B-lymphocytes have long been recognized for their critical contributions to adaptive immunity, providing defense against pathogens through cognate antigen presentation to T cells and antibody production. More recently appreciated is that B cells are also integral in securing self-tolerance; this has led to interest in their therapeutic application to down-regulate unwanted immune responses such as transplant rejection. In this study, we found that PMA and ionomycin-activated mouse B cells acquire regulatory properties following stimulation through Toll-like receptor (TLR)4/TLR9 receptors (Bregs-TLR). Bregs-TLR efficiently inhibited T cell proliferation in vitro and prevented allograft rejection. Unlike most reported Breg activities, the inhibition of alloimmune responses by Bregs-TLR relied on the expression of TGF-βand not IL-10. In vivo, Bregs-TLR interrupted donor-specific T cell expansion and induced regulatory T cells in a TGF-β dependent manner. RNA-seq analyses corroborated the involvement of TGF-β pathways in Breg-TLR function, identified potential gene pathways implicated in preventing graft rejection, and suggested new targets to foster Breg regulation.
Kang Mi Lee, Qiang Fu, Guoli Huai, Kevin Deng, Ji Lei, Lisa Kojima, Divyansh Agarwal, Peter Van Galen, Shoko Kimura, Naoki Tanimine, Laura Washburn, Heidi Yeh, Ali Naji, Charles G. Rickert, Christian LeGuern, James F. Markmann
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality after transplant. Despite GVHD prophylaxis, 30-70% of patients develop GVHD resulting in susceptibility to infections, relapse and secondary malignancies. Regulatory T-cells (Tregs) have shown efficacy in preventing GVHD, but variably suppressive at high doses. To enhance in vivo suppressor function, murine Treg were transduced to express an anti-human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated hCD19 transgenic recipients for allo-HSCT. As compared to recipients receiving controlled transduced Tregs, those receiving hCAR19 Tregs had a significant decrease in acute GVHD lethality. GVHD amelioration was accomplished with not only maintenance but potentiation of the graft-versus tumor (GVT) response, as recipient hCD19 B-cells and murine hCD19TBL12luc lymphoma cells were both cleared by allogeneic hCAR19 Tregs. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19- murine TBL12luc cells in vitro in a perforin-dependent, granzyme B-independent manner. Importantly, cyclophosphamide treated hCD19 transgenic mice given hCAR19 cytotoxic T-lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity, whereas hCAR19 Tregs avoided this severe complication. In conclusion, CAR19 Tregs are a novel and effective strategy to suppress GVHD without loss of GVT responses.
Sara Bolivar-Wagers, Michael L. Loschi, Sujeong Jin, Govindarajan Thangavelu, Jemma H. Larson, Cameron S. McDonald-Hyman, Ethan A. Aguilar, Asim Saha, Brent H. Koehn, Mehrdad Hefazi, Mark J. Osborn, Michael C. Jensen, John E. Wagner, Christopher A. Pennell, Bruce R. Blazar.
Regulatory CD4+Foxp3+ T cells (Treg) restrain inflammation and immunity. However, the mechanisms underlying Treg suppressor function in inflamed non-lymphoid tissues remain largely unexplored. Here, we restricted immune responses to non-lymphoid tissues and used intravital microscopy to visualize Treg suppression of rejection by effector T cells (Teff) within inflamed allogeneic islet transplants. Despite their elevated motility, Treg preferentially contact antigen-presenting cells (APCs) over Teff. Interestingly, Treg specifically target APCs that are extensively and simultaneously contacted by Teff. In turn, Treg decrease MHC-II expression on APCs and hinder Teff function. Lastly, we demonstrate that Treg suppressor function within inflamed allografts requires ecto-nucleotidase CD73 activity, which generates the anti-inflammatory adenosine. Consequently, CD73-/- Treg exhibit reduced contacts with APCs within inflamed allografts compared to wt Treg, but not in spleen. Overall, our findings demonstrate that Treg suppress immunity within inflamed grafts through CD73 activity and suggest that Treg-APC direct contacts are central to this process.
Hehua Dai, Andressa Pena, Lynne Bauer, Amanda Williams, Simon c. Watkins, Geoffrey Camirand
Dominant infectious tolerance explains how brief tolerance-inducing therapies result in lifelong tolerance to donor antigens and “linked” third-party antigens, while recipient sensitization and ensuing immunological memory prevent the successful induction of transplant tolerance. In this study, we juxtapose these 2 concepts to test whether mechanisms of dominant infectious tolerance can control a limited repertoire of memory T and B cells. We show that sensitization to a single donor antigen is sufficient to prevent stable transplant tolerance, rendering it unstable. Mechanistic studies revealed that recall antibody responses and memory CD8+ T cell expansion were initially controlled, but memory CD4+Foxp3– T cell (Tconv) responses were not. Remarkably, naive donor-specific Tconvs at tolerance induction also acquired a resistance to tolerance, proliferating and acquiring a phenotype similar to memory Tconvs. This phenomenon of “linked sensitization” underscores the challenges of reprogramming a primed immune response toward tolerance and identifies a potential therapeutic checkpoint for synergizing with costimulation blockade to achieve transplant tolerance in the clinic.
Michael S. Andrade, James S. Young, Jared M. Pollard, Dengping Yin, Maria-Luisa Alegre, Anita S. Chong
Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.
Sydney Felker, Archana Shrestha, Jeff Bailey, Devin M Pillis, Dylan Siniard, Punam Malik
Erythropoietin (EPO) has multiple non-erythropoietic functions including immune modulation, but EPO’s effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using wild type (WT) and conditional EPO receptor (EPOR) knockout mice as recipients. In WT controls, peri-transplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive regulatory T cells (TREG, P < 0.01 for each) vs. CTLA4-Ig alone. Studies performed in conditional EPOR knockout recipients showed that each of these differences required EPOR expression in myeloid cells, but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, anti-inflammatory, regulatory and pro-efferocytosis genes, and downregulated selected pro-inflammatory genes. Together, the data support the conclusion that EPO promotes TREG-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes TREG expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.
Julian K. Horwitz, Sofia Bin, Robert L. Fairchild, Karen S, Keslar, Zhengzi Yi, Weijia Zhang, Vasile I. Pavlov, Yansui Li, Joren C. Madsen, Paolo Cravedi, Peter S. Heeger
Long-term impairment in T cell mediated adaptive immunity is a major clinical obstacle following treatment of blood disorders with Hematopoietic Stem Cell Transplantation (HSCT). Though T cell development in the thymus has been extensively characterized, there are significant gaps in our understanding of pre-thymic processes which influence early T cell potential. We have uncovered a Notch-IL21 signaling axis in Bone Marrow (BM) Common Lymphoid Progenitor (CLP) cells. IL21r expression is driven by Notch activation in CLPs, and in vivo treatment with IL21 induces Notch-dependent CLP proliferation. Taking advantage of this novel signaling axis, we have generated T cell progenitors ex vivo which better repopulate the thymus and peripheral lymphoid organs of mice in an allogeneic transplant model. Importantly, Notch and IL21 activation is equally effective in the priming and expansion of human Cord Blood (CB) cells toward the T cell fate, confirming the translational potential of the combined treatment.
Kilian Sottoriva, Na Yoon Paik, Zachary White, Thilinie Bandara, Lijian Shao, Teruyuki Sano, Kostandin Pajcini
Monocytes play an important role in the regulation of alloimmune responses after heart transplantation (HTx). Recent studies have highlighted the importance of immunometabolism in the differentiation and function of myeloid cells. While the importance of glucose metabolism in monocyte differentiation and function has been reported, a role for fatty acid β-oxidation (FAO) has not been explored. Heterotopic HTx was performed using hearts from Balb/c donor mice implanted into C57Bl/6 recipient mice and treated with etomoxir (eto), an irreversible inhibitor of carnitine palmitoyltransferase 1 (Cpt1), a rate-limiting step of FAO, or vehicle control. FAO inhibition prolonged HTx survival, reduced early T cell infiltration/activation and reduced dendritic cell (DC) and macrophage infiltration to heart allografts of eto-treated recipients. ELISPOT demonstrated eto-treated HTx were less reactive to activated donor antigen presenting cells. FAO inhibition reduced monocyte-to-DC and monocyte-to-macrophage differentiation in vitro and in vivo. FAO inhibition did not alter the survival of heart allografts when transplanted into Ccr2-deficient recipients, suggesting the effects of FAO inhibition were dependent on monocyte mobilization. Finally, we confirmed the importance of FAO on monocyte differentiation in vivo using conditional deletion of Cpt1a. Our findings demonstrate that targeting FAO attenuates alloimmunity after HTx, in part through impairing monocyte differentiation.
Yuehui Zhu, Hao Dun, Li Ye, Yuriko Terada, Leah P. Shriver, Gary J. Patti, Daniel Kreisel, Andrew E. Gelman, Brian W. Wong
Type 1 diabetes is an autoimmune disease characterized by insulin-producing beta-cell destruction. While islet transplantation restores euglycemia and improves patient outcomes, an ideal transplant site remains elusive. Brown adipose tissue (BAT) is a highly vascularized and anti-inflammatory microenvironment. As these tissue features can promote islet graft survival, we hypothesize that islets transplanted into BAT will maintain islet graft and BAT function, while delaying immune-mediated rejection. We performed syngeneic and allogeneic islet transplants into BAT or under the kidney capsule of streptozotocin (STZ)-induced diabetic NOD.Rag and NOD mice to investigate islet graft function, BAT function, metabolism, and immune-mediated rejection. Islet grafts within BAT restored euglycemia similarly to kidney capsule controls. Islets transplanted in BAT maintained expression of islet hormones, transcription factors, and were vascularized. Compared to kidney capsule and euglycemic mock surgery controls, no differences in glucose or insulin tolerance, thermogenic regulation, or energy expenditure were observed with islet grafts in BAT. Immune profiling of BAT revealed enriched anti-inflammatory macrophages and T cells. Compared to kidney capsule, islets transplanted in BAT demonstrated significant delays in autoimmune and allograft rejection, possibly due to increased anti-inflammatory immune populations. Our data support BAT as an alternative islet transplantation site that may improve graft survival.
Jessica D. Kepple, Jessie M. Barra, Martin E. Young, Chad S. Hunter, Hubert M. Tse
Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Several investigators have reported that adaptive NKG2C+ NK cells persistently expand during CMV reactivation. In our study, two cohorts were enrolled to explored the relationships among the NKG2C genotype, NKG2C+ NK cell reconstitution, and CMV infection. Multivariate analysis showed that donor NKG2C gene deletion was an independent prognostic factor for CMV reactivation and refractory CMV reactivation. Furthermore, the quantitative, qualitative reconstitution and anti-CMV function of adaptive NKG2C+ NK cells after transplantation was significantly lower in patients grafted with NKG2Cwt/del donor cells than in those grafted with NKG2Cwt/wt donor cells. The quantitative reconstitution of NKG2C+ NK cells at day 30 after transplantation was significantly lower in patients with treatment-refractory CMV reactivation than in those in the no-CMV-reactivation and CMV-reactivation groups. In humanized CMV-infected mice, we found that compared with those from NKG2Cwt/del donors, adaptive NKG2C+ NK cells from NKG2Cwt/wt donors induced earlier and stronger expansion of NKG2C+ NK cells and earlier and stronger CMV clearance in vivo. In conclusion, donor NKG2C homozygosity contributes to CMV clearance by promoting the quantitative and qualitative reconstruction of adaptive NKG2C+ NK cells after haploidentical allo-HSCT.
Xing-Xing Yu, Qian-Nan Shang, Xue-Fei Liu, Mei He, Xu-Ying Pei, Xiao-Dong Mo, Meng Lv, Ting-Ting Han, Ming-Rui Huo, Xiaosu Zhao, Ying-Jun Chang, Yu Wang, Xiao-Hui Zhang, Lan-Ping Xu, Kai-Yan Liu, Xiangyu Zhao, Xiaojun Huang
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