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In clinical organ transplantation, donor and recipient ages may differ substantially. Old donor organs accumulate senescent cells that have the capacity to induce senescence in naïve cells. We hypothesized that the engraftment of old organs may induce senescence in younger recipients, promoting age-related pathologies. When performing isogeneic cardiac transplants between age-mismatched C57BL/6 old donor (18 months) mice and young and middle-aged C57BL/6 (3- or 12- month-old) recipients , we observed augmented frequencies of senescent cells in draining lymph nodes, adipose tissue, livers, and hindlimb muscles 30 days after transplantation. These observations went along with compromised physical performance and impaired spatial learning and memory abilities. Systemic levels of the senescence-associated secretory phenotype factors, including mitochondrial DNA (mt-DNA), were elevated in recipients. Of mechanistic relevance, injections of mt-DNA phenocopied effects of age-mismatched organ transplantation on accelerating aging. Single treatment of old donor animals with senolytics prior to transplantation attenuated mt-DNA release and improved physical capacities in young recipients. Collectively, we show that transplanting older organs induces senescence in transplant recipients, resulting in compromised physical and cognitive capacities. Depleting senescent cells with senolytics, in turn, represents a promising approach to improve outcomes of older organs., (Copyright © 2023 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Mouse kidney allografts are spontaneously accepted in select, fully mismatched donor-recipient strain combinations, like DBA/2J to C57BL/6 (B6), by natural tolerance. We previously showed accepted renal grafts form aggregates containing various immune cells within 2 weeks posttransplant, referred to as regulatory T cell-rich organized lymphoid structures, which are a novel regulatory tertiary lymphoid organ. To characterize the cells within T cell-rich organized lymphoid structures, we performed single-cell RNA sequencing on CD45 + sorted cells from accepted and rejected renal grafts from 1-week to 6-months posttransplant. Analysis of single-cell RNA sequencing data revealed a shifting from a T cell-dominant to a B cell-rich population by 6 months with an increased regulatory B cell signature. Furthermore, B cells were a greater proportion of the early infiltrating cells in accepted vs rejecting grafts. Flow cytometry of B cells at 20 weeks posttransplant revealed T cell, immunoglobulin domain and mucin domain-1 + B cells, potentially implicating a regulatory role in the maintenance of allograft tolerance. Lastly, B cell trajectory analysis revealed intragraft differentiation from precursor B cells to memory B cells in accepted allografts. In summary, we show a shifting T cell- to B cell-rich environment and a differential cellular pattern among accepted vs rejecting kidney allografts, possibly implicating B cells in the maintenance of kidney allograft acceptance., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: I.R. and R.B.C. are consultants of eGenesis. R.B.C. is also a consultant of Sangamo. Other authors declare no competing financial interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antibody-mediated rejection is a major cause of long-term graft loss in kidney transplant patients. T follicular helper (Tfh) cells are crucial for assisting B cell differentiation and are required for an efficient antibody response. Anti-thymocyte globulin (ATG) is a widely used lymphocyte-depleting induction therapy. However, less is known about how ATG affects Tfh cell development and donor-specific antibody (DSA) formation. We observed an increase in circulating Tfh cells at 6 months after kidney transplant in patients who received ATG. Using an NP-OVA immunization model, we found that ATG-treated mice had a higher percentage of Tfh cells, germinal center B cells, and higher titers of antigen-specific antibodies compared to controls. ATG-treated animals had lower levels of IL-2, a known Bcl-6 repressor, but higher levels of IL-21, pSTAT3 and Bcl-6, favoring Tfh differentiation. In a mouse kidney transplant model, ATG-treated recipients showed an increase in Tfh cells, DSA and C4d staining in the allograft. Although ATG was effective in depleting T cells, it favored the expansion of Tfh cells following depletion. Concomitant use of IL-2, tacrolimus, or rapamycin with ATG was essential to control Tfh cell expansion. In summary, ATG depletion favors Tfh expansion, enhancing antibody-mediated response., (© 2022 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO., (© 2022 by The American Society of Hematology.)

Following solid organ transplantation, a substantial proportion of chronic allograft loss is attributed to the formation of donor-specific antibodies (DSAs) and antibody-mediated rejection (AbMR). The frequency and phenotype of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells is altered in the setting of kidney transplantation, particularly in patients who develop AbMR. However, the roles of Tfh and Tfr cells in AbMR after solid organ transplantation is unclear. We developed mouse models to inducibly and potently perturb Tfh and Tfr cells to assess the roles of these cells in the development of DSA and AbMR. We found that Tfh cells are required for both de novo DSA responses as well as augmentation of DSA following presensitization. Using orthotopic allogeneic kidney transplantation models, we found that deletion of Tfh cells at the time of transplantation resulted in less severe transplant rejection. Furthermore, using inducible Tfr cell deletion strategies we found that Tfr cells inhibit de novo DSA formation but only have a minor role in controlling kidney transplant rejection. These studies demonstrate that Tfh cells promote, whereas Tfr cells inhibit, DSA to control rejection after kidney transplantation. Therefore, targeting these cells represent a new therapeutic strategy to prevent and treat AbMR., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The immune system is capable of generating robust antibody responses to foreign antigens during infection and vaccination, while simultaneously limiting antibodies to self-antigens. T follicular regulatory (Tfr) cells are a subset of follicular T cell with specialized roles in regulating humoral immunity. Although Tfr cells have been studied for the past 10 years, their roles have remained elusive. In this review we discuss the current understanding of Tfr cell functions in autoimmunity and how Tfr cells simultaneously control foreign and autoantigen specific antibody responses. We highlight new tools that enable in-depth study of Tfr cells in vivo and recent data suggesting an important role for Tfr cells in limiting participation of autoreactive B cells in germinal centers., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Patient outcomes for steroid-dependent or -refractory chronic graft-versus-host diesease (cGVHD) are poor, and only ibrutinib has been US Food and Drug Administration (FDA) approved for this indication. cGVHD is often driven by the germinal center (GC) reaction, in which T follicular helper cells interact with GC B cells to produce antibodies that are associated with disease pathogenesis. The transcriptional corepressor B-cell lymphoma 6 (BCL6) is a member of the Broad-complex, Tramtrack, and Bric-abrac/poxvirus and zinc finger (BTB/POZ) transcription factor family and master regulator of the immune cells in the GC reaction. We demonstrate that BCL6 expression in both donor T cells and B cells is necessary for cGVHD development, pointing to BCL6 as a therapeutic cGVHD target. A small-molecule BCL6 inhibitor reversed active cGVHD in a mouse model of multiorgan system injury with bronchiolitis obliterans associated with a robust GC reaction, but not in cGVHD mice with scleroderma as the prominent manifestation. For cGVHD patients with antibody-driven cGVHD, targeting of BCL6 represents a new approach with specificity for a master GC regulator that would extend the currently available second-line agents., (© 2019 by The American Society of Hematology.)

DEPTOR is an evolutionarily conserved cell-intrinsic binding partner of mTOR that functions as a negative regulator of signaling responses. In this study, we show that DEPTOR is expressed within CD4 + T cells, and we observed that its relative level of expression modulates differentiation as well as glucose utilization within CD4 + T effectors in vitro. Using knock-in mice, we also find that induced expression of DEPTOR within CD4 + T regulatory cells stabilizes Foxp3 expression, shifts metabolism toward oxidative phosphorylation, and increases survival and suppressive function. In vivo, fully MHC mismatched cardiac allograft survival is significantly prolonged in knock-in recipients and sustained recipient expression of DEPTOR in combination with costimulatory blockade induces long-term graft survival. Furthermore, we show that the induced expression of DEPTOR in CD4 + T effectors fails to inhibit acute allograft rejection. Rather, prolonged survival is dominantly mediated via induced expression and function of DEPTOR within recipient CD4 + T regulatory cells. These collective findings identify DEPTOR as a novel protein that functions in CD4 + T cells to augment immunoregulation in vitro and in vivo., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Programmed death ligand-1 (PD-L1) plays a critical role in T-cell regulatory function. Here, we report a newly discovered effect of PD-L1 on angiogenesis. We demonstrate that PD-L1 and its receptor CD80, but not PD-1, are expressed by primary murine lung and heart vascular endothelial cells and the miscrovascular endothelial cell line (MS1) at both the mRNA and protein levels in vitro. The inhibition of PD-L1 or CD80 expression in MS1 cells, by small-interfering RNA transfection, led to a significant up-regulation of vascular endothelial growth factor receptor 2 expression and cell proliferation levels in MS1 cells. Furthermore, MS1 cells were found to have a significantly lower proliferation and vascular endothelial growth factor receptor 2 expression levels when they were co-cultured with PD-L1-expressing normal corneal epithelial cells, as compared to MS1 cells co-cultured with PD-L1(-/-) corneal epithelial cells. In a suture-induced corneal angiogenesis model, we observed a significantly higher level of angiogenic response in PD-L1(-/-) knockout mice as compared to wild-type mice, although there was no significant difference in the expression of inflammatory cytokines (interleukin-1α, interleukin-1β, or tumor necrosis factor-α) or the infiltration of innate immune cells (neutrophils and macrophages) between the two groups. We conclude that the expression of PD-L1 in both vascular endothelial cells and corneal epithelial cells regulates corneal angiogenesis., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen-presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC-mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4-Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long-term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1-deficient endothelium hearts had significant higher frequency of IFN-γ-producing alloreactive cells as well as higher frequency of CD8(+) effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)