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

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.

indirect donor specificity (Indirect; i) in one-way ELISPOT-the frequency of circulating IFN-γ secreting T cells is presented as scatter plots. * P < 0.05; one-way Student's t test. (j) ADL infusions significantly suppressed IL-17 protein levels in supernatants of donor-stimulated PBLs collected at intervals posttransplant from Cohort C recipients when compared with IL-17 levels in posttransplant Cohort B MLRs. (k-l) Relative numbers of circulating Tbet + B cells before and at 3, 6, and 12 months posttransplant (k) in PBLs and CD20 + B cells (l) in PBLs, LMNCs, and LNs cells at time of termination in islet allograft recipients from Cohort B (n=4 to 7, red) and Cohort C (n= 3 to 5, blue) (l) Fold-Change in percentage of circulating non-regulatory CD4 + T cells with indirect donor MHC class I specificity among Cohort B (n=3) and C (n=2) monkeys. *P < 0.05 and **P < 0.001, ***P < 0.001, Student's t test (panels l), non-parametric Mann-Whitney U test followed by post-hoc analysis with the Holm-Sidak method (panel m) and non-parametric Wilcoxon Rank Sum test (all other panels). Gating strategy for identification of MDSC. Singlets were gated first to eliminate doublets and dead cells were excluded. Based on CD33 + and CD11b + coexpression, MDSCs were identified in gated CD14 + cells within the Lin -HLA-DRpopulation and with Lindepicting CD3 -CD20 -cells.
Representative FACS profiles from one Cohort B (Red) and one Cohort C (Blue) monkeys are shown. (b) Significant increase in percentage of circulating MDSC among Cohort C monkey compared to Cohort B monkey. Fold-change in MFI of circulating (c) HLA-DR + CD11b + dendritic cells, (d) HLA-DR + CD14 + monocytes and (e) HLA-DR + CD20 + B cells among Cohort B and C monkeys. *P < 0.01, **P < 0.001, non-parametric Mann-Whitney U test followed by post-hoc analysis with the Holm-Sidak method.  B Two sections of pancreas were examined. Islets appeared normal in size to slightly smaller than normal and many islet cells were smaller than normal. A few scattered insulin positive cells were present in the islets and in the exocrine pancreas. 13EP8 B One section of pancreas was examined. Islets appeared normal in size to slightly smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. A few scattered insulin positive cells were present in the islets and in the exocrine pancreas. 14HP26 B One section of pancreas was examined. Islets appeared normal in size to slightly smaller than normal and many islet cells were smaller than normal. Rare scattered insulin positive cells were present in the islets and in the exocrine pancreas. 14HP31 B Three sections of pancreas were examined. Islets appeared normal in size to slightly smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. A few insulin positive cells were present in the islets and in the exocrine pancreas. 14HP34 B One section of pancreas was examined. Islets appeared smaller than normal and many islet cells were smaller than normal. Rare scattered insulin positive cells were present in the islets and in the exocrine pancreas.

Supplementary
15CP3 B Three sections of pancreas were examined. Islets appeared normal in size to slightly smaller than normal. Most islet cells were smaller than normal with reduced cytoplasm. No insulin positive cells were present. 15CP6 B One section of pancreas was examined. Islets appeared normal in size to moderately smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. Rare insulin positive cells were present in the islets and in the exocrine pancreas. 13EP5 C One section of pancreas was examined. Islets appeared normal in size to slightly smaller than normal and many islet cells were smaller than normal. A few scattered insulin positive cells were present in the islets and in the exocrine pancreas. 14HP24 C One section of pancreas was examined. Islets appeared slightly to moderately smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. Rare insulin positive cells were present in the islets or exocrine pancreas. 14HP33 C Two sections of pancreas were examined. Islets appeared normal in size to slightly smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. A few insulin positive cells were present in the islets and in the exocrine pancreas. 15CP1 C One section of pancreas was examined. Islets appeared slightly to moderately smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. Rare insulin positive cells were present in the islets or exocrine pancreas. 15CP4 C One section of pancreas was examined. Islets appeared slightly to moderately smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. Rare insulin positive cells were present in the islets and in the exocrine pancreas. 15FP1 D Three sections of pancreas were examined. Islets appeared moderately smaller than normal with reduced and minimally vacuolated cytoplasm. Rare insulin positive cells were present in the islets and in the exocrine pancreas. 15FP2 D One section of pancreas was examined. Islets appeared moderately smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. Rare insulin positive cells were present in the islets and in the exocrine pancreas. 15FP3 D One section of pancreas was examined. Islets appeared moderately smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. Rare insulin positive cells were present in the islets and in the exocrine pancreas. 13EP3 E One section of pancreas was examined. Islets appeared normal in size to slightly smaller than normal. Many islet cells were smaller than normal with reduced cytoplasm. Only rare insulin positive cells were present in the islets and low numbers of scattered isolated insulin-positive cells were present between exocrine acini. The pancreas was otherwise within normal limits. 14HP21 E Two sections of pancreas were examined. Islets appeared normal in size to slightly smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. A few scattered insulin positive cells were present in the islets and in the exocrine pancreas.
14HP29 E Two sections of pancreas were examined. Islets appeared normal in size to slightly smaller than normal. Many islet cells were smaller than normal with reduced and minimally vacuolated cytoplasm. A few scattered insulin positive cells were present in the islets and in the exocrine pancreas.
15FP13 E Three sections of pancreas were examined. Islets appeared normal in size to slightly smaller than normal. Many islet cells were smaller than normal with reduced cytoplasm. Rare insulin positive cells were present in the interstitium of the exocrine pancreas.