Islet transplantation is an appropriate treatment for selected individuals with type 1 diabetes mellitus and hypoglycaemia unawareness. The gold standard of maintenance immunosuppression to prevent allograft rejection is calcineurin inhibitor-based therapy; however, side effects warrant exploration of alternative immunosuppression. The co-stimulatory blocker belatacept and the mammalian target of rapamycin inhibitor sirolimus have demonstrated clinical benefit in solid organ transplantation, but efficacy in human islet transplantation is unknown. We conducted a non-randomised, phase 2, multi-centre, open-label clinical study. Participants with type 1 diabetes receiving at least one islet transplant were treated with belatacept/sirolimus (bela/siro) and compared with a contemporaneous cohort treated with tacrolimus/mycophenolate mofetil (tac/MMF). The primary outcome was freedom from hypoglycaemia, with positive C-peptide and HbA1c <53 mmol/mol (7.0

Human Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal lung disease with unknown etiology and lacking efficient treatments. Here, we reported that human urine stem cells (hUSCs) significantly alleviated pulmonary fibrosis via inhibiting macrophage-myofibroblast transition (MMT), which was identified as a pivotal pathological process in IPF, with the strong interaction among infiltrated macrophages, damaged alveolar epithelial cells, and myofibroblasts via single-nucleus RNA sequencing data analysis and co-immunostaining. In addition, hUSCs significantly alleviated pulmonary fibrosis by attenuating alveolar epithelial cell damage, reducing monocyte-derived macrophage infiltration, and suppressing MMT in the bleomycin-induced pulmonary fibrosis mouse model. Furthermore, we demonstrated hUSCs inhibited monocyte recruitment and MMT via paracrine actions in the macrophage-alveolar epithelial cell co-culture system. Mechanistically, DKK1, which was highly secreted by hUSCs and identified by Venn diagram analysis between the luminex assay in supernatants of THP1 treated with hUSC-CM and antibody array of hUSC-CM, might contribute to preventing MMT via suppressing the Wnt/β-catenin signaling pathway in macrophages. In summary, hUSCs exerted multifaceted protective effects against pulmonary fibrosis, at least in part through paracrine mechanisms involving DKK1 and its modulation of Wnt/β-catenin-associated fibrotic responses in MMT. Therefore, hUSCs might provide a potential therapeutic strategy for IPF clinically.

ABSTRACT Aim CD8 + regulatory T cells (Tregs) are cross‐protective across multiple animal models of autoimmunity. Recently, specific peptides from a yeast‐peptide‐major histocompatibility complex library that expanded CD8 + Tregs in murine experimental multiple sclerosis were reported. Whether these peptides also expand CD8 + Tregs and protect against Heymann nephritis (HN), an experimental model of membranous nephropathy is unknown. We aimed to assess the efficacy of peptide vaccination to induce CD8 + Tregs in HN. Methods Lewis rats were immunised with Fx1A/complete Freund's adjuvant to induce HN and received peptide vaccination 1 week before (prevention vaccination) or 1 week after disease induction (treatment vaccination). To understand whether the effect of peptide vaccination was mediated by CD8 + Tregs, we adoptively transferred CD8 + T cells 1 week after peptide vaccination into HN rats. Results Prevention vaccination, but not treatment vaccination, significantly reduced anti‐Fx1A autoantibody levels and serum creatinine. Both prevention and treatment vaccination reduced histological kidney injury. mRNA expression of Helios, the major CD8 + Treg transcription factor, was upregulated in both the spleen and kidney with prevention vaccination and in the kidney with treatment vaccination. Adoptive transfer of CD8 + T cells after peptide vaccination significantly reduced serum creatinine, proteinuria, histological kidney injury, anti‐Fx1A autoantibody levels, germinal centre formation, and mRNA expression of markers of T follicular helper cells (Bcl6, interleukin‐21), T helper 1 cells (interferon‐γ, Tbet) and T helper 17 cells (interleukin‐6, interleukin‐17). Conclusions Peptide vaccination induces CD8 + Tregs that ameliorate induction of experimental membranous nephropathy which may represent a further peripheral regulation of autoimmunity.

Background and hypothesis Recent advances in membranous nephropathy treatment have focused on B cell depletion, which is incompletely effective, potentially due to persistent autoantibody-producing plasma cells or alternative pathways of injury. T cell costimulatory blockade [cytotoxic-T-lymphocyte-associated antigen 4 (CTLA4)-Ig] to prevent T cell-dependent B cell activation and short-course proteasome inhibition (bortezomib) to deplete plasma cells may represent a complementary form of treatment.Methods Lewis rats were immunized with Fx1A and complete Freund's adjuvant to induce experimental membranous nephropathy (Heymann nephritis or HN) and treated with CTLA4-Ig alone or CTLA4-Ig plus a short-course of bortezomib. Serum creatinine, proteinuria, kidney histology, serum anti-Fx1A levels, kidney and spleen messenger RNA expression, and flow cytometry on splenocytes were evaluated at 12 weeks.Results CTLA4-Ig-treated and CTLA4-Ig plus bortezomib-treated rats had significant and similar reductions in serum creatinine and proteinuria, with less histological kidney injury compared with untreated HN rats. Glomerular IgG deposition was reduced in CTLA4-Ig-treated and CTLA4-Ig plus bortezomib-treated rats compared with untreated HN rats but there were no significant differences in serum anti-Fx1A levels. CTLA4-Ig-treated and CTLA4-Ig plus bortezomib-treated rats exhibited significantly reduced T helper (Th)-17 cell cytokines (interleukin-6, -17, -21) and regulatory T cell (Foxp3, TGF-beta) expression in the kidney but not the spleen. Immunohistochemical staining of CD4+ and intracellular STAT3+ cells was reduced in CTLA4-Ig plus bortezomib-treated and CTLA4-Ig-treated compared with untreated HN rats. On flow cytometry, CTLA4-Ig reduced B cells and plasma cells but not T cell subsets.Conclusions CTLA4-Ig ameliorated induction of experimental membranous nephropathy, potentially through suppression of Th17 cells in the kidney, and may represent an effective adjunct treatment in membranous nephropathy. 10.1093/ndt/gfaf030 Video Abstract Watch the video abstract of this contribution at https://academic.oup.com/ndt/pages/author_videos gfaf030Media1 6371561009112

CD4(+)Foxp3(+) regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127(hi) Tregs with memory features were found in lymphoid organs, presented in high proportions within long -surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory -like CD127(hi) Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag(-/-) hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL -7 to the CD127(hi)Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory -like CD127(hi) Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.

Introduction: CD4+Foxp3+regulatory T-cells (Tregs) are essential for islet xenotransplant tolerance. We identified a novel memory-like CD127+/highTreg subset in the spleen of tolerant mice following CTLA4-Fc/MR-1 induction and demonstrated their potent suppressive capacity in an adaptive-transfer mouse model. Our aims were to: (1) Further characterise CD127+/high Tregs. (2) Investigate transcriptional profile of CD4+Foxp3+Treg and non-Foxp3 CD4+ subsets in transplant tolerance. Methods:DEpletion of REGulatory T-cells (DEREG) mice carrying a GFP transgene under a Foxp3 promoter were used as recipients in a porcine NICC xenotransplant model. Cell-subsets were selected with BD Influx Cell Sorter based on expression of CD4, GFP, and CD127 or CD45, CD4 and GFP. Bulk RNA-Seq and flow-cytometry profiled the transcriptomes and phenotypes of Treg subsets (CD127highTreg, CD127-/lowTreg, and all Treg) from the spleen (sp), draining lymph node (DLN) and grafts of tolerant-mice (day-100) compared to non-transplant mice. Quantitative PCR (qPCR) was used to assess expression of Il10/Tgfb1/Ifnγ/Il2/Il7//Il18//Il33/Ctla4 in spleen, ALN, DLN and graft cells of transplant mice (CTLA4-Fc/MR-1-treatment), rejection mice (no treatment) at day-8/100 and non-transplant mice. Imaging mass cytometry (17 antibodies) was used to evaluate the graft-infiltrating immune cells in tolerant-(day-8, 20, 100) and rejection-(day-8, 20) groups. Results: A high proportion of CD127highTregs was observed in tolerant-grafts compared to tolerant-spleens [25.6±3.1% vs 14.8±0.4%]. Ebi3(IL-35), Il-10 and Blimp-1 expression were upregulated in splenic CD127highTregs of day-100 transplant-mice compared to naïve-Tregs. We identified 1740 differentially expressed genes (DEGs)(FDR<0.05) from 15 pairwise-comparisons that distinguished between CD45+CD4-, Foxp3-CD4+T-cells, and Treg subsets with: similar expression patterns between naive and tolerant CD45+CD4- cells; minor differences in Foxp3-CD4+T cells; and notable differences across Treg subsets where transcriptional profile was not uniform. Between different Treg subsets, 9 paired cross-comparisons identified 427 DEGs. In tolerant mice, both sp- and DLN-CD127+Tregs showed an effector/memory Treg profile. Compared to naïve-Treg or CD127-/lowTreg subsets, graft-Treg and CD127highTregs displayed upregulated DEGs including Il7r, Kctd12, Cxcr6, Ctla2a, Anxa1, H2-Ab, Klrk1, Klrg1, Ccl5, Id2, Ccr2, Adam8, Il18r1, Il1rl that have been reported in multiple tissue/tumour Treg subsets with memory features. Additionally, measured by qPCR, increased Il7 expression and decreased Il2 expression in day-100 vs day-8 tolerant-graft suggest activated Tregs may preferentially utilise IL-7 or IL-35 over IL-2 (more broadly acting). Conclusion: Memory-like CD127+/highTregs are critical for maintaining tolerance and may share a transcriptional trajectory with other tissue/tumour Tregs. National Health and Medical Research Council (NHMRC; Grants: GNT1013185 & GNT1125456). JDRF/Australian Research Council (Grant: 4-SRA-2016-265-M-B). Diabetes Australia (Grant: Y16G-HUMI). Westmead Scientific Platforms, Westmead Institute for Medical Research. Department of Animal Care at Westmead Hospital. Cancer Institute New South Wales. Ian Potter Foundation. Ramaciotti Facility for Human Systems Biology. Sydney Cytometry, University of Sydney.

Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell–depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ–/– mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.

Xenotransplantation using porcine donors is rapidly approaching clinical applicability as an alternative therapy for treatment of many end-stage diseases including type 1 diabetes. Porcine neonatal islet cell clusters (NICC) have normalised blood sugar levels for relatively short periods in the preclinical diabetic rhesus model but have met with limited success in the stringent baboon model. Here we report that NICC from genetically modified (GM) pigs deleted for αGal and expressing the human complement regulators CD55 and CD59 can cure diabetes long-term in immunosuppressed baboons, with maximum graft survival exceeding 22 months. Five diabetic baboons were transplanted intraportally with 9,673 – 56,913 islet equivalents (IEQ) per kg recipient weight. Immunosuppression consisted of T cell depletion with an anti-CD2 mAb, tacrolimus for the first 4 months, and maintenance with belatacept and anti-CD154; no anti-inflammatory treatment or cytomegalovirus (CMV) prophylaxis/treatment was given. This protocol was well tolerated, with all recipients maintaining or gaining weight. Recipients became insulin-independent at a mean of 87 ± 43 days post-transplant and remained insulin-independent for 397 ± 174 days. Maximum graft survival was 675 days. Liver biopsies showed functional islets staining for all islet endocrine components, with no evidence of the inflammatory blood-mediated inflammatory reaction (IBMIR) and minimal leukocytic infiltration. The costimulation blockade-based immunosuppressive protocol prevented an anti-pig antibody response in all recipients. In conclusion, we demonstrate that genetic modification of the donor pig enables attenuation of early islet xenograft injury, and in conjunction with judicious immunosuppression provides excellent long-term function and graft survival in the diabetic baboon model.

Introduction: Renal transplantation improves the life expectancy and quality of life of end-stage renal patients. Due to the development of immunosuppressive drugs, the results of short-term renal transplantation have improved significantly, but the long-term survival rate of renal transplantation has hardly changed in the past few decades. Interstitial fibrosis is an important cause of graft loss in chronic allograft renal injury. CD40-CD154 costimulatory pathway block can inhibit T cell activation and prolong the survival of allografts in a variety of transplantation models. In this study, we found that the single use of MR1 (CD154 antibody) can significantly reduce the fibrosis of transplanted kidney. Method: The kidney of BALB/c mice was transplanted into C57BL6/J recipient by orthotopic transplantation. The contralateral kidney of the recipient was removed 1-3 days after transplantation. The MR1 treatment group was intraperitoneally injected with 500ug of MR1 antibody on the day of operation, and the control group was given normal saline at the same time. After 30 days, serum was collected to measure the blood creatinine. The kidney was obtained after cardiac perfusion. PAS staining was used to detect renal injury. The number of T lymphocytes and macrophages and the expression of a-SMA in the transplanted kidney were analyzed by flow cytometry. Sirius red and gomori trichrome staining were used to detect fibrosis. The colocalization of macrophages and a-SMA was detected by multiple immunofluorescence. Results: It was found that serum creatinine from MR1 treatment group was significantly lower than control group. Compared with control group, MR1 treated mice also showed milder renal histological lesions that the loss of brush border of proximal tubules, tubular necrosis, tubular atrophy, protein casting and cell infiltration at the cortical medullary junction were much lower. Sirius red and gomori trichrome staining showed that MR1 treatment group significantly reduced the fibrosis of transplanted kidney. RT-PCR showed that the expressions of collagen deposition related proteins such as ColI and FN mRNA were up-regulated in the control group. Further immunohistochemistry analysis showed that the number of infiltrated macrophages in the MR1 treatment group decreased significantly compared to control group. Through flow cytometry analysis, it was found that the number of macrophages secreting a-SMA in control group were significantly higher than those in MR1 treatment group. The FACS results showed that 92.7%± 2.8% of the infiltrating macrophages were M2 type, and they were all derived from the recipients. Immunofluorescence experiment also confirmed this result. Conclusion: MR1 significantly reduced the fibrosis in kidney graft which may due to less infiltration of M2 macrophages and less a-SMA+ macrophages. National key research and development program 2019YFA0110703. Natural Science Foundation of Hunan Province, China (Grant No.:2021JJ31018).