Long-term tolerance of islet allografts in nonhuman primates induced by apoptotic donor leukocytes

Abstract

Immune tolerance to allografts has been pursued for decades as an important goal in transplantation. Administration of apoptotic donor splenocytes effectively induces antigen-specific tolerance to allografts in murine studies. Here we show that two peritransplant infusions of apoptotic donor leukocytes under short-term immunotherapy with antagonistic anti-CD40 antibody 2C10R4, rapamycin, soluble tumor necrosis factor receptor and anti-interleukin 6 receptor antibody induce long-term (≥1 year) tolerance to islet allografts in 5 of 5 nonsensitized, MHC class I-disparate, and one MHC class II DRB allele-matched rhesus macaques. Tolerance in our preclinical model is associated with a regulatory network, involving antigen-specific Tr1 cells exhibiting a distinct transcriptome and indirect specificity for matched MHC class II and mismatched class I peptides. Apoptotic donor leukocyte infusions warrant continued investigation as a cellular, nonchimeric and translatable method for inducing antigen-specific tolerance in transplantation.

Fig. 1

Apoptotic donor leukocytes (ADLs) induce abortive expansion of donor-specific T and B cells. a Experimental schema describing the ADL administration protocol and the transient immunosuppression used in this Cohort A of major histocompatibility complex class I-disparate, one Mamu-DR matched, nondiabetic, nontransplanted monkeys (n = 3). b Relative frequencies of myeloid-derived suppressor cells, c Ki67 expression in peripheral blood lymphocytes (PBLs) of Cohort A given ADLs IV on days −7 and 0. Fluorescence-activated cell sorting analysis demonstrates peak proliferation at day −5 or day −4 followed by subsequent contraction of proliferating cells—CD4⁺ T cells (Ki67⁺CD4⁺ T cells; open circle), CD8⁺ T cells (Ki67⁺CD8⁺ T cells; open square), and CD20⁺ B cells (Ki67⁺CD20⁺ B cells; open triangle). d Frequency of interferon-gamma- and interleukin-10-positive CD4⁺ T cells collected at the indicated time points from ADL-infused Cohort A (n = 3) and restimulated in vitro with donor antigen. e–g Fold-change in proliferation of carboxyfluorescein diacetate succinimidyl ester-labeled recipient PBLs in response to irradiated donor (donor-specific) and third-party (3rd party) PBLs in a 6-day mixed lymphocyte reaction relative to proliferation of PBLs in naive animals (Pre-ADLs) and at the indicated time points relative to intended transplant on day 0. Data represents Mean ± SD of 3 monkeys—e proliferating CD4⁺ T cells, f proliferating CD8⁺ T cells, and g proliferating CD20⁺ B cells. h Frequency of Mamu A*0427-41 Mamu DR03a tetramer⁺ circulating CD4⁺ T cells collected from ADL-treated Cohort A. i Line graphs represent the mean ± SD of 3 monkeys (n = 3). j Clonality of the T cells determined by high-throughput TCRβ sequencing at the individual time points. The frequency of clonal expansion was calculated by dividing the frequency of the clone at individual time points over the average frequency of all the identified mapped T cell receptor (TCR) clones. The frequencies of TCR clones at various time points are presented. Statistical analysis using paired t test was used to analyze whether a significant reduction was observed after ADL infusions when compared to naive animals. *P < 0.05; **P  < 0.005; ***P < 0.0005; unpaired t test with Welch’s correction. Source data are provided as a Source Data file

Fig. 2

Apoptotic donor leukocyte (ADL) infusions facilitate stable tolerance of islet allografts. a Immunotherapy protocols including treatment products, dosages, routes, and timelines in Cohort B and C monkeys. sTNFR soluble tumor necrosis factor receptor (etanercept), anti-IL-6R anti-IL-6 receptor (tocilizumab), IE islet equivalent. b Kaplan–Meir estimates of rejection-free islet allograft survival confirmed by histology show superior sustained allograft survival in Cohort C (ADLs; n = 5; blue solid) compared with Cohort B (no ADLs; n = 7; red dashed; P = 0.021, Mantel–Cox). c–j Example of stable tolerance to islet allograft following peritransplant intravenous (IV) infusions of ADLs under the cover of transient immunosuppression (Monkey #13EP5; Cohort C). c Preprandial and postprandial BG (lines) and daily insulin (bars). Restoration of normoglycemia after intraportal transplant of 7-day-cultured islets (5547 IE kg⁻¹ by DNA). Maintenance of normoglycemia despite discontinuation of insulin and immunosuppression at day +21 posttransplant. d Glycated hemoglobin (HbA1c). Restoration of near-normal HbA1c levels throughout the 1-year follow-up. e Weight. Continued weight gain posttransplant, indicating that posttransplant euglycemia is not due to a malabsorptive state. f C-peptide. Positive and stable C-peptide levels (fasted, random, and mixed meal-stimulated) during the 1-year follow-up. g BG and Kg levels. BG before and after IV infusion of 0.5 g glucose kg⁻¹ (IV glucose tolerance test) and Kg levels before and after diabetes induction and 28, 91, 153, and 271 days posttransplant. Normal Kg levels posttransplant. h Acute C-peptide response to IV glucose (0.5 g kg⁻¹). Robust C-peptide increases to IV glucose during follow-up. i Intact, non-infiltrated, insulin-stained donor islet in recipient liver at 1 year posttransplant (a representative example of five animals studied, anti-insulin; ×40). j Native, insulin negative islet in pancreas at necropsy at 1 year posttransplant (a representative example of five animals studied, anti-insulin; ×40). Source data are provided as a Source Data file

Fig. 3

Apoptotic donor leukocyte infusions suppress effector cell expansion and function. a Percentage of CD4⁺ T effector memory (TEM) cells in the peripheral blood lymphocytes (PBLs) measured longitudinally before and at 3, 6, and 12 months posttransplant in recipients from Cohorts B (n = 7; red) and C (n = 5; blue); see also Supplementary Fig. 7a, b. b Percentage of CD4⁺ TEM cells in PBLs, liver mononuclear cells (LMNCs), and lymph nodes (LNs) at termination in recipients from Cohorts B (n = 3–7, red) and C (n = 2–5, blue). c Fold change in proliferation (compared to pretransplant levels; naive) of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4⁺ T cells in Cohorts B and C in response to irradiated donor PBLs (donor-specific) and third-party PBLs (3rd Party; Supplementary Fig. 7c) before and at the indicated intervals posttransplant in a 6-day mixed lymphocyte reaction (MLR). d Percentage of circulating CD8⁺ TEM cells in PBLs from Cohorts B (n = 7, red) and C (n = 5, blue); see also Supplementary Fig. 8d, e. e Percentage of CD8⁺ TEM cells in PBLs, LMNCs, and LNs at termination in recipients from Cohorts B (n = 3–7, red) and C (n = 3–5, blue). f Fold change in proliferation (compared to pretransplant levels; naive) of CFSE-labeled CD8⁺ T cells in Cohorts B and C in response to irradiated donor PBLs (donor-specific) and third-party PBL (3rd Party; Supplementary Fig. 7f) before and at the indicated intervals posttransplant in a 6-day MLR. Percentage of circulating g follicular helper cells (Tfh), h PD-1⁺CD4⁺ T cells, and i PD-1⁺CD8⁺ T cells. j Percentage of circulating CD20⁺ B cells (see also Supplementary Fig. 7j, k). k–l Recipient IgG antibody levels against their respective donors (donor-specific antibody) expressed in mean fluorescence intensity (MFI) in recipients from k Cohorts B (n = 7, red) and l C (n = 5, blue) at the indicated time points. Unpaired t test (b, e) and non-parametric Mann–Whitney U test followed by post hoc analysis with the Holm–Sidak method for comparisons between two groups. (all other panels). *P < 0.05 and **P < 0.01. Source data are provided as a Source Data file

Fig. 4

Apoptotic donor leukocyte infusions increase the frequency and function of regulatory cells. Relative numbers of circulating cells in Cohort B (n = 7, red) and Cohort C (n = 5, blue) monkeys. a Circulating Tr1 cells, b Tr1 cells in peripheral blood lymphocytes (PBLs), liver mononuclear cells (LMNCs), and lymph nodes (LNs) at the time of termination, c natural suppressor cells, circulating Treg (d), Breg and B10 cells (e, g) and in PBLs, LMNCs and LNs at the time of termination (f, h). i Circulating myeloid-derived suppressor cells (MDSCs) and j MDSCs in PBLs, LMNCs and LNs at termination. *P < 0.05 and **P < 0.01, unpaired t test (b, f, h, j) and non-parametric Mann–Whitney U test followed by post hoc analysis with the Holm–Sidak method for comparisons between two groups (all other panels). k Depletion of Tr1, Treg, and Breg cells in PBLs of Cohort C (n = 3) collected at 12 months posttransplant restored donor-specific proliferation of T and B cells in carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reaction. l Passive transfer of flow-sorted Tr1 cells from Cohort C recipients (n = 3) at 12 months posttransplant abrogated the donor-specific proliferation of T and B cells in naive PBLs with no discernible effect on third-party responses. m Tr1 cells in a contact-independent manner regulated donor-specific immune responses and n addition of neutralizing anti-IL-10 antibody effectively abrogated the suppression of donor-specific proliferation of T and B cells by sorted Tr1 cells; bars represent the mean ± SD from three Cohort C monkeys. *P < 0.05 and **P < 0.01; unpaired t test with Welch’s correction. Heat map showing the z-score gene expression of o immune signaling intermediates and q metabolic pathways in Cohorts B and C monkeys. Scatter plots of transcription levels of p XBP1, SUMO2, and SH2D2 in PBL (at termination) and the relative expression profile of r NDUFS4 and NDUFS5 in PBLs (at termination) in Cohort B and C recipients. Heat map shows the differentially expressed genes with adjusted P value <0.05 between the Cohort B and C monkeys. s RNA silencing of SH2D2 in Tr1 cell incapacitate its suppressive capacity. Fold change in donor-specific proliferation of T and B cells without Tr1 cells, Tr1cells plus vehicle, and Tr1 cells treated with small interfering RNA targeting SH2D2 transcription molecules compared to donor-treated recipient PBLs only. Source data are provided as a Source Data file

Fig. 5

Graft survival and immune mechanisms in fully mismatched and sensitized monkeys. Preprandial and postprandial BG (lines) and daily insulin (bars) in a 15FP03 and b 15FP02. Fully major histocompatibility complex (MHC) mismatched monkeys (Cohort D; n = 3) poorly control T cell response and failed to sustain expanded regulatory T cells (c–f). Percentage of c CD4⁺ and CD8⁺ T effector memory (TEM) cells and d Treg cells and Tr1 cells. Fold change in proliferation (compared to pretransplant levels; naive) of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled e CD4⁺ T cells and f CD8⁺ T cells in response to donor peripheral blood lymphocytes (PBLs) (donor-specific (DS)) and third-party PBL (3P) at a given time point. g Donor-specific suppression of T and B cells proliferation by Tr1 cells. Relative number of proliferating T and B cells when flow-sorted Tr1 cells from fully MHC-mismatched monkeys (Cohort D; n = 2) at the time of sacrifice (sac) added back to donor-stimulated naive recipient PBLs or depleted from PBLs obtained from the time of sacrifice. h–k Preprandial and postprandial BG (lines) and daily insulin (bars) in four sensitized islet allograft monkeys from Cohort E. h 15FP13, i 13EP3, j 14HP21, and k 14HP29. Sensitized islet allograft monkeys (Cohort E; n = 4) demonstrated expanded circulatory frequency of l CD4⁺ and CD8⁺ TEM cells. m Poor expansion of Treg cells and Tr1 cells. n–p Fold-change proliferation (compared to pretransplant levels; naive) of CFSE-labeled n CD4⁺ T cells and o CD8⁺ T cells in response to donor PBLs (DS) and third-party PBL (3P) at a given time point. p Comparative CD4⁺ and CD8⁺ T cell proliferative responses in Cohorts E (n = 4) and C (n = 4) recipients with and without apoptotic donor leukocytes. Data bars represent the mean ± SD. Unpaired t test with Welch’s correction. *P < 0.05 and **P < 0.01. Source data are provided as a Source Data file

Fig. 6

Tracking of antigen-specific CD4⁺ T cells. Tracking of non-regulatory CD4⁺ T cells, Tr1, and Treg cells with indirect specificity for self (shared) major histocompatibility complex (MHC)-II, mismatched donor MHC-II, and mismatched donor MHC-I peptides among Cohort B (n = 5), C (n = 3 or 4), and D (n = 3) monkeys. a–c At baseline, the percentage of Tr1 cells within tetramers + CD4⁺ T cells with indirect specificities for a self (shared) MHC II, b mismatched donor MHC-II and c mismatched donor MHC-I in Cohorts B–D (Supplementary Fig. 14, Online Methods). d–f Compared with baseline, fold change in frequency of d non-regulatory CD4⁺ T cells, e Treg cells, and f Tr1 cells with indirect specificity for self (shared) MHC-II peptide among Cohorts B–D. g–i No substantial fold changes in tetramer⁺ g non-regulatory CD4⁺ T cells or regulatory h Treg cells and i Tr1 cells frequency specific for mismatched donor MHC-II peptide in Cohort D. j–l Compared with baseline, fold-change frequency of j non-regulatory CD4⁺ T cells, k Treg cells, and l Tr1 cells with indirect specificity for mismatched donor MHC-I peptide among cohorts B–D. Source data are provided as a Source Data file