Extended Data Figure 5: Disease reversal by pMHC–NPs is driven by the T_R1 cytokines IL-21, IL-10 and TGF-β and involves several downstream cellular targets.

a, Changes in blood glucose levels in diabetic NOD mice (>11 mM) treated with IGRP_4–22/IA^g7–NPs and blocking anti-IL-10, anti-IFNγ or anti-TGF-β mAbs or anti-HRPN rat-IgG (n = 4–6 per group). b, c, Percentages of tetramer⁺CD4⁺ T cells in the spleens (b), and proliferation of CFSE-labelled 8.3-CD8⁺ T cells in the PLNs verus MLN of the mice from Fig. 3a at the end of follow up (c). d, Changes in blood glucose in hyperglycaemic NOD, NOD Il10^−/− and NOD Ifng^−/− mice (n = 3−6 per group) in response to 2.5mi/IA^g7–NPs. e, f, Percentages of tetramer⁺CD4⁺ T cells in the spleens (e), and proliferation of CFSE-labelled 8.3-CD8⁺ T cells in the PLNs versus MLN of the mice from d at the end of follow up (f). g, EAE scores of mice treated with pMHC–NPs and rat-IgG or blocking mAbs (n = 4 per group). h, LFB/H&E staining of the cerebellum of HLA-DR4-IE-transgenic C57BL/6 IAb^null mice from g, highlighting differences in inflammation and demyelination in mice treated with hPLP_175–192/DR4-IE–NPs and rat-IgG versus blocking anti-IL-10, anti-TGF-β or anti-IL-21R mAbs. i, Changes in the average body weights of HLA-DR4-IE-transgenic C57BL/6 IAb^null mice from g. j, Percentage of tetramer⁺CD4⁺ T cells in spleen, blood and inguinal LNs of mice from g (n = 4 per group). k, l, Changes in the average EAE scores (k) and body weights (l) of C57BL/6 Il27r^−/− mice immunized with pMOG_35–55 and treated with pMOG_38–49/IA^b–NPs or uncoated nanoparticles starting on the day when mice reached a score of 1.5 (to synchronize the groups for disease activity) (n = 7 and 4, respectively). m, Representative IBA1 and LFB/H&E stainings of the cerebellum and the corresponding relative rank scores of mice from k (n = 3 and 4, respectively). n, Percentage of tetramer⁺CD4⁺ T cells in spleen, blood, inguinal LNs and bone marrow of mice from k (left), and representative CD49b and LAG-3 staining profiles of tetramer⁺ versus tetramer⁻ cells (right). o, Percentage of B220⁺ cells in the PLNs or MLNs of 2.5mi/IA^g7–NP- or HEL_14-22/IA^g7–NP-treated mice (n = 4 per group). p, Correlation between the percentages of PLN and splenic B220⁺ cells and 2.5mi/IA^g7 tetramer⁺CD4⁺ T cells in additional cohorts of mice treated with 2.5mi/IA^g7–NPs, over a range of total pMHC dose (0.75–25 μg of total pMHC) (n = 24–28). q, Left, in vitro proliferation of CFSE-labelled BDC2.5 CD4⁺ T cells against 2.5mi or GPI_282–292 peptide-pulsed B cells purified from the PLNs or MLNs of untreated NOD mice or mice treated with 2.5mi/IA^g7–NPs (n = 5–6 per group). Right, representative CFSE dilution profiles. Briefly, profiles show the extent of CFSE dilution in CFSE-labelled BDC2.5 CD4⁺ T cells cultured in the presence of 2.5mi or GPI_282–292 peptide-pulsed B cells purified from the PLNs or MLNs of untreated or 2.5mi/IA^g7–NP-treated NOD mice. r, PLN-derived B cells (10⁵) from 2.5mi/IA^g7–NP-treated mice secrete IL-10 ex vivo in response to LPS (1 μg ml⁻¹). Data correspond to 6 pMHC-treated and 5 untreated NOD mice. s, t, Changes in the percentages of 2.5mi (PKH26-labelled) compared with GPI_282–292 peptide-pulsed (CFSE-labelled) B cells (s) or DCs (t) 7 days after transfer (at 1:1 ratio) into untreated or 2.5mi/IA^g7–NP-treated NOD mice. Histograms show averaged ratios for each cell type and condition (n = 3–4 mice per cell type and condition). u, Percentages of CD5⁺CD1d^hieGFP⁺B220⁺ cells in mice treated as in Fig. 3b plus blocking Abs (n = 4 each). v, LPS-stimulated PLN B cells from NOD mice treated with 10 doses of 2.5mi/IA^g7–NPs suppress the proliferation of CFSE-labelled BDC2.5 CD4⁺ T cells by 2.5mi peptide-pulsed DCs in vitro, as compared to LPS-stimulated PLN B cells from untreated controls. x, Percentage of CD19⁺CD3⁻ cells in blood before and after 3 doses of 250 μg of anti-CD20 mAb (n = 4). y, 2.5mi/IA^g7–NP-induced upregulation of IL-21 and IL-10 mRNA in memory eGFP⁻ BDC2.5 CD4⁺ T cells from BDC2.5-TCR-transgenic NOD Foxp3-eGFP donors in NOD Thy1^a hosts (n = 5). P values were calculated by Pearson correlation, Mann–Whitney U-test or two-way ANOVA. Data are averages ± s.e.m.