
Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β-cells driven primarily by autoreactive T cells1,2. Recent advances in stem cell-derived islet transplantation have offered a potential cure for diabetes3-6. However, most recipients remain dependent on life-long immunosuppressive regimens, which over time increase the risk of nephrotoxicity7, infection8, and malignancy9. By contrast, antigen-specific immunotherapy (ASI) aims to selectively silence pathogenic T cell reactivity in target tissues while sparing the majority of the T cell repertoire10,11. This offers a precision approach to restoring peripheral T cell tolerance in autoimmune patients in the absence of indiscriminate immunosuppression. In T1D and other T cell-mediated autoimmune diseases, precision delivery of autoantigen peptide for antigen-specific immunotherapy via tolerogenic vehicles such as nanoparticles has emerged as a versatile method to induce antigen-specific peripheral tolerance without eliciting broad immunosuppression12-15.

A natural mechanism of peripheral tolerance is mediated by antigen-presenting cell (APC) uptake and presentation of self-peptides without co-stimulatory signals such as CD80 and CD4016,17. Early efforts to therapeutically harness this pathway demonstrated that conjugating autoantigenic peptides to apoptotic cells could suppress autoreactive T cell responses and prevent autoimmunity18,19. To improve translational potential through greater stability and uniformity, biodegradable poly(lactic-co-glycolic acid) (PLG) nanoparticles (NPs) were engineered to mimic apoptotic cells as tolerogenic, antigen-carrying vehicles20. Epitope-conjugated PLG nanoparticles have since been shown to induce anergy in effector T cell responses and to promote regulatory T cell expansion in multiple autoimmune disease models21-23.

Identifying the specificities of islet-infiltrating T cells has been pivotal for understanding disease progression and guiding the selection of ASI target peptides. Genetic susceptibility in both human T1D and the non-obese diabetic (NOD) mouse is predominantly linked to certain MHC class II alleles24, highlighting the central role of autoreactive CD4+ T cells in disease initiation and progression25. Early work in the NOD mouse identified a panel of CD4+ T cell clones, including the prototype T cell clone BDC-2.5, that could infiltrate islets and markedly accelerate disease26. These clones enabled the identification of cognate autoantigens for islet-specific CD4+ T cells, culminating in the discovery of a novel class of post-translationally modified neoantigens, termed hybrid insulin peptides (HIPs)27. HIPs are generated by covalent fusion of proinsulin C-peptide fragments to other β-cell granule-derived peptides27,28. Among the HIPs identified in NOD islets, the 2.5HIP epitope (Ins2 /ChgA ) elicits a potent pathogenic CD4+ T cell response. 2.5HIP-reactive T cells are enriched in the islets of prediabetic NOD mice, increase in frequency during disease progression29, and have been targeted in other preclinical antigen-specific immunotherapies15,30,31. HIPs have also been detected in human islets, and HIP-reactive CD4+ T cells, including those specific for the human analog of the 2.5HIP, have been identified in patients with T1D27,32-40.

CD4+ T cells typically act as helpers, supporting the activation, expansion, and differentiation of cytolytic CD8+ T cells through both direct and indirect mechanisms41-44 including the licensing of dendritic cells (DCs). However, the specific pathways through which HIP-specific CD4+ T cells influence the fate of autoreactive CD8+ T cells, and how this instructional role is altered following tolerance induction, is not entirely understood. Recent studies have shown that antigen-specific autoreactive CD8+ T cells undergo a hierarchical differentiation process, beginning as self-renewing, stem-like precursors in pancreatic lymph nodes and progressing into terminal effectors that accumulate within the islets45,46. The two populations can be readily distinguished by the expression of the self-renewal transcription factor, TCF1. Other work has demonstrated that a proportion of bulk CD8+ T cells in islets remain TCF1hi and can differentiate into TCF1lo effectors in situ47,48. Some studies have shown that many islet-specific CD8+ T cells are restrained by an exhaustion-like, terminal differentiation program akin to that seen in cancer and chronic viral infection49,50. This exhaustion-like program can be induced in islet-specific CD8+ T cells as demonstrated by extra-islet expression of the CD8+ T cell epitope, IGRP51. However, an unanswered question has been how antigen-specific immunotherapy with a CD4+ T cell epitope influences the differentiation fate in the CD4+ T cells targeted by the therapy and in other islet-specific T cells, including cytotoxic CD8+ effectors. It has been proposed that tolerance to CD4+ epitopes may constrain CD8+ T cell effector function, and therefore tissue differentiation, via a form of dominant tolerance mediated by the suppression of antigen-presenting cells52-55. The APCs may lack proper CD4+ licensing, generating a helpless CD8+ fate and/or could be actively suppressed by regulatory T cells. In our previous transplant tolerance study56 and in another tolerance induction study during spontaneous disease progression57, initial evidence of suppression in CD8+ T cells following CD4-targeted antigen-specific immunotherapy has been observed, but the precise nature of the defect in antigen-specific CD8+ T cells and the mechanism by which this peripheral tolerance occurs remains undefined.

Here, we use antigen-specific immunotherapy with a single CD4+ T cell hybrid insulin peptide neoepitope, the 2.5HIP, to mechanistically determine how peripheral tolerance shapes the tissue differentiation fate and function of islet-specific CD8+ T cells in a polyclonal, autoimmune syngeneic islet graft setting.

Results
Antigen-specific CD4+ and CD8+ T cells undergo stem-like to effector differentiation in islet grafts.

Antigen-specific CD8+ T cells exhibiting stem-like features occupy the pancreatic lymph node and terminally differentiate into effector cells upon infiltration into islets where antigen is abundant45,46. This anatomical compartmentalization of cell states can be delineated by the expression of the self-renewal transcription factor, T cell factor 1 or TCF1 (gene Tcf7). Stem-like T cells express high levels of TCF1 (TCF1hi) and terminally differentiated T cells lose TCF1 (TCF1lo), to adopt an effector-like program in tissue. We hypothesized that antigen-specific CD8+ and CD4+ T cells infiltrating islet grafts undergo the same stem-to-effector transition. To test this hypothesis, we used an autoimmune transplant model in which spontaneously diabetic NOD mice receive syngeneic islet transplants under the kidney capsule from NOD.scid donors (Fig. 1a). Normoglycemia is transiently restored until the graft is destroyed by recurrent T cell mediated autoimmunity58-60 and animals revert to hyperglycemia (Fig. 1b). Peptide-loaded MHC tetramers were used to examine antigen-specific T cells recognizing immunodominant CD4+ (2.5HIP presented by I-Ag7)29 and CD8+ (IGRP presented by H2-Kd)61 T cell epitopes in the islet graft and the kidney-draining lymph node (kLN).

Around the time of graft failure at day 8-9 post-transplant, tetramer-positive T cells from islet grafts and kLN were assessed for antigen encounter and effector differentiation by flow cytometry (gating strategy in Fig. S1a). Both 2.5HIP-tetramer+ CD4+ (2.5HIP ) and IGRP-tetramer+ CD8+ (IGRP ) T cells were more abundant in the graft than in the draining lymph node, in frequency and absolute number. (Fig. 1c). Similarly, 2.5HIP and IGRP T cells (>70%) were TCF1hi in the kLN whereas tetramer-positive T cells in the graft were predominantly TCF1lo, terminally differentiated effector T cells (Fig. 1d). These data indicate that both antigen-specific CD4+ and CD8+ T cells undergo a similar stem-like-to-effector transition from draining lymph node to graft. Levels of inhibitory receptors PD1 and TIM3 (as proxies for TCR activation and effector differentiation) and the proliferation marker Ki67 were higher in graft-infiltrating antigen-specific CD4+ and CD8+ T cells compared to the same specificities in the kLN, more evidence indicative of antigen encounter and subsequent differentiation in islet graft tissue (Fig.1e).

To further understand the kinetics of effector differentiation leading up to graft destruction, the proportion and number of TCF1hi/lo 2.5HIP and IGRP T cells were examined in islet grafts on day 3, 6, and 9 post-transplant (Fig.1f-g). There was no significant change in the proportion of 2.5HIP T cells of total CD4+ T cells, or as a fraction of TCF1hi T cells, over the course of graft destruction. Yet, there is a significant accumulation in the absolute number of both TCF1hi and TCF1lo 2.5HIP T cells as the graft progresses toward destruction. By contrast, IGRP T cells increase as a proportion of total CD8+ T cells over time. Few IGRP T cells can be detected at day 3 post-transplant, most of them being TCF1lo effectors. By day 6, there is a reasonable percentage of (~15%) of TCF1hi IGRP T cells. While the absolute number of TCF1hi IGRP is established by day 6 and does not increase by day 9, there is a steady and significant accumulation of IGRP TCF1lo effector T cells between all timepoints. Together, these data demonstrate that both 2.5HIP and IGRP T cells are predominately TCF1hi in the graft draining lymph node and differentiate into TCF1lo effectors in islet grafts, aligning with previous observations of antigen-specific CD8+ T cells in pancreatic islets and the draining lymph node45,46.

Induction of tolerance to a hybrid insulin peptide restrains functional effector differentiation of graft-infiltrating, antigen-specific T cells.

Graft infiltration of antigen-specific T cells is a critical step in the destruction of transplanted islet grafts62-64. We hypothesized that tolerance induction to a dominant CD4 neoepitope, the 2.5HIP (Ins2 /ChgA ), would diminish the magnitude and quality of effector fates in graft-infiltrating, antigen-specific CD4+ and CD8+ T cells. To test this hypothesis, tolerogenic PLG nanoparticles (NPs), covalently conjugated to either the 2.5HIP or an irrelevant control peptide from hen egg lysozyme (HEL ) (Fig. 2a), were administered to spontaneously diabetic NOD mice before and after syngeneic islet transplantation (-7d, +1d, +7d relative to transplant) (Fig. 2b). A separate group of 2.5HIP NP tolerized mice were maintained on 2.5HIP NP treatment administered every 10 days for a total of 9 injections to determine if continued therapeutic treatment could extend graft survival.

As previously demonstrated56, three doses of 2.5HIP NPs were able to transiently extend graft survival (~45 days) over that of HEL NP control treated mice (~10 days) (Fig. 2c). Continued treatment with 2.5HIP NPs every 10 days until day 67 (9 total doses), increased graft survival even further to ~100 days and nearing the cured time-point in mice. This result indicates that graft survival can be extended commensurate with the duration of continued antigen-specific therapy.

To understand how tolerance was altering effector differentiation, non-survival mice were euthanized 8-9 days post-transplant, and graft-infiltrating 2.5HIP (target of nanoparticle therapy) and IGRP (immunodominant CD8+ T cell epitope) T cells were analyzed from islet grafts and kidney-draining lymph nodes (kLN). Treatment with 2.5HIP-NPs significantly reduced the frequency and absolute number of 2.5HIP and IGRP T cells in islet grafts (Fig. 2d). In the kLN, however, 2.5HIP-NP treatment slightly increased the proportion of 2.5HIP T cells with no significant differences observed in IGRP T cells (Fig. S2a,b). To assess the impact of antigen-specific tolerance on effector differentiation, we examined the stem-like (TCF1hi) and effector-like (TCF1ˡᵒ) subsets among 2.5HIP and IGRP T cells in both the kLN (Fig. S2.c) and islet grafts (Fig. 2e). In islet grafts from 2.5HIP-NP tolerized animals, a greater proportion of antigen-specific T cells retained TCF1 expression compared to HEL-NP controls (Fig. 2e). 2.5HIP-NP treatment markedly reduced the number of TCF1⁻ effector cells in both 2.5HIP and IGRP T cells, despite similar absolute numbers of TCF1⁺ cells (Fig. 2f). In comparison, there were no changes in the kLN in the portion of TCF1hi cells for either specificity in the kLN (Fig. S2c), or in the fraction of TCF1hiCD62L+ T cells (Fig. S2d), a subset present in LNs thought to have the most stem-like properties46. These data suggest the effects of tolerance induction by 2.5HIP-NPs are manifested as a lack in the absolute numbers of TCF1lo graft-infiltrating, antigen-specific T cells with minimal impact on the same population in the draining lymph node at this equivalent timepoint.

To assess antigen encounter and exhaustion-like differentiation fates in islet grafts, expression levels of inhibitory receptors PD1 and TIM3 and Ki67 were measured within TCF1hi and TCF1ˡᵒ subsets. The inhibitory receptor, PD1 was highly expressed in nearly all graft-infiltrating antigen-specific T cells and was significantly decreased in both TCF1hi/lo 2.5HIP populations and TCF1hi IGRP T cells following tolerance induction. By contrast, TIM3 surface expression was reduced exclusively on 2.5HIP T cells in grafts upon 2.5HIP-NP tolerance induction, with little change in IGRP T cells (Fig. 2g,h). These data may indicate that 2.5HIP-NP-treatment limits terminal effector differentiation and functional antigen-encounter in antigen-specific CD4+ and CD8+ T cells, as opposed to inducing an exhaustion-like program characterized by higher IR expression. Ki67 expression did not significantly differ between treatments for either specificity (Fig. S2e), suggesting local proliferation in the graft was unaffected at this timepoint. To examine how tolerance induction impacted the functional capacity of T cells, suspensions from grafts were stimulated ex vivo with PMA and ionomycin and stained for inflammatory cytokine accumulation. In our previous study56, we observed impaired cytokine production upon graft-failure but did not resolve whether stem-like, effector, or both T cell populations were functionally disrupted, nor did we examine responses at equivalent timepoints. Antigen-specific T cells were gated on TCF1 by TIM3 to examine how functional capacity changes through progression to terminal differentiation. Tolerance induction reduced IFNγ⁺TNF⁺ double-producing cells among both 2.5HIP (Fig. 2i,j) and IGRP T cells (Fig. 2k,l). The significance and magnitude of these differences were most exaggerated in terminally differentiated (TCF1loTIM3hi) T cells compared to their stem-like counterparts. This indicates that, as T cells differentiate within a tolerogenic environment, they are progressively diverted toward an altered, hypofunctional terminal effector state with reduced inhibitory receptor expression, distinct from an exhaustion-like phenotype and exhibiting limited differentiation. Together, these findings demonstrate that 2.5HIP-NP-induced tolerance reduces the number and functional differentiation of TCF1lo 2.5HIP and IGRP T cells in islet grafts.

IGRP-specific CD8+ T cells display a reduced interferon-stimulated gene (ISG) signature and cytolytic effector fate in grafts following 2.5HIP-NP tolerance induction.

To further interrogate how 2.5HIP tolerance reshapes graft-infiltrating antigen-specific CD8+ T cell effector fates, IGRP T cells were sorted (gating shown in Fig. S1b) from islet grafts of 2.5HIP-NP- and HEL-NP-treated NOD mice eight days post-transplant and subjected to single-cell RNA sequencing (Fig. 3a). Dimensionality reduction by UMAP on all sequenced cells, excluding TCR genes, revealed eight transcriptionally discrete clusters (Fig. 3b, Fig. S3a) annotated within the framework of effector differentiation, dominant gene signatures, and inferred function using differences in cluster-defining genes (Fig. 3c, Fig. S3b). Briefly, we found two stem-like clusters characterized by high expression of self-renewal genes (Tcf7, Ccr7, Slamf6), the larger Tcf7_hi cluster and a smaller subset, Tcf7_ISG, which additionally exhibited an interferon-stimulated gene (ISG) signature (Ifit3, Isg15, Ly6a). The six Tcf7 low clusters included four effector clusters Eff_ISG, Eff_Ifng, Eff_early, Eff_late characterized by high inhibitory receptor (Lag3, Pdcd1) and chemokine receptor expression (Ccr5, Cxcr6), a proliferating cluster Prolif (Mki67, Pclaf, Cdk1), and recently activated cluster Activated (Nfatc1, Nfatc3, Tox). While Tox transcripts were detected at high levels only in the Activated cluster, Tox protein was expressed at high levels in all graft-infiltrating IGRP CD8⁺ T cells and at higher levels than in IGRP-tetramer-negative CD8⁺ T cells (Fig. S3k). Further description of the differences between the four effector clusters are elaborated in Figure legend 3.

Notably, the Eff_ISG cluster showed elevated ISG expression, including high expression of the surface molecule Ly6a, and the highest levels of Gzmb, a key cytolytic effector molecule (Fig. 3d).The Eff_ISG cluster scored significantly above all other clusters for cytolytic gene modules (Fig. S3c,d) and ISG modules (except for the small Tcf7_ISG cluster), which were positively correlated together (Fig. S3e,f). Collectively, these data indicate that cytolytic and ISG signatures may represent a linked cell fate during the functional differentiation of graft-infiltrating IGRP T cells.

Following cluster annotation, we determined how treatment impacted IGRP T cells in grafts. Mirroring flow cytometry results (Fig. 2e,f), 2.5HIP-NP treatment led to a significant proportional increase in the Tcf7_hi stem-like cluster (Fig. S3g,h). There was also a concomitant reduction in the relatively small Tcf7_ISG and Activated clusters (Fig. S3g,h). To broadly assess treatment-induced transcriptional changes, pseudo-bulk differential expression revealed that approximately 30% of genes downregulated upon 2.5HIP-NP treatment were ISGs (Ly6a, Ifit3, Ifitm2, Ifitm1), alongside significant decreases in the main cytolytic effector molecules Gzma and Gzmb (Fig. 3e). This indicates the arrest of the aforementioned highly functional ISG/cytolytic IGRP CD8 fate by 2.5HIP-NP treatment. Gene set enrichment analysis (GSEA) confirmed marked depletion of ISG activation in IGRP T cells with significant reduction in an Interferon Induced Antiviral Module and significant reduction in an Antigen Response pathway (Fig. 3f, Fig. S3i). Single-cell module scoring demonstrated bulk and cluster-specific reductions in Ly6a and Gzmb expression following 2.5HIP tolerance induction (Fig. 3g, Fig. S3j).

To validate the transcriptional reduction in the ISG signature in IGRP T cells, Ly6A surface expression, readily detected by flow cytometry compared to other ISGs, was measured on IGRP T cells from grafts of control (HEL-NP) and tolerized (2.5HIP-NP) mice (Fig. 3i). Relative levels of Ly6A progressively increased on IGRP T cells from spleen to pancreatic lymph node and peaked in the graft, indicating Ly6A may serve as a marker of effector differentiation and survival in graft tissue (Fig. 3 h). Reflecting the scRNAseq data, Ly6A surface levels were significantly lower in both TCF1lo and TCF1hi IGRP T cells following 2.5HIP tolerance induction (Fig. 3i). To further dissect how peripheral tolerance alters CD8+ T cell fate, we applied Monocle3 pseudotime analysis65 to infer differentiation trajectories of IGRP T cells within the graft (Fig. 3j). In control mice, cells progressed through the highly functional Eff_Ifng cluster and predominantly terminated in the cytolytic Eff_ISG state. By contrast, following 2.5HIP-NP treatment, cells followed an alternative trajectory through the Eff_early cluster and instead terminated part way into the less functional Eff_late state, largely bypassing the Eff_Ifng and Eff_ISG branches. These findings suggest that 2.5HIP-induced peripheral tolerance diverts autoreactive CD8+ T cells away from a cytolytic/ISG fate toward a hypofunctional differentiation state.

IGRP-specific CD8+ T cells have a low-avidity TCR repertoire following 2.5HIP-NP tolerance induction.

Single-cell RNA sequencing analysis revealed that 2.5HIP-NP-mediated tolerance induction dampens TCR-driven transcriptional programs in IGRP-specific CD8 T cells. Expression of Rln3, a downstream target of the TCR-regulated transcription factor Nur7766,67, was significantly reduced (Fig. 3e). In parallel, expression of Cd5, a gene whose surface expression on T cells correlates with cumulative TCR signal strength and apparent TCR avidity68, was also decreased in tolerized cells (Fig. 3e, Fig. 4a). Additionally, GSEA revealed a decrease in an Antigen Response pathway following 2.5HIP-NP tolerance induction (Fig. 3.f, Fig. S3i). Together, these findings suggest that induction of tolerance to the 2.5HIP modifies the TCR signaling axis in graft-infiltrating IGRP T cells. Therefore, we hypothesized that 2.5HIP-NP-induced tolerance might impact on the relative avidity/affinity of the IGRP TCR repertoire in islet grafts.

Surface staining of IGRP T cells confirmed treatment-induced reduction in CD5 levels (Fig. 4b) that were observed in the transcriptional results. Tetramer staining intensity for similar levels of surface TCR expression approximates the relative avidity of TCRs69,70. Indeed, IGRP T cells from 2.5HIP-NP tolerized grafts had a lower average IGRP-H2-Kd tetramer gMFI than IGRP T cells from control, HEL-NP treated grafts (Fig. 4c). To ensure that down-regulation of surface TCR levels was not responsible for the apparent reduction in tetramer avidity, we examined the total level of the TCR on IGRP T cells and found no significant differences in the gMFI for surface TCR levels (Fig. 4d). These results indicate that the repertoire of IGRP T cells present in 2.5HIP-NP tolerized grafts may have a lower apparent avidity than the CD8 T cells in control grafts.

To assess whether 2.5HIP-NP tolerance alters the diversity and public nature of the IGRP T cell TCR repertoire, we analyzed TCR sequences from 5' scRNA-seq data. Analysis of expanded clones revealed highly restricted V gene usage, with a single Vα (TRAV16N) and single Vβ (TRBV13-3) comprising over 50% of variable chain usage across all animals regardless of treatment (Fig. S4a), consistent with prior reports of islet-infiltrating IGRP T cells48,71. Every animal, whether treated with 2.5HIP-NPs or not, had 3 to 4 highly expanded TCR clones occupying ~60-90% of their respective repertoire (Fig. 4e). Expanded clones were largely private among control animals, with minimal sharing observed (Fig. 4e,f). By contrast, a single public clone comprised over 50% of the repertoire in two of the 2.5HIP-NP-treated animals (HIP_1, HIP_2) and was significantly expanded in the third (HIP_3) (~10%) (Fig. 4e,f). The combined α and β CDR3 sequences of this treatment-expanded clone (Fig. S4b) included at least two distinct nucleotide variants across all three 2.5HIP-NP-treated animals (Fig. S4c), indicating convergent TCR recombination. Repertoire diversity and public TCRs were assessed by Shannon Diversity index and Jaccard similarity index, respectively. While not statistically significant, IGRP T cells from 2.5HIP-NP tolerized grafts trended towards lower diversity (Fig. S4d) and Jaccard similarity index between all animals indicated more sharing between tolerized animals (Fig. S4e). 2.5HIP NP treatment seemed to restrict the repertoire of IGRP T cells to a single shared clonotype.

To further assess TCR repertoire changes induced by tolerance induction we examined the sequences of expanded clones from either treatment. Weighted logo plot comparisons of CDR3α and CDR3β sequences revealed strong consensus in CDR3α across treatments, and near-consensus in CDR3β except for variation at two central residues (positions 6 and 7) (Fig. 4h). After aligning the CDR3 sequence of the expanded clones, the most abundant clone from control animals, from animal HEL_4, shared all but the two central amino acids (PG) in the CDR3β region with the expanded clone (SQ) found in 2.5HIP-NP tolerized animals (Fig. S4f). To validate the difference in apparent avidity, the HEL_4 TCR and HIP_1 TCRs were reconstructed from 5’ scRNA-sequencing data and expressed in immortalized 5KC mouse T cells (gating shown in Fig. S1c). Cells were then stained with the IGRP tetramer versus total TCRβ surface expression. The total level of TCR on the surface was no different between the two TCRs (Fig4. h). At a given median total TCR gMFI slice, there is higher binding of the IGRP tetramer to HEL_4 TCR expressing cells than to the HIP_1 expressing cells. This demonstrates the expanded clone in HIP tolerized grafts has a lower affinity than an equivalent, highly expanded clone in HEL control grafts (Fig. 4i, Fig. S4g). These data suggest that 2.5HIP-NP tolerance induction limits the repertoire of IGRP clones present in treated grafts to a low-avidity population.

IL-10+ 2.5HIP T cells are expanded in islet grafts following 2.5HIP-NP tolerance induction.

To investigate how 2.5HIP-NPs may actively suppress CD8 T cell effector differentiation within islet grafts, we tracked the regulatory fate of graft-infiltrating 2.5HIP T cells (Fig. 5a) in response to treatment. We focused on two key regulatory subsets: FOXP3⁺ T regulatory cells (Tregs) and FOXP3⁻CD25⁻ IL-10-producing Tr1-like cells. Tolerance induction with 2.5HIP-NPs significantly increased the frequency of FOXP3⁺ 2.5HIP⁺CD4⁺ T cells within the graft (Fig. 6b) consistent with a trend we previously observed at the same timepoint56, though this population comprised only ~10% of the total antigen-specific CD4⁺ T cells.

Previous studies employing antigen-specific therapies in NOD mice have demonstrated the critical role of IL-10 producing Tr1-like FOXP3⁻CD25⁻ regulatory T cells in establishing peripheral tolerance72. Additionally, Tr1 cells have been associated with positive outcomes in transplantation73. In our previous study56, we also observed that the majority of 2.5HIP T cells adopted an anergic phenotype, which may give rise to IL10-producing Tr1-like T cells. To assess IL-10 expression in both regulatory subsets following treatment, we utilized the tiger (interleukin-ten ires gfp-enhanced reporter)74 mouse backcrossed to the NOD mouse (NOD/Tiger)75 for islet transplant and tolerance induction experiments. To detect IL-10GFP/mRNA without fixation, surrogate surface markers for FOXP3⁺ Tregs (CD25⁺CD127⁻) and conventional CD4 T cells (CD25⁻) were validated (Fig. S5a) and used to phenotype graft-infiltrating 2.5HIP⁺ CD4 T cells following tolerance induction. Diabetic NOD/Tiger mice were treated as before with 2.5HIP-NPs or HEL-NPs and graft-infiltrating 2.5HIP T cells were examined for levels of GFP (Fig. 5c). CD4 T cells comprised ~80% of IL-10GFP/mRNA⁺ cells in islet grafts from both 2.5HIP-NP-tolerized and HEL-NP control mice (Fig. 5d), suggesting they are the primary source of IL-10 at this time point. Graft-infiltrating IL-10+CD25- CD4 T cells expressed higher levels of canonical markers of Tr1 CD4 T cells (Lag3, and CD49b) than their IL-10- CD25- CD4 counterparts (Fig. 5e), indicating these cells have a Tr1-like surface phenotype.

When examining these populations in response to treatment, tolerance induction to the 2.5HIP significantly increased the proportion of CD25+CD127- Tregs cells expressing IL-10 (Fig. 5f). 2.5HIP-NP treatment also expanded a population of IL-10+ CD25- Tr1-like CD4 T cells (Fig. 5g). However, as a fraction of all 2.5HIP T cells, CD25-IL-10+ Tr1-like cells accounted for nearly half of the total 2.5HIP T cells in the graft following treatment, 3 to -4-fold more than their FOXP3+ counterparts. This indicates CD25-IL10+ 2.5HIP T cells are the predominant regulatory population expanded by 2.5HIP-NP tolerance induction in islet grafts. To replicate these findings in a different model, pre-diabetic NOD/Tiger mice were treated with 2.5HIP- or HEL-NPs to determine if islet-infiltrating 2.5HIP T cells exhibited the same increase in IL-10mRNA/GFP expression (Fig. S5b). Indeed, both CD25+CD127- Treg and CD25- populations of 2.5HIP T cells were expanded in the islets (Fig. S5c,d), lymph nodes (only CD25- 2.5HIP T cells recovered), and spleens of 2.5HIP-NP tolerized animals (Fig. S5d).

Taken together, these data demonstrate a robust expansion of IL-10⁺ 2.5HIP T cells in 2.5HIP-NP-tolerized grafts, with the majority comprising Tr1-like suppressive cells and a smaller FOXP3⁺ Treg subset.

IL-10 blockade compromises prolonged graft survival, promotes effector differentiation of antigen-specific CD8 T cells, and restores CD40 expression on intra-graft cDC1s.

Increased IL-10 production by 2.5HIP T cells may affect differentiation of IGRP T cell effector fate directly76 and/or indirectly via suppression of antigen-presenting cells such as DCs77,78. To investigate whether IL-10 is required for prolonged graft survival following 2.5HIP-NP tolerance induction, IL-10 was depleted using an IL-10 monoclonal antibody during induction of tolerance (Fig. 6a). Blockade of IL-10 abrogated the prolonged graft survival afforded by 2.5HIP-NP-induced tolerance, with the time to disease recurrence mirroring that of HEL-NP isotype control and HEL-NP IL-10 treated animals (Fig. 6b,c). To determine if IL-10 blockade restored the effector fate of graft-infiltrating, antigen-specific T cells, grafts were isolated at 8-9 days post-transplant from HEL-NP or 2.5HIP-NP tolerized mice and were treated with IL-10 antibody or an isotype control Ab. Inhibiting IL-10 during tolerance induction had no impact on the expansion of FOXP3+ 2.5HIP Tregs in islet grafts (Fig. 6d). There was also no apparent effect on the fraction of TCFhi 2.5HIP T cells (Fig. 6e) or absolute numbers of TCF1lo terminal effectors (Fig. 6f). By contrast, blockade of IL-10 restored the fraction of IGRP T cells to levels similar to those found in HEL-NP isotype control grafts (Fig. 6g). IL-10 blockade reduced the proportion of TCF1⁺ IGRP T cells to levels seen in HEL-NP controls (~10–20%) (Fig. 6h), which was accompanied by an increase in TCF1⁻ effector cell numbers (Fig. 6i). The elevated ISG signature (measured by Ly6A surface expression) (Fig. 6j,k) and relative TCR avidity (measured by normalized IGRP staining) (Fig. 6l) were also restored to control levels upon IL-10 blockade in IGRP T cells. Collectively, these data indicate that IL-10 is essential for arrest of IGRP T cell effector differentiation induced by 2.5HIP tolerance, but that this effect occurs downstream of both the expansion of FOXP3⁺ 2.5HIP regulatory T cells and the arrest of graft-infiltrating effector 2.5HIP T cells.

Given the reduced ISG signature, altered TCR repertoire of IGRP T cells, and the known inhibition of antigen presentation by IL-1077,78, we next asked whether 2.5HIP-NP therapy impairs APC licensing, thereby indirectly limiting CD8 T cell effector fates. Professional APCs, especially CD4-licensed dendritic cells, are necessary for robust CD8 effector differentiation79,80. CD4 T cell mediated licensing of DCs by CD40-CD40L ligation is critical to achieve full cytolytic potential in CD8 T cells79 81. To determine if 2.5HIP-NP tolerance induction had any impact on APC populations, prediabetic NOD mice (<12 weeks old) were treated with 2.5HIP- or HEL-NPs and APC subsets were examined in pancreatic islets and the pancreatic lymph node (pLN) (Fig. S6a, gating strategy in Fig. S6b). Flow cytometry revealed there was a significant decrease in CD40 levels exclusively on migratory cDC1s (CD11c MHCIIhiXCR1+) in the pancreatic LN of 2.5HIP-NP tolerized mice but not in resident cDC1s or migratory cDC2s (Fig. S6d). These cells likely migrated from the islets, indicating that 2.5HIP-NP tolerance induction suppresses cDC1 activation in islets.

Next, we assessed whether DCs tolerized in situ by 2.5HIP-NPs can independently modulate IGRP T cell activation. DCs were isolated from the islets and spleens of NOD.scid mice adoptively transferred with 2.5HIP-specific transgenic T cells (BDC-2.5 TCR-Tg) and tolerized with either 2.5HIP- or HEL-conjugated nanoparticles (Fig. S7a). When co-cultured with transgenic IGRP-specific CD8 T cells (NY8.3), islet-derived DCs from either treatment were able to activate NY8.3 T cells based on the increased proportion of CD25+CD69+ double-positive T cells (Fig. S7b). However, there was a significant decrease in the gMFI of CD69 and Ly6A on NY8.3 T cells incubated with islet-derived DCs from 2.5HIP-NP treated animals (Fig. S7c,d). This difference was not observed with spleen-derived DCs from either treatment, indicating that 2.5HIP-NP tolerance induction can specifically impact the ability of intra-islet DCs to activate IGRP-specific T cells and express Ly6A.

Finally, we determined if DCs are suppressed by 2.5HIP tolerance induction in islet grafts and whether this suppression is IL-10 dependent. The cDC1 and cDC2 subsets (Fig. 6m) were examined in the grafts of HEL-NP or 2.5HIP-NP-tolerized mice, with or without IL-10 blockade at day 9 post-transplant. Tolerance induction selectively reduced the fraction of CD40hi intra-islet cDC1s but not cDC2 cells (Fig. 6n). Following IL-10 blockade, CD40hi cDC1s were restored to levels founds in control grafts. In summary, these findings demonstrate that 2.5HIP-specific tolerance reshapes the graft microenvironment by inducing IL-10-dependent suppression of DC activation, thereby impairing the ability of DCs to license CD8 T cells and promote ISG/cytolytic differentiation.

Fate of T cellsand dendritic cells during tolerance and following graft destruction in 2.5HIP NP treated mice.

While therapeutic induction of tolerance with 2.5HIP NPs during islet transplant prolongs graft survival (~45 days), and continued treatment extends this protection (~90 days) (Fig. 2c), eventually the grafts are destroyed. In order to understand where tolerance breaks, antigen-specific T cells were examined in tolerized grafts and the draining lymph node at a timepoint when treated grafts are protected (day 15) and again upon graft failure (~day 45) (Fig. 7a).

In the kLN, we observed a numeric accumulation of 2.5HIP T cells at d15 (Fig. 7b), higher than the number at graft failure and d9 (Fig. S2b). This was true for 2.5HIP tetramer-negative CD4 T cells as well (Fig. S8a) and continued a trend observed in tolerized grafts at d9 (Fig. S2b). At d15 kLN 2.5HIP T cells were similar or less TCF1hi compared to the same cells at graft failure (Fig 7c). Conversely in the graft, there was a non-significant increase in the total number of 2.5HIP T cells upon graft failure compared to d15 (Fig 7b), as with 2.5HIP tetramer-negative CD4 T cells (Fig. S8a). Interestingly, upon graft failure 2.5HIP T cells were equally or more TCF1hi compared to the equivalent cells at d15 (Fig 7c). IGRP T cells exhibited a very similar phenotype to the 2.5HIP T cells at both timepoints, albeit muted in magnitude. At d15, there was a trending accumulation of IGRP T cells in the kLN (Fig 7d), with a significant increase for IGRP tetramer-negative CD8 T cells (Fig S8a). Most of the IGRP T cells in the kLN were TCF1hi (Fig 7e) at either timepoint. Upon graft failure there was a near-significant increase of IGRP T cells in the islet graft compared to d15 (Fig 7d); this was also the case for IGRP tetramer-negative CD8 T cells (Fig. S8a). At graft failure, IGRP T cells were significantly more TCF1hi than at d15. Collectively, there was an accumulation of antigen-specific, and non-specific T cells in the kLN at d15 when grafts were protected. Upon graft destruction, there was an increase in the number of antigen-specific and non-specific CD4 and CD8 T cells in the graft, which may indicate epitope spread to T cells of other specificities, including insulin B-chain. Interestingly, TCF1hi cells were more abundant in antigen-specific CD4 and CD8 T cells upon graft failure. This is possibly the result of the accumulation of stem-like TCF1hi cells during the time that the graft is still protected. Tolerance may induce a helpless fate82 in CD8 T cells that promotes memory formation at the expense of effector function, resembling a tertiary lymphoid structure at the graft.

To determine if treatment-induced regulatory 2.5HIP T cells were maintained in the tissue, we assessed the proportion of FOXP3+ 2.5HIP T cells at the d15 tolerance point and upon graft failure. While there was no difference in percentage of FOXP3+ 2.5HIP T cells in the kLN, there was a significant decrease in regulatory 2.5HIP T cells in the graft upon failure (Fig. 7f), returning to levels found in d9 control grafts (Fig. 5b). This indicates the graft regulatory landscape induced by 2.5HIP NP tolerance induction is eroded upon graft failure, with the regulatory cells likely dependent on continued exogenous antigen-NP treatment.

Finally, dendritic cells were assessed to determine if CD40hi DC are suppressed at d15 and return upon graft failure. At d15, the percentage of CD40hi cDC1s was similar to that found at d9 tolerized grafts, but returned to significantly higher levels upon graft failure, similar to those found in d9, control grafts (Fig. 6n). Without continued active suppression by regulatory 2.5HIP T cells, intra-graft CD40hi cDC1s are re-established, driving functional antigen-presentation to CD8 T cells within the islet graft. We posit the graft ultimately fails due to lack of treatment-dependent, regulatory 2.5HIP T cells in the graft, allowing for effective antigen-presentation to CD8 T cells of multiple specificities, eventually reaching a threshold of sufficient number and function of cytotoxic T cells that destroy the graft.

The mechanistic basis of the dominant tolerance observed in treated islet grafts, whereby CD4 antigen-specific therapy restrains CD8 T cell effector differentiation by limiting effective antigen presentation by cDC1s, is summarized in the graphical abstract (Fig. 8).

Methods
Animal studies
All animal study experimental designs, treatments, husbandry, group numbers, statistical cut offs, and power calculations were submitted to the IACUC at the University of Colorado Anschutz Medical Campus for approval prior to beginning experiments. All mice were maintained and bred in a specific pathogen-free facility at the University of Colorado Anschutz Medical Campus Vivarium (Aurora, CO, USA) (12/12 light/dark cycles, temperature 18-26°C, humidity 30-70%). Experimental and control animals were co-housed and littermates were used for different treatments when possible. Mice were euthanized using carbon dioxide (CO₂) inhalation followed by secondary method of cervical dislocation in accordance with institutional guidelines. NOD.ShiLtJ (Jackson lab #001976), NOD.scid (Jackson Lab #001303), and NOD.BDC-2.5 TCR-transgenic (Tg/BDC-2.5) (Jackson Lab #004460) mice were obtained from The Jackson Laboratory for breeding and new breeders were ordered every 4-5 generations. NOD/tiger (interleukin-ten ires gfp-enhanced reporter74 (Jackson Lab #034192) backcrossed onto the NOD.ShiLtJ background for >10 generations) and NOD/NY8.3 TCR-transgenic (Tg/NY8.3) (Jackson Lab #005868) breeders were obtained from Dr. Rachel Friedman, and were bred as heterozygous animals with NOD.ShiLtJ mice from The Jackson Laboratory. NOD/BDC-2.5 TCR-transgenic (Tg/BDC-2.5) mice were also bred heterozygously with NOD.ShiLtJ. All mice were maintained on standard chow and water ad libitum except NOD.scid mice, which were maintained on a 50/50 mixture of standard chow and antibiotic chow ad libitum to prevent infection in the immunocompromised strain.

Polyclonal NOD and NOD/Tiger mice were monitored for diabetes by weekly random urine glucose measurements; high readings were confirmed for hyperglycemia by tail vein blood glucose readings >250 mg/dl. Following onset of hyperglycemia, mice were maintained on daily exogenous insulin, Humulin R (0.5-1U/day) until transplant to prevent overt metabolic disease. On the indicated days, mice were transplanted by inserting under the kidney capsule 500 islet equivalents (~300 individual islets) from a NOD.scid mouse. A single experiment is defined as the diabetic recipient mice receiving an islet transplant on the same day from the same pool of NOD.scid islets. For transplant surgery, mice were given Buprenorphine SR (1.0 mg/kg by body weight) analgesic subcutaneously, ~30 minutes prior to surgery. Under anesthesia (isoflurane, 3-5% induction maintained with 1-2.5% oxygen) an incision was made to expose the left kidney. An opening was made in the kidney capsule with a needle and the islets expelled beneath the capsule. The retroperitoneal wall was closed with dissolving sutures and the skin closed with autoclips. Animals were closely monitored for recovery in a temporary cage on a heating pad until determined to be bright, alert and responsive. If needed, post-op fluid supplementation (0.5-1mL saline solution) was given subcutaneously to facilitate recovery. After transplanting, mice were monitored by random blood glucose measurements for recurrence of disease (>250 mg/dl). For IL-10 antibody depletion experiments, diabetic mice were injected intraperitoneally with 100 g/dose of IL-10 mAb (JES5-2A5 or an isotype control, Bioxcell) on the indicated days. Treatment with IL-10 was given 4 hours before NP treatment if given on the same day.

For injections of nanoparticles and transgenic T cells, nanoparticles or cells were resuspended and filtered with 45m mesh before retro-orbital intravenous (i.v.) injection into mice under isoflurane anesthesia. For islet transplant experiments, diabetic female mice were used as recipients based on the sex-specific increased disease incidence. For prediabetic and transgenic adoptive transfer experiments, age-matched (8-12-weeks-old) male mice were used as donors.

Nanoparticle synthesis, peptide conjugation, and treatment
Biodegradable poly(lactic-co-glycolic acid) nanoparticles (NPs) were provided by the Miller lab generated using a single emulsion followed by solvent evaporation as previously described20. Nanoparticles were then washed three times with PBS and conjugated covalently to a soluble form of the 2.5HIP (RGG-LQTLALWSRMD-GGR) or the HEL (AMKRHGLDNYRGYSL) peptide by incubating with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (ECDI) and then washed three times again. Nanoparticles were prepared fresh for each experiment and immediately administered by retro-orbital i.v. injection (2.5 mg of nanoparticles in 200 l of sterile PBS) on indicated days.

Flow cytometry MHC-tetramer and antibody staining
For tetramer and antibody staining for flow cytometry analysis, single cell suspensions were generated from various organs. Lymph nodes and spleen were minced with scissors and filtered through a 70 m cell strainer with RBC lysis if needed. Grafts were resected from the kidney and digested in Dispase II (Gibco 17105-041) at 2 U/ml for 10-15 minutes with intermittent mixing by pipetting. Following digestion, graft suspensions were also filtered through a 70 m cell strainer. After a wash, cells were resuspended in complete media and stained with MHC tetramers at 37ºC for 20 minutes with the 2.5HIP I-Ag7 tetramer or IGRP (206-214) H2-Kd tetramer in PE or APC. Surface antibody cocktails were then added to the tetramer stain and incubated for a further 40 minutes. The following antibodies were used: rat IgG2b anti-mouse CD3 APC/Fire 750, clone:17A2, Biolegend, cat #100267, 1:400; mouse IgG1 anti-rat RT1B (MHCII) FITC, clone:OX-6, Biolegend, cat #205305, 1:600; mouse IgG2b anti-mouse/rat XCR1 APC, clone:ZET, Biolegend, cat #148205, 1:200; rat IgG1 anti-mouse CD172a (SIRP), PE clone:P84, Biolegend, cat #124629, 1:400; rat IgG2a anti-mouse F4/80 BV711, clone:BM8, Biolegend, cat #103057, 1:200; rat IgG2a anti-mouse CD40 PE/Dazzle 594, clone:3/23, Biolegend, cat #124629, 1:400; Armenian hamster IgG anti-mouse CD11c BV705, clone: N418, Biolegend, cat #117333, 1:200; Armenian hamster IgG anti-mouse CD11c BB700, clone:HL3, BD Biosciences, cat #745899; rat IgG2a anti-mouse CD19 BB700, clone:1D3, BD Biosciences, cat #566412; rat IgG2b anti-CD11b BB700, clone:M1/70, BD Biosciences cat #566416; rat IgG2a anti-mouse Ly6G BB700, clone:1A8, BD Biosciences cat #566435; rat IgG2a anti-mouse CD5 PE, clone:53-7.3, Biolegend, cat #100607, 1:400; rat IgG2a anti-mouse Ly6A/E BV421, clone:D7, Biolegend, cat #108127, 1:400; Armenian hamster IgG anti-mouse PD1 BUV615, clone:J43, BD Biosciences, cat #752299, 1:200; hamster anti-mouse CD279 (PTD-1) BUV615, clone:J43, Biolegend 1:200; rat IgG2a anti-mouse CD8a PerCP-eFluor710, clone:53-6.7, eBioscience, cat #46-0081-82, 1:400; rat IgG2a anti-mouse CD8a BB515, clone:53-6.7, BD Biosciences, cat #564459; recombinant human IgG1 anti-human/mouse TOX PE, clone:REA473, Miltenyi Biotec, cat #130-120-785, 1:200; rat IgG1 anti-mouse CD25 BV650, clone:PC61, Biolegend, cat #102038, 1:200; rat IgG2a anti-mouse CD127 PE Cy7, clone:A7R34, Biolegend, cat #135013, 1:400; rat IgG1 anti-mouse LAG3 BV786, clone:C9B7W, Biolegend, cat #125219; rat IgG2a anti-mouse CD4 BV605 and BV421, clone:RM4-5, Biolegend, cat #100547, #116023, 1:400; rat IgG2a anti-mouse CD366 (TIM3) BV785, clone:RMT3-23, Biolegend, cat #119725, 1:200; rat IgM anti-mouse CD49b APC Fire750, clone:DX5, Biolegend, cat #108925, 1:200; rat IgG2a anti-mouse/human CD44 BV771, clone:IM7, Biolegend, cat #103057, 1:400; rat IgG2a anti-mouse CD62L FITC, clone:MEL-14, Biolegend, cat #104406, 1:400; rat IgG2a anti-mouse Ki67 FITC, clone:SolA15, Biolegend, cat #151211, 1:600; rabbit IgG anti-TCF1/TCF7 AlexaFluor 647, clone:E601K, Cell Signaling Technologies, cat #83268S, 1:400; rabbit IgG anti-TCF1/TCF7 AlexaFluor488, clone:E601K Cell Signaling Technologies, cat #50639S, 1:400; rat IgG2a anti-mouse FOXP3 eFluor, 450 clone:FJK-16s, Invitrogen, cat #48-5773-82, 1:200; rat IgG2a anti-mouse IFN eFluor, 450 clone:XMG1.2, eBioscience, cat #48-7311-80 1:400; rat IgG1 anti-mouse, TNF PE Cy7, clone:MP6-XT22, Biolegend cat #506323, 1:400; hamster anti-mouse TCR BV605, clone:H57-597, Biolegend, cat #109241, 1:400; and rat IgG2a anti-mouse CD16/32 (Fc-block), clone:93, Biolegend, cat #101302, 1:500.

Cells were washed, stained with viability dye in PBS and fixed with the fixation buffer from the FOXP3 eBioscience Intracellular Staining Kit (#00-5523-00). Following overnight fixation, cells were permeabilized and stained for intracellular targets. After two hours, cells were washed, resuspending in FACS buffer (2% FBS, 1mM EDTA in PBS). Cells were then run on a 5 laser Cytek Aurora Spectral flow Cytometer and analyzed using Flowjo v10.10. To ensure reproducibility, cells were stained in the same antibody concentrations, incubated for the same period of time, and analyzed on the same cytometer.

5’ scRNA-sequencing of IGRP T cells
IGRP T cells were sorted by FACS from day 8 islet grafts (lymphocytes, live, CD45+, lineage-(CD11c, CD11b, CD19, Ly6G, CD4), IGRP tetramer+). After sorting, cells from individual animals were barcoded with has htag antibodies to deconvolute after sequencing (Biolegend: TotalSeq -C0301 anti-mouse Hashtag 1 ACCCACCAGTAAGAC cat #155861, TotalSeq -C0301 anti-mouse Hashtag 2 GGTCGAGAGCATTCA cat #155863, TotalSeq -C0301 anti-mouse Hashtag 3 CTTGCCGCATGTCAT cat #155865, TotalSeq -C0301 anti-mouse Hashtag 4 AAAGCATTCTTCACG cat #155867 all 1:100). Single cell gene expression profile, cell surface proteomics profile, and T cell receptor (TCR) profile were obtained using the Chromium Single-Cell 5’ NextGEM Library and Gel Bead Kit from 10X Genomics. Sorted and hashed cells were partitioned on the 10X Genomics Chromium X instrument for single cell capture, followed by amplification of cDNA and library preparation to recover 10,000 - 12,000 cells per sample. Individual libraries corresponding to gene expression, cell surface protein, and TCR were quantified, pooled, and sequenced on the Illumina NovaSeq 6000 sequencer at a depth of 50,000 reads/cell for gene expression, 5,000 reads/cell for cell surface protein, and 5,000 reads/cell for TCR, respectively. Following sequencing, standard quality control, filtering, and normalization were applied using default CellRanger pipelines. TCR sequences were annotated using the scRepertoire package. All cells from both treatments were used to generate a UMAP reduction. Clustering was then performed using SNN nearest-neighbor analysis while masking TCR genes from the clustering. Pseudobulk differential gene expression (DEG) analysis was used to generate the Volcano plots with the ProjecTIL R package to compare between treatments after excluding TCR genes. Gene set enrichment analysis (GSEA) was run on differentially expressed gene (also after excluding TCR genes) using the fgsea R package from the molecular signature database (MSigDB) C2 curated gene sets, subcategory CGP, with a normalized enrichment score NES cutoff of 1 and adjusted p value (q) of 0.05. All packages required for data processing and analysis and version number: scProportionTest (0.0.4), ggseqlogo (0.2), immunarch (0.9.1), tidyverse (2.0.0), SingleCellExperiment (1.30.1), Seurat (5.3.0), Matrix (1.6-5), scale (1.2.1), cowplot (1.2.0), RCurl (1.98-1.16), AnnotationHub (3.16.1), AnnotationDbi (1.64.1), ensembldb (2.26.0), harmony (1.2.3), ggpubr (0.6.0), gridExtra (2.3), clusterProfiler (4.10.1), Startrac (0.1.0), ggplot2 (4.0.0), msigdbr (7.5.1), scales (1.4.0), fgsea (1.28.0), tradeSeq (1.22.0), viridis (0.6.5), SignatuR (0.3.0), UCell (2.8.0), circlize (0.4.16), ggrepel (0.9.5), ggsci (3.2.0), gprofiler2 (0.2.3), MetBrewer (0.2.0), wesanderson (0.3.7), scRepertoire (2.4.0), SCpubr (2.0.0), dittoSeq (1.16.0), EnhancedVolcano (1.20.0), SeuratExtend (1.2.5), Nebulosa (1.14.0), impute (1.78.0), ComplexHeatmap (2.24.1), sscVis (0.4.0), ggsignif (0.6.4), Biostrings (2.76.0), dplyr (1.1.4), SeuratWrapper (0.4.0), monocle3 (1.4.26), vegan (2.8.0), psych (2.4.6), ProjecTIL (2.4.1), metap (1.11).

TCR reconstruction and expression on TCRko 5KC mouse T cell line
The top expanded IGRP-specific clones from each treatment (HEL_4, HIP_1) were reconstructed and expressed in the immortalized, TCRko 5KC mouse T cell line. Briefly, exact nucleotides sequences were reconstructed from the standard 10X Genomics 5’ scRNAseq TCR library for each the alpha and beta of each TCR. Sequences from 5 separate barcoded cells IDs were aligned for each TCR to ensure correct and consensus sequences. The alpha and beta chain were connected by a P2A and cloned into the pMIGII96 vector (addgene #52107) by restriction enzymes and grown up in chemically competent E. coli. Gamma-retroviral stocks containing the IGRPTCR-pMIGII plasmid were packaged using Platinum-E cells. 5KC were transduced using polybrene spinfection. Resulting cells were cultured in Iscove’s Modification of DMEM with 10% fetal calf serum, 1% pen/strep, and 50M 2-ME at 37ºC in 5% CO . Cells were split daily and assessed after 5 days by flow cytometry for IGRP tetramer staining and total surface TCR expression.

Tg/BDC-2.5 T cell adoptive transfer, NP treatment, and dendritic cell isolation
BDC-2.5 transgenic CD4 T cells were isolated from the spleen and lymph nodes (pancreatic, inguinal, axillary, submandibular) by negative selection (CD4 mojo sort Biolegend). Single cell suspensions of T cells were stimulated in complete RPMI media (10% FBS, 1% pen/strep, Na pyruvate, glutamine, 2-ME, and NEAA) with 20 U/ml recombinant murine IL2 (Peprotech # 212-12-20UG) and CD3/CD28 coated-beads (dynabeads Thermo Fischer #11452D) at a 1:2 ratio (T cell:bead). Following three days of stimulation, beads were removed, and cells were washed and counted. Tg/BDC-2.5 T cells (2.5 million) were adoptively transfer IV to 8-12-week-old NODscid recipients. One day after adoptive cell transfer mice received 2.5mg of either HEL- or 2.5HIP-NPs i.v.; four days later mice were euthanized and dendritic cells isolated by negative selection (EasySep pan-DC #19863) from islets and spleen.

DC and Tg/NY8.3 T cell co-culture
Isolated, primary DCs were incubated in complete RMPI and pulsed with 2ug/ml IGRP for 4 hours. DCs were washed 4 times before incubation with Tg/NY8.3 T cells to remove residual peptide. Tg/NY8.3 were isolated by CD8 negative selection (Biolegend #480008) and stimulated as Tg/BDC-2.5 T cells with the addition of recombinant murine IL7 (PeproTech # 217-17-50UG) for three days. Beads were removed and Tg/NY8.3 T cells were washed two times and incubated with un-pulsed or peptide-pulsed DCs isolated from the spleen or islets of HEL- or 2.5HIP-NP treated mice at a 1:1 ratio.

Statistical analysis
Sample sizes were determined based on previous tolerance and transplant studies as well as preliminary experiments, and on an expected effect size of 1.2 (difference in means standardized by pooled standard deviation), a significance level (α) of 0.05, and a desired power of 80% (β = 0.2). Power analysis was performed using G*Power v3.1.9.7. Data collection was stopped after appropriate numbers of n were analyzed based on the aforementioned power calculations. Data from transplanted animals were excluded if recipient mice did not normalize to <250 mg/dl within two days post-transplant. Flow cytometry data with 200 events of intact cells or less were excluded from analysis. Transplant experiments were repeated 3-6 times with 2-3 transplanted animals per experiments. Experiments with prediabetic and transgenic mice were repeated at least 2 times with 3-4 animals per experiment. Data are presented as mean ± s.e.m. for continuous variable or mean with box and whisker plots for interquartile range (IQR) and 1.5*IQR, respectively. Analysis was performed in GraphPad Prism v10.4.2 or RStudio version 2023.12.1+402. Shaprio-Wilk test was used to test for normality in n<20 datasets. Statistical significance was determined by a non-parametric unpaired two-tailed T test for comparison of a single continuous variable between two group with n<20. For continuous variable with n>20 the Wilcoxon rank-sum test was used with Bonferroni correction if necessary. The Kaplan-Meier method and Multiple Log-Rank-test were used to determine statistical significance for survival analysis with Bonferroni correction for three groups. One-way analysis of variance (ANOVA) was computed to test statistical differences between two or more groups. Levene’s test was used to determine the variances between multiple groups. Šídák’s multiple comparison test was computed when performing pairwise-comparison between cell subsets of different treatments (i.e., TCF1lo IGRP in 2.5HIP-NP vs HEL-NP). Tukey Honest Significant Differences was used to compare multiple means from different treatments groups (i.e., HEL-NP isotype vs 2.5HIP-NP isotype vs 2.5HIP-NP IL10). Details and statistical tests and analysis for specific experiments can be found in figure legends. The alpha value for threshold of significance was 0.05 including corrected p-values.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The work presented in the current manuscript is essentially an extension of a previous study by the same group of authors (Miller Haskins Baker Delisio etc) (PMID: 35007324) published in Diabetes in 2022. (reference # 51 in the current paper).\nTheir previous work has already shown:\n1: 2.5 HIP NPs can protect syngeneic islet grafts\n2: Reduced effector function of both 2.5HIP specific CD4 and IGRP specific CD8 T cells\n(Same is reproduced in this paper- with added markers TCF1, TIM3 and PD1 further defining stem like and effector like\nsubsets)\n3: 2.5 HIP NPs result in expansion of 2.5 HIP specific CD4 T cells in spleen. The previous study reported no change in the\nproportion of HIP CD4s or IGRP CD8’s in the graft (Different from what is presented here)\n4: 2.5 HIP NPs result in increased Ag-specific Foxp3 Tregs in the graft (replicated here), as well as an increase in Ag-\nspecific anergic CD4 T cells (CD73+ FR4+ CD44+) (Reproduced in the current paper as IL-10 producing cells using an IL-\n10 reporter mouse- which in effect are TR1 like anergic CD4 T cells described in their previous study)\nIn summary, previous results have been reproduced and polished with addition of sc-RNA seq, IL-10 blockade and TCR seq\ndata to provide a mechanistic explanation for the observed transient graft protection.\nOther Comments\n1. All figure legends state the presented data is compiled from 4-6 independent experiments. The summary bar graphs for\nmost panels have 3-4 dots. What do the dots represent?\n2. Key features of differentiation of autoreactive T cells in the NOD model have not been noted by the authors in the\nIntroduction. Not all T cells infiltrating the islets during spontaneous disease in NOD mice are TCF-/Slamf6low effector like\ncells. A predominance of stem like TCF1+ CD8+ T cells within the islets has been reported by several studies, (Zakharov P\net.al 2020, Ciecko A et.al 2022, Selck C et.al 2024)- which can give rise to TCF1 - effector-like cells. Moreover, effector-like\ncells that destroy the islets are restrained to an extent by an exhaustion program within the islets similar to cancer and\nchronic infection.\n3. All analyses of Ag-specific T cells in the graft and kLN for most figures have been done at d8-9 post graft (a time point\nwhere control grafts reject). Data in figure 1 panels d,e represents the peak of graft destruction where all cells become TCFlo\nin the graft compared to kLN. What are the TCF levels on T cells in graft during the intervening period say d3-7 post graft?\nare there TCF1+/hi cells in the control grafts at this time? What are the kinetics of HIP and IGRP specific T cell\naccumulation/differentiation between d0 and d9?\n- % PD1 calculation in figure 1e does not make sense. All IGRP and HIP tetramer binding cells in kLN and graft are CD44+,\nas per flow plots in figure 1 c and thus should also be PD1 +/hi. The cut off for PD1 positivity in the histograms (middle panel\n1e) is wrongly placed for kLN- they also should be > 80% PD1 +.\n4. In figure 2 data is presented from HIP2.5 NP treated, or HEL NP treated graft recipients. Why are there no survival curves\nshown at this stage? this data is only presented later in figure 6. All FACS analyses are done at d9 (to coincide with control\ngraft rejection) Does this represent the peak of tolerogenic response? Based on the survival curves and blood glucose levels\n(n=4) in figure 6- mice receiving tolerizing NP start rejecting islet grafts around d 26 post-transplant, thus some\ncharacterization of both graft and kLN in the intervening period say d15-20 would provide more insights into Ag-specific T\ncell differentiation.\n- Authors state there is no impact of 2.5HIP NP tolerance in the kLN (all data in Fig S2), again this is all done at d9, and there\nis some expansion of HIP tetramer+ CD4 T cells (panel S2a)- what are the numbers, proportions and phenotype (TCF, TIM3,\nKi67) of HIP2.5 reactive T cells at later time points?\n- The grafts are eventually destroyed by d40- It would be of interest to know the frequency and phenotype of HIP2.5 and\nIGRP reactive T cells at or shortly before this stage.\n- Do other Ag-specific T cells such as Ins B9-23 specific CD4+ T cells or InsB15-23 reactive CD8+ T cells play any role in\ngraft destruction of HIP NP treated recipients?i.e. why do you eventually get rejection\n- A significant increase in TCF1+ Ag specific CD4 and CD8 in the graft bearing mice receiving HIP-NPs is shown in Figure\npanels 2 d-g. There are some production errors here- shaded areas in Figure 2, d, e, f and g show TCF lo populations, these\nare wrongly labelled as Tcf hi in the summary graph panels across 3 figures.\n- Rather than MFI, the proportions of TCF and IR + populations in panels 2 f and g would be more informative. (as the main\nobservation here is that there are increased TCF1+ tetramer + T cells in HIP NP treated grafts)\n- Indeed in general it would be helpful for the format of Figures 1 and 2 to be similar for ease of interpretation.\n- Reduced polyfunctionality of HIP and IGRP reactive T cells from HIP NP treated grafts is shown in figure 2h-k. There is a\nsizeable TCF-TIM3- population - do these produce effector cytokines? The TCF+ TIM3- stem like cells (green contour plots,\nfig 2 h,k) also have reduction in cytokine production- hard to believe that this is a non-significant difference (consistent 10-12\n% reduction, with tight grouping)\n5. In the sc-RNAseq experiments only IGRP tetramer + CD8 T cells have been examined. Inclusion of HIP 2.5 tetramer +\nCD4+ T cells as well as other immune cell populations would have provided more insights into mechanism behind the\ntolerogenic effects described here. Looking at the annotated cluster defining genes in the dot plot (fig 3 c) there is absence of\nTox in Tcf hi cluster- which is surprising (This is different from what is observed in Tcf+ cells found in the islets- Selck,\nZakharov,Ciecko papers) and suggests that differentiation trajectory of graft infiltrating T cells is different to spontaneous\ndiabetes.\n6. In the IL-10 blockade experiments (data pertaining to figure 6)- a HEL-anti IL-10 cohort is missing, based on the scheme\ndepicted in fig 6a both HEL -NP and HIP-NP groups should have received isotype and anti-IL10 Ab. All plots in figure 6\nshow data from 3 treatment groups. Also, there is a labelling error in fig 6c- HIP anti-iso in red should read HEL-anti-iso.", "reviewer_2": "This manuscript by DiLisio et al explore the mechanisms by which nanoparticles linked to HIP peptides induce tolerance in\nNOD Tx model. This approach allows the investigators to explore the autoreactive response to islets due to the transfer of\nislets from an NOD SCID mouse. The findings demonstrate that CD25-IL10+ 2.5HIPCD4 T cells are the predominant\nregulatory population expanded by these nano-particles, and further that these then altered CD8 GAD specific T cells in the\npancreatic islet, but not the KLN. The mechanisms by which this occurs is via IL-10 production and DC licensing. This study\nis significant as it sheds light on the mechanisms through which this form of antigen specific therapy acts.\nAreas that would be helpful to address.\nThe discussion here is about tolerance induction, but the data demonstrating the efficacy of this intervention in the model\nused is limited and shown only in the final figure. It would be helpful to show early on outcome based on glucose in this\nmodel compared to Hel- NP particularly since this is an intervention that appears to be short lived. Understanding this\nearly will give better context to the study for its readers. It also needs to be made clear that this is a transient form of\ntolerance.\nAdditionally, it would be quite interesting to see if reversal of the altered T cell and DC phenotypes occurs when the disease\nrecures in treated animals, similar to what is show with the use of IL-10 blockade. This additional experiment would add\nessential context to how one would address the question of how to enhance this therapy, or if repeated treatments would be\nuseful.\nThe discussion of TCR avidity and sharing raised several questions for this reviewer. First it would be helpful to understand\nthe level of TCR expression on the cell surface may differ between HIP and HEL- mp treated mice- as an interaction with\nantigen or altered cytokine environment could alter expression. Similarly TCR sharing is interesting but with so few animals\nthis data is hard to interpret and it would be helpful to understand the degree of sharing at baseline with no therapy\nadministered. It is also unclear how this short period of time would result in such significant changes in repertoire.\nMinor issue there is a missing description of figure 2k", "reviewer_3": "The manuscript “Antigen-specific immunotherapy with a CD4 neoepitope restrains CD8 T cell differentiation in islet grafts”\nexplores the mechanisms through which tolerance is induced by PLGA NPs delivering a CD4 neopeptide for treatment of\nT1D through islet transplant. The authors demonstrate that NP administration inhibits the transition from stem-like-to-effector\nT cells in islet grafts, but not islet-draining LNs. Systemically-delivered NPs alter the CD8 T cell repertoire and arrest CD8\neffector differentiation in grafts, a process dependent on IL-10 production by regulatory cells and DC subsets. These findings\nexpand on mechanisms through which biomaterials can drive infectious tolerance – helping to close persistent gap in our\nunderstanding of tolerance-inducing strategies to tackle autoimmunity.\nThis work that builds on previously published studies by this group represents a significant advance in that work, and our\nmechanistic understanding of how engineered strategies to induce antigen-specific tolerance impact autoimmunity.\nExperiments are controlled, mouse models are well-utilized, figures are clear, data interpretation is aligned with the results,\nand the manuscript is well-written. Only minor comments are noted.\n1. In the introduction, the authors introduce the role of different immune cell types (e.g., APC, CD4 and CD8). Could the\nauthors expand on the crosstalk mechanisms between the cells explored in this manuscript should be provided?\nAlternatively, or in addition, a graphical abstract or cartoon illustrating the mechanistic relationship between immune\npathways elucidated in this work could also be helpful.\n2. The transition from stem-like to effectors is an interesting way to characterize what is going on in the graft. Are tetramer\nnegative T cells also going through this transition? In other words, is the phenomenon ag-specific?\n3. Throughout the manuscript, can the authors expand rationale for examining specific markers and their functional\nsignificance in the context of islet transplant and tolerance (e.g., Ly6A, TIM3)?\n4. One outstanding question for ASIs is whether or not disease-relevant antigens are required to drive potent efficacy. Here,\nstudies elucidate mechanisms through which biomaterials can drive infectious tolerance – helping to close persistent gap in our\nunderstanding. The authors should further highlight the implications their findings in the context of infectious tolerance,\nwhich help alleviate some concern over whether ASI will translate to the clinic.\n5. Work in this manuscript focuses on tolerizing against transplanted islet antigen, which is elegant, but are similar DC and\nCD8-related mechanisms occurring in pancreas in the absence of transplant? If this mechanism applies to transplanted\nislets only, do the authors speculate they are similar during syn- and allogeneic responses?\n6. Other: The x axis of Fig. 1B need a label. PLGA is misspelled (PGLA) in the legend of Fig. 2. Histograms in Fig. 4C look\nclipped (maybe it is the just biexponential axis?). Please provide gating strategies for staining in Fig. 4."}, "author_response": "All three reviewers have given robust and meaningful critiques of the manuscript that have\nstrengthen this revised submission and we thank them for their time and consideration.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe work presented in the current manuscript is essentially an extension of a previous\nstudy by the same group of authors (Miller Haskins Baker Delisio etc) (PMID: 35007324)\npublished in Diabetes in 2022. (reference # 51 in the current paper).\nTheir previous work has already shown:\n1: 2.5 HIP NPs can protect syngeneic islet grafts\n2: Reduced effector function of both 2.5HIP specific CD4 and IGRP specific CD8 T cells\n(Same is reproduced in this paper- with added markers TCF1, TIM3 and PD1 further\ndefining stem like and effector like subsets)\n3: 2.5 HIP NPs result in expansion of 2.5 HIP specific CD4 T cells in spleen. The previous\nstudy reported no change in the proportion of HIP CD4s or IGRP CD8’s in the graft\n(Different from what is presented here)\n4: 2.5 HIP NPs result in increased Ag-specific Foxp3 Tregs in the graft (replicated here), as\nwell as an increase in Ag-specific anergic CD4 T cells (CD73+ FR4+ CD44+) (Reproduced in\nthe current paper as IL-10 producing cells using an IL-10 reporter mouse- which in effect\nare TR1 like anergic CD4 T cells described in their previous study)\nIn summary, previous results have been reproduced and polished with addition of sc-RNA\nseq, IL-10 blockade and TCR seq data to provide a mechanistic explanation for the\nobserved transient graft protection.\nOther Comments\n1. All figure legends state the presented data is compiled from 4-6 independent\nexperiments. The summary bar graphs for most panels have 3-4 dots. What do the dots\nrepresent?\nEach dot represents a transplanted mouse. Because of the spontaneous nature of NOD\ndiabetic onset and our colony size, we are only able to treat and transplant 1-3 mice at a\ntime. In our colony with 100-200 female NOD mice, only 1-3 become diabetic every week.\nIn order to get a total n of 3-4 for each group, this often requires 4-6 individual transplant\nexperiments. We define an “experiment” as the recipient mice that are treated at the same\n2025_12_17\ntime and receive the same pool of NOD.scid donor islets; this has been clarified in the\nanimal studies section of Materials and Methods.\n2. Key features of differentiation of autoreactive T cells in the NOD model have not been\nnoted by the authors in the Introduction. Not all T cells infiltrating the islets during\nspontaneous disease in NOD mice are TCF-/Slamf6low effector like cells. A predominance\nof stem like TCF1+ CD8+ T cells within the islets has been reported by several studies,\n(Zakharov P et.al 2020, Ciecko A et.al 2022, Selck C et.al 2024)- which can give rise to TCF1\n- effector-like cells. Moreover, effector-like cells that destroy the islets are restrained to an\nextent by an exhaustion program within the islets similar to cancer and chronic infection.\nThe work on the presence of TCF1+ T cells in islets has been added to the Introduction with\nthe corresponding citation. We have also added the observations of an exhaustion-like\nprogram in autoreactive T cells, reflecting that of cancer and chronic viral infection. In\naddition, we have compared our data with that of an exhaustion-like program in the\nResults section for Fig. 2 and Fig. 3.\n3. All analyses of Ag-specific T cells in the graft and kLN for most figures have been done at\nd8-9 post graft (a time point where control grafts reject). Data in figure 1 panels d,e\nrepresents the peak of graft destruction where all cells become TCFlo in the graft\ncompared to kLN. What are the TCF levels on T cells in graft during the intervening period\nsay d3-7 post graft? are there TCF1+/hi cells in the control grafts at this time? What are the\nkinetics of HIP and IGRP specific T cell accumulation/differentiation between d0 and d9?\n- % PD1 calculation in figure 1e does not make sense. All IGRP and HIP tetramer binding\ncells in kLN and graft are CD44+, as per flow plots in figure 1 c and thus should also be PD1\n+/hi. The cut off for PD1 positivity in the histograms (middle panel 1e) is wrongly placed for\nkLN- they also should be > 80% PD1 +.\nTo further understand the kinetics of HIP- and IGRP-specific T cell accumulation and\ndifferentiation in grafts we have examined tet+ cells at d3, d6, and added that to d9 data in\nFig.1 e. These results demonstrate that there are very few TCF1+ HIP- or IGRP-specific T\ncells in the graft at d3, and by d6, expansion peaks for both 2.5HIP and IGRP T cells, with a\nslight decline in TCF1+ HIP T cells between d6 and d9. Similarly, when looking at absolute\nnumbers of TCF1+ and TCF1- HIP and IGRP T cells, there are few cells of either fate at d3.\nBy d6 there is a proportional increase of TCF1+ and TCF1- T cells of both specificities\ncompared to d3, and little significant increase is observed by d9. This demonstrates that\nthe proportion of TCF1+/TCF1- and absolute number of antigen-specific T cells is\nestablished by d6 with little further change by d9.\nPD1 cutoff for positivity has been placed to include all PD1-staining cells and is reflected in\nthe example staining and bar graph in Fig. 1. This gating was then also used in Fig. 2.\n4. In figure 2 data is presented from HIP2.5 NP treated, or HEL NP treated graft recipients.\n2025_12_17\nWhy are there no survival curves shown at this stage? this data is only presented later in\nfigure 6. All FACS analyses are done at d9 (to coincide with control graft rejection) Does\nthis represent the peak of tolerogenic response? Based on the survival curves and blood\nglucose levels (n=4) in figure 6- mice receiving tolerizing NP start rejecting islet grafts\naround d 26 post-transplant, thus some characterization of both graft and kLN in the\nintervening period say d15-20 would provide more insights into Ag-specific T cell\ndifferentiation.\nWe have added blood glucose and survival data into figure 2 (Fig. 2c). We have also\nincluded new data where mice were continued on 2.5HIP NPs every 10 days for 9x total\ntreatments. This demonstrates continued graft protection.\nWe chose d9 to analyze antigen-specific T cells for a direct comparison at the same\ntimepoint because the control grafts have begun to fail by d9 and treated grafts are\nprotected.\nIn order to address what occurs after d9 in 2.5HIP tolerized animals, we have added a new\nfigure (Fig. 8) including analysis of 2.5HIP and IGRP T cells in the kLN and graft at d15\n(protected) and upon graft failure. Interestingly we see an accumulation of tet+ T cells in\nthe kLN at d15 which is gone by the time of graft failure. This aligned with the data at d9\nwith even more total cells in the kLN. Conversely there are still few tet+ T cells in the graft\nat d15, but there is an increase in the number of both tet+ and tet- T cells in transplanted\ntissue upon graft failure.\n- Authors state there is no impact of 2.5HIP NP tolerance in the kLN (all data in Fig S2),\nagain this is all done at d9, and there is some expansion of HIP tetramer+ CD4 T cells\n(panel S2a)- what are the numbers, proportions and phenotype (TCF, TIM3, Ki67) of HIP2.5\nreactive T cells at later time points?\nData and analysis of T cells in the kLN are presented in the new Fig. 8. Interestingly, there\nseemed to be accumulation of 2.5HIP and IGRP T cells in the kLN at d15 in both number\nand %, compared to d9 and graft failure. The cells are at a slightly smaller percentage of\nTCF1+ in the kLN for both 2.5HIP and IGRP T cells at d15, then return to the ~90% level\nupon graft failure. We did not see any change in the 2.5HIP FOXP3+ Treg population in the\nkLN at any timepoint.\n- The grafts are eventually destroyed by d40- It would be of interest to know the frequency\nand phenotype of HIP2.5 and IGRP reactive T cells at or shortly before this stage.\nUpon graft failure we see a large accumulation of total T cells, including bulk and 2.5HIP\nand IGRP T cells (Fig. 8, S8). Interestingly, the cells are proportionally more TCF1+ at\nfailure. This is potentially a consequence of continuous “helpless” antigen activation over\nthe length of graft protection because conventional 2.5HIP CD4 T cells are suppressed and\n2025_12_17\nregulatory 2.5HIP CD4 T cells are actively inhibiting functional DC antigen presentation.\nThere was a reduction of the 2.5HIP FOXP3+ Treg population in grafts upon failure,\nreturning to levels found in control grafts. This was accompanied by an increase in\nlicensed cDC1 in the graft.\n- Do other Ag-specific T cells such as Ins B9-23 specific CD4+ T cells or InsB15-23 reactive\nCD8+ T cells play any role in graft destruction of HIP NP treated recipients?i.e. why do you\neventually get rejection\nInsulin-reactive CD4 and CD8 T cells are likely involved in the eventual destruction of islet\ngrafts. Analysis of 2.5HIP and IGRP T cells demonstrates that there are few TCF- effectors\nof either specificity in the graft tissue at the time of graft failure. However, there is a much\ngreater number of total T cells in the graft. This may indicate that epitope spread has\noccurred and that there is expansion of islet-specific T cells of other specificities, such as\nthose recognizing insulin B chain. In Fig. 8 Results and in the Discussion, we posit that B-\nchain and other T cell specificities may then collectively contribute to the eventual graft\ndestruction following the collapse in regulatory T cell suppression of cDC1 antigen\npresentation, an explanation that warrants further investigation.\n- A significant increase in TCF1+ Ag specific CD4 and CD8 in the graft bearing mice\nreceiving HIP-NPs is shown in Figure panels 2 d-g. There are some production errors here-\nshaded areas in Figure 2, d, e, f and g show TCF lo populations, these are wrongly labelled\nas Tcf hi in the summary graph panels across 3 figures.\nThis has been corrected in Fig. 2.\n- Rather than MFI, the proportions of TCF and IR + populations in panels 2 f and g would be\nmore informative. (as the main observation here is that there are increased TCF1+ tetramer\n+ T cells in HIP NP treated grafts)\n- Indeed in general it would be helpful for the format of Figures 1 and 2 to be similar for\nease of interpretation.\nThe IR data in Fig. 2 has been changed to match that of Fig. 1 for ease of interpretation.\n- Reduced polyfunctionality of HIP and IGRP reactive T cells from HIP NP treated grafts is\nshown in figure 2h-k. There is a sizeable TCF-TIM3- population - do these produce effector\ncytokines? The TCF+ TIM3- stem like cells (green contour plots, fig 2 h,k) also have\nreduction in cytokine production- hard to believe that this is a non-significant difference\n(consistent 10-12 % reduction, with tight grouping)\n2025_12_17\nWe have added all three subpopulations (TCF+TIM3-, TCF-TIM3-, and TCF-TIM3+) to the\ncytokine staining and included the absolute p value for data nearing .05 significance (Fig. 2\ni-l). These data indicate the progressive loss of function in cells differentiating in a\ntolerogenic graft setting. We have included the absolute p value for data nearing .05\nsignificance for Fig. 2 and throughout the manuscript. This demonstrates how close (.06)\nthe tolerized TCF+TIM3- IGRP T cells are to a significant decrease in polyfunctionality.\n5. In the sc-RNAseq experiments only IGRP tetramer + CD8 T cells have been examined.\nInclusion of HIP 2.5 tetramer + CD4+ T cells as well as other immune cell populations\nwould have provided more insights into mechanism behind the tolerogenic effects\ndescribed here. Looking at the annotated cluster defining genes in the dot plot (fig 3 c)\nthere is absence of Tox in Tcf hi cluster- which is surprising (This is different from what is\nobserved in Tcf+ cells found in the islets- Selck, Zakharov,Ciecko papers) and suggests\nthat differentiation trajectory of graft infiltrating T cells is different to spontaneous\ndiabetes.\nWe agree that sequencing of more cell types would have been more informative. We\nattempted to sequence all CD45+ cells from control and tolerized grafts, but this\nexperiment failed as we were unable to deconvolute different samples due to poor\namplification of the hashtag barcodes. Currently we do not have the budget to undertake\nanother scRNAseq experiment.\nWhile the scRNAseq displays a single small cluster with high TOX transcript expression,\nflow cytometry reveals that both TCF1+ and TCF1+ tetramer+ IGRP CD8 T cells express\nrelatively high levels of TOX in the graft, compared to tetramer-negative CD8 T cells, as\nseen below.\n2025_12_17\nanimal 1 animal 2\n6. In the IL-10 blockade experiments (data pertaining to figure 6)- a HEL-anti IL-10 cohort is\nmissing, based on the scheme depicted in fig 6a both HEL -NP and HIP-NP groups should\nhave received isotype and anti-IL10 Ab. All plots in figure 6 show data from 3 treatment\ngroups. Also, there is a labelling error in fig 6c- HIP anti-iso in red should read HEL-anti-iso.\nWe have added the HEL-NP anti-IL10 treatment group to Fig. 6 and corrected the labeling\nerror in Fig 6c.\nReviewer #2 (Remarks to the Author):\nThis manuscript by DiLisio et al explore the mechanisms by which nanoparticles linked to\nHIP peptides induce tolerance in NOD Tx model. This approach allows the investigators to\nexplore the autoreactive response to islets due to the transfer of islets from an NOD\nSCID mouse. The findings demonstrate that CD25-IL10+ 2.5HIPCD4 T cells are the\npredominant\nregulatory population expanded by these nano-particles, and further that these then\naltered CD8 GAD specific T cells in the pancreatic islet, but not the KLN. The mechanisms\nby which this occurs is via IL-10 production and DC licensing. This study is significant as it\nsheds light on the mechanisms through which this form of antigen specific therapy acts.\n2025_12_17\nAreas that would be helpful to address.\nThe discussion here is about tolerance induction, but the data demonstrating the efficacy\nof this intervention in the model used is limited and shown only in the final figure. It would\nbe helpful to show early on outcome based on glucose in this model compared to Hel- NP\nparticularly since this is an intervention that appears to be short lived. Understanding this\nearly will give better context to the study for its readers. It also needs to be made clear that\nthis is a transient form of tolerance.\nWe have added blood glucose readings and survival curves to Fig. 2. An exact time of graft\nfailure has been denoted in Fig. 2 to make clear the duration of graft protection.\nAdditionally, it would be quite interesting to see if reversal of the altered T cell and DC\nphenotypes occurs when the disease recures in treated animals, similar to what is show\nwith the use of IL-10 blockade. This additional experiment would add essential context to\nhow one would address the question of how to enhance this therapy, or if repeated\ntreatments would be useful.\nTo add context to how the graft eventually fails, we have carried out the experiment\nsuggested by the reviewer and have added a new figure (Fig. 8) which is an analysis of T cell\nand DC populations upon graft failure. Interestingly, the T cell TCF phenotypes did not\nreverse, but this may be a consequence of the time the graft has survived and a “helpless”\nmemory fate in CD8 T cells, as suggested by the increase in total number of TCF1hi T cells.\nHowever, the Treg and DC altered phenotypes did revert to control levels upon graft failure,\nand may be indicative of why grafts fail.\nIn regard to whether repeated treatments would be useful, we have added a continued\ntreatment group (every 10 days for 9 total injections) to survival data in Fig. 2. Indeed,\nprolonged graft survival was observed with extended 2.5HIP-nanoparticle dosing.\nThe discussion of TCR avidity and sharing raised several questions for this reviewer. First it\nwould be helpful to understand the level of TCR expression on the cell surface may differ\nbetween HIP and HEL- mp treated mice- as an interaction with antigen or altered cytokine\nenvironment could alter expression. Similarly TCR sharing is interesting but with so few\nanimals this data is hard to interpret and it would be helpful to understand the degree of\nsharing at baseline with no therapy administered. It is also unclear how this short period of\ntime would result in such significant changes in repertoire.\nThese are all valid points. We have repeated the d9 experiments and stained for total TCR-\nbeta vs tetramer and added this to Fig. 4. We did not see a statistical difference in the\nsurface levels of TCR-beta on IGRP tet+ or total CD8 T cells (Fig. 4 d). We have also\nexpressed the most abundant TCR from the HIP or HEL treated groups as avatars on 5KC\n2025_12_17\ncells and compared their avidity for the IGRP tetramer at a given level of total TCR\nexpression to address this valid question directly (Fig. 4 I, Fig. S4g).\nWe agree that the number of mice is not powered well for assessing TCR levels of sharing\nor public TCRs. We have therefore focused the interpretation of the results on the\ndifferences observed in apparent avidity and the single expanded clone, and less so on the\ndifferences in repertoire, while stating there are limited n. We have changed the section\ntitle to only mention the apparent avidity observed.\nMinor issue there is a missing description of figure 2k\nThis description has been added.\nReviewer #3 (Remarks to the Author):\nThe manuscript “Antigen-specific immunotherapy with a CD4 neoepitope restrains CD8 T\ncell differentiation in islet grafts” explores the mechanisms through which tolerance is\ninduced by PLGA NPs delivering a CD4 neopeptide for treatment of T1D through islet\ntransplant. The authors demonstrate that NP administration inhibits the transition from\nstem-like-to-effector T cells in islet grafts, but not islet-draining LNs. Systemically-\ndelivered NPs alter the CD8 T cell repertoire and arrest CD8 effector differentiation in\ngrafts, a process dependent on IL-10 production by regulatory cells and DC subsets. These\nfindings expand on mechanisms through which biomaterials can drive infectious tolerance\n– helping to close persistent gap in our understanding of tolerance-inducing strategies to\ntackle autoimmunity.\nThis work that builds on previously published studies by this group represents a significant\nadvance in that work, and our mechanistic understanding of how engineered strategies to\ninduce antigen-specific tolerance impact autoimmunity. Experiments are controlled,\nmouse models are well-utilized, figures are clear, data interpretation is aligned with the\nresults, and the manuscript is well-written. Only minor comments are noted.\n1. In the introduction, the authors introduce the role of different immune cell types (e.g.,\nAPC, CD4 and CD8). Could the authors expand on the crosstalk mechanisms between the\ncells explored in this manuscript should be provided? Alternatively, or in addition, a\ngraphical abstract or cartoon illustrating the mechanistic relationship between immune\npathways elucidated in this work could also be helpful.\nThis is a good suggestion for adding context to interpreting and summarizing the study. We\nhave expanded on the crosstalk mechanism in the Introduction and have included in the\nResults section a graphical abstract, Fig. 7. There is an explanation in the legend to help\nillustrate the mechanistic relationship between the cell types, with and without 2.5HIP\ntolerance induction.\n2025_12_17\n2. The transition from stem-like to effectors is an interesting way to characterize what is\ngoing on in the graft. Are tetramer negative T cells also going through this transition? In\nother words, is the phenomenon ag-specific?\nThere are tetramer- (IGRP-neg and 2.5HIP-neg) negative T cells that are TCF1lo cells at d9,\nbut these are at a lower number than the tetramer-positive cells. We theorize the majority\nof TCFlo T cells are islet-specific in the graft and that the TCFhi memory cells are composed\nof both islet-specific memory cells and some other non-islet-specific T cells attracted by\nchemokines but not undergoing differentiation due to lack of productive, antigen\nencounter.\n3. Throughout the manuscript, can the authors expand rationale for examining specific\nmarkers and their functional significance in the context of islet transplant and tolerance\n(e.g., Ly6A, TIM3)?\nTIM3 is used as a marker of antigen-encounter and terminal differentiation of both 2.5HIP\nand IGRP tetramer+ T cells, often used in studies of exhaustion. This rationale has been\nexpanded upon in Figs. 1 and 2.\nLy6A is one of many interferon signature genes that came up in our scRNAseq experiment\nas being decreased in IGRP CD8 T cells upon tolerance induction and was highly\nexpressed in many cells including the functional, ISG/cytotoxic cluster. Ly6A is one of the\nfew ISG for which there is a reliable antibody for validation of the scRNAseq observation by\nflow. This explanation has been extended in Figs. 3 and 6.\n4. One outstanding question for ASIs is whether or not disease-relevant antigens are\nrequired to drive potent efficacy. Here, studies elucidate mechanisms through which\nbiomaterials can drive infectious tolerance – helping to close persistent gap in our\nunderstanding. The authors should further highlight the implications their findings in the\ncontext of infectious tolerance, which help alleviate some concern over whether ASI will\ntranslate to the clinic.\nWe have elaborated on this point further with regard to the implications for human therapy\nin the Discussion to emphasize that tolerance induction to a dominant CD4 epitope may\nbe sufficient to induce infectious tolerance in cytotoxic CD8 T cells of distinct islet\nspecificities.\n5. Work in this manuscript focuses on tolerizing against transplanted islet antigen, which is\nelegant, but are similar DC and CD8-related mechanisms occurring in pancreas in the\n2025_12_17\nabsence of transplant? If this mechanism applies to transplanted islets only, do the\nauthors speculate they are similar during syn- and allogeneic responses?\nWe posit this form of infectious tolerance also occurs in the pancreatic environment during\ntreatment of primary disease progression. Prediabetic mice treated with 2.5HIP\nnanoparticles had reduced CD40 expression on migratory sDC1s from the pancreatic\nlymph node (Fig. S6d); we predict the same mechanism likely applies to induction of\ntolerance in the prediabetic phase of spontaneously developing disease.\nWe have elaborated upon this observation in the Discussion to address how this\nmechanism may also function in the pancreas and its draining lymph node as well as\nincluding the speculation that this may also suppress alloreactive cells.\n6. Other: The x axis of Fig. 1B need a label. PLGA is misspelled (PGLA) in the legend of Fig.\n2. Histograms in Fig. 4C look clipped (maybe it is the just biexponential axis?). Please\nprovide gating strategies for staining in Fig. 4.\nThe x axis in Fig. 1B has been labeled. The spelling has been corrected for PLGA in the Fig. 2\nlegend. Fig. 4C is pre-gated on IGRP tetramer-positive cells, and this has been clarified in\nthe figure legend. Gating strategies for Fig. 4 are the same as for Fig. 1; this has also been\nclarified in the figure legend.\n2025_12_17\nResponse to reviewers\nReviewer #1 (Remarks to the Author):\nThank you for the responses.\n1: In the revised figure 2 ( summary panels 2J, 2 l and associated flow plots showing\nIFNgamma/TNFa ) population 3 is wrongly labelled as Tim3 hi TCFhi. It should read Tim3hi\nTCFlo.\nThis has been corrected in the figure.\n2: The representative Flow plots showing Tox expression in IGRP cells in the rebuttal should\nbe added as supplementary data.\nThese representative flow plots have been added and referenced in the Results section.\nWe have also included additional responses and manuscript edits in regard to the\ncomments from the first round of reviews as follows:\nReviewer #1 (Remarks to the Author):\nThe work presented in the current manuscript is essentially an extension of a previous\nstudy by the same group of authors (Miller Haskins Baker Delisio etc) (PMID: 35007324)\npublished in Diabetes in 2022. (reference # 51 in the current paper).\nTheir previous work has already shown:\n1: 2.5 HIP NPs can protect syngeneic islet grafts\nIn our previous study (Jamison, DiLisio et al, Diabetes 2022; ref #56 in current revision),\nwe demonstrated that treatment with 2.5HIP nanoparticles could transiently protect\nsyngeneic islet grafts from autoimmune rejection. In the present manuscript revision,\nwe describe new data regarding this finding by showing that continued post-transplant\nadministration of 2.5HIP nanoparticles (nine total treatments) markedly prolongs graft\nsurvival. This result directly addresses the durability of tolerance induction and\ndemonstrates that graft protection is not intrinsically short-lived but can be extended\nwith sustained antigen-specific therapy. This distinction has now been explicitly stated\nand referenced in the Results section.\n2: Reduced e]ector function of both 2.5HIP specific CD4 and IGRP specific CD8 T cells\n(Same is reproduced in this paper- with added markers TCF1, TIM3 and PD1 further\ndefining stem like and e]ector like subsets)\nThe earlier study also reported reduced e]ector function of both 2.5HIP-specific CD4 T\ncells and IGRP-specific CD8 T cells, based primarily on cytokine production assessed\nat the time of graft failure. However, that work did not define the underlying nature of the\ndi]erentiation defect in these T cells. In the current study, we perform direct\ncomparisons between treated and control grafts at matched time points and provide\nsubstantially deeper phenotypic and transcriptional resolution. Using additional\ndi]erentiation markers (TCF1, TIM-3, PD-1), single-cell RNA sequencing, and TCR\nsequencing, we demonstrate that tolerance induction limits terminal e]ector\ndi]erentiation, leads to progressively increased dysfunction as CD8 T cells\ndi]erentiate, suppresses interferon-stimulated and cytolytic gene programs, and\nreduces apparent TCR avidity within graft-infiltrating IGRP-specific CD8 T cells. These\nfindings reveal a previously unrecognized reprogramming of CD8 T cell fate rather than a\nsimple reduction in e]ector output. We compare these findings to our previous finding\n(Jamison, DiLisio et al 2022) both in the Introduction and in the Results.\n3: 2.5 HIP NPs result in expansion of 2.5 HIP specific CD4 T cells in spleen. The previous\nstudy reported no change in the proportion of HIP CD4s or IGRP CD8’s in the graft\n(Di]erent from what is presented here)\nOur prior publication reported no significant change in the proportion of antigen-\nspecific CD4 or CD8 T cells within grafts. However, those analyses were performed\nexclusively at the time of graft failure, and the CD8 tetramer staining protocol used at\nthat time likely underestimated total IGRP-specific populations due to excessive\nproteolysis during graft digestion. In the present study, we optimized graft processing\nand tetramer staining and directly compared treated and control grafts at equivalent\npost-transplant time points. The di]erences in timepoints of comparison have been\nclarified in the Results.\n4: 2.5 HIP NPs result in increased Ag-specific Foxp3 Tregs in the graft (replicated here),\nas well as an increase in Ag-specific anergic CD4 T cells (CD73+ FR4+ CD44+)\n(Reproduced in the current paper as IL-10 producing cells using an IL-10 reporter\nmouse- which in e]ect are TR1 like anergic CD4 T cells described in their previous\nstudy)\nAlthough the prior study (Diabetes, 2022) reported increased absolute numbers of\n2.5HIP-specific FOXP3⁺ regulatory T cells in the spleen, we were unable in that study to\ndetect statistically significant enrichment of FOXP3⁺ antigen-specific CD4 T cells within\ngrafts at the time points examined, only trends. As a result, the relative contribution of\nFOXP3⁺ Tregs to local graft protection remained unresolved, although we did reference\nthe trend observed in our previous publication within the Results of the current study.\nWith respect to regulatory CD4 T cells, our prior study demonstrated a marked\nexpansion of antigen-specific anergic CD4 T cells (CD73⁺FR4⁺CD44⁺) within islet\ngrafts following 2.5HIP-NP treatment. These cells were phenotypically consistent with a\ntolerant, hyporesponsive state; however, we did not assess IL-10 production, nor did we\npropose or test whether these anergic cells actively contributed to the suppression of\nautoreactive T cell responses, including IGRP-specific CD8 T cells. This has been added\nto the Results section of the present study.\nAlso in the present study, we extend these findings by demonstrating statistically\nsignificant expansion of both FOXP3⁺ and Tr1-like antigen-specific CD4 T cell\npopulations within islet grafts using IL-10 reporter mice. Notably, Tr1-like cells represent\nthe dominant regulatory population induced by treatment and are the primary source of\nIL-10 in the graft. We further show that IL-10 blockade reverses suppression of dendritic\ncell licensing and restores pathogenic CD8 T cell di]erentiation, establishing a\nfunctional, IL-10-dependent mechanism of dominant suppression and providing a new\nand important addition to previous findings. We now explicitly state that the anergic\nCD4 T cells identified in our prior work may represent precursors to the Tr1-like\npopulation defined here, a mechanistic link that was neither proposed nor\nexperimentally tested in the previous study.\nIn summary, previous results have been reproduced and polished with addition of sc-\nRNA seq, IL-10 blockade and TCR seq data to provide a mechanistic explanation for the\nobserved transient graft protection.\nWhile this study builds upon our prior work, it provides substantial new insight by\ndemonstrating prolonged graft survival with continued treatment, defining the\ndi]erentiation trajectory and dysfunction of dominantly tolerized IGRP-specific CD8 T\ncells at high resolution, and establishing an IL-10-dependent mechanism by which Tr1-\nlike CD4 T cells suppress dendritic cell licensing and restrain pathogenic CD8 T cell\nfates. We believe these findings go well beyond replication and polishing of earlier\nresults, but instead provide a mechanistic explanation for the previously observed graft\nprotection and dominant suppression of antigen-specific CD8 T cells.\nReviewer #2 (Remarks to the Author):\nThe authors have thoroughly addressed the concerns of this reviewer and the manuscript is\nmuch improved with the additional data and figures.\nReviewer #3 (Remarks to the Author):\nThe authors have made thoughtful revisions to the manuscript that has been strengthened\nby the addition of both data and discussion. I would like to recommend acceptance for\npublication."}]

Decision letter