Immune suppressive donor regulatory T cells (Tregs) can prevent graft-versus-host disease (GVHD) or solid organ allograft rejection. We previously demonstrated inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report human pSTAT3-inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor anti-leukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing CLA antigen, immune suppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduce alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection, and impair infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provokes a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, co-treatment with coenzyme Q10 (coQ10) restores OxPhos in pSTAT3-inhibited iTregs and augments their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3-inhibited iTregs to control alloreactive T cells.
Kelly Walton, Mario R. Fernandez, Elizabeth M. Sagatys, Jordan Reff, Jongphil Kim, Marie Catherine Lee, John Kiluk, Jane Yuet Ching Hui, David McKenna, Meghan Hupp, Colleen Forster, Michael A. Linden, Nicholas J. Lawrence, Harshani R. Lawrence, Joseph Pidala, Steven Z. Pavletic, Bruce R. Blazar, Said M. Sebti, John L. Cleveland, Claudio Anasetti, Brian C. Betts
Epstein-Barr Virus (EBV) is a ubiquitous virus linked to a variety of lymphoid and epithelial malignancies. In solid organ and hematopoietic stem cell transplant recipients, EBV is causally associated with posttransplant lymphoproliferative disorder (PTLD), a group of heterogeneous lymphoid diseases. EBV+ B cell lymphomas that develop in the context of PTLD are generally attributed to the immunosuppression required to promote graft survival, but little is known regarding the role of EBV genome diversity in the development of malignancy. We deep-sequenced the EBV genome from the peripheral blood of 18 solid organ transplant recipients, including 6 PTLD patients. Sequences from 6 EBV+ spontaneous lymphoblastoid B cell lines (SLCL) were similarly analyzed. The EBV genome from PTLD patients had a significantly greater number of variations than EBV from transplant recipients without PTLD. Importantly, there were 15 nonsynonymous variations, including 8 in the latent cycle gene EBNA3C that were associated with the development of PTLD. One of the nonsynonymous variations in EBNA3C is located within a previously defined T cell epitope. These findings suggest that variations in the EBV genome can contribute to the pathogenesis of PTLD.
Eden M. Maloney, Vincent A. Busque, Sin Ting Hui, Jiaying Toh, Marcelo Fernandez-Vina, Sheri M. Krams, Carlos O. Esquivel, Olivia M. Martinez
Acute Graft-Versus-Host Disease (aGVHD) is a T cell mediated immunological disorder and the leading cause of non-relapse mortality in patients who receive allogeneic hematopoietic cell transplants. Based on recent observations that PRMT5 and arginine methylation is upregulated in activated memory T cells, we hypothesized that PRMT5 is involved in the pathogenesis of aGVHD. Here, we show that PRMT5 expression and enzymatic activity is upregulated in activated T cells in vitro and in T cells from mice developing aGVHD after allogeneic transplant. PRMT5 expression is also upregulated in T cells of patients who developed aGVHD after allogeneic hematopoietic cell transplant compared to those who did not develop aGVHD.PRMT5 inhibition using a selective small-molecule inhibitor (C220) significantly reduces mouse and human allogeneic T cell proliferation and inflammatory IFN-γ and IL-17 cytokine production. Administration of PRMT5 small-molecule inhibitors significantly improves survival, reducing disease incidence and clinical severity in mouse models of aGVHD without adversely affecting engraftment. Importantly, we show that PRMT5 inhibition retains the beneficial graft versus leukemia (GVL) effect by maintaining cytotoxic CD8 T cell responses. Mechanistically, we show that PRMT5 inhibition potently reduces STAT-1 phosphorylation as well as transcription of pro-inflammatory genes including Interferon Stimulated Genes (ISG) and IL-17. Additionally, PRMT5 inhibition deregulates cell-cycle in activated T cells and disrupts signaling by impacting ERK1/2 phosphorylation. Thus, we have identified PRMT5 as a regulator of T cell responses and as a therapeutic target in aGVHD.
Katiri Snyder, Nina C. Zitzer, Yandi Gao, Hannah K. Choe, Natalie E. Sell, Lotus Neidemire-Colley, Anora Ignaci, Charuta Kale, Raymond D. Devine, Maria G. Abad, Maciej Pietrzak, Min Wang, Hong Lin, Yang W. Zhang, Gregory K. Behbehani, Jane E. Jackman, Ramiro Garzon, Kris Vaddi, Robert A. Baiocchi, Parvathi Ranganathan
BACKGROUND RNA sequencing (RNA-Seq) is a molecular tool to analyze global transcriptional changes, deduce pathogenic mechanisms, and discover biomarkers. We performed RNA-Seq to investigate gene expression and biological pathways in urinary cells and kidney allograft biopsies during an acute rejection episode and to determine whether urinary cell gene expression patterns are enriched for biopsy transcriptional profiles.METHODS We performed RNA-Seq of 57 urine samples collected from 53 kidney allograft recipients (patients) with biopsies classified as acute T cell–mediated rejection (TCMR; n = 22), antibody-mediated rejection (AMR; n = 8), or normal/nonspecific changes (No Rejection; n = 27). We also performed RNA-Seq of 49 kidney allograft biopsies from 49 recipients with biopsies classified as TCMR (n = 12), AMR (n = 17), or No Rejection (n = 20). We analyzed RNA-Seq data for differential gene expression, biological pathways, and gene set enrichment across diagnoses and across biospecimens.RESULTS We identified unique and shared gene signatures associated with biological pathways during an episode of TCMR or AMR compared with No Rejection. Gene Set Enrichment Analysis demonstrated enrichment for TCMR biopsy signature and AMR biopsy signature in TCMR urine and AMR urine, irrespective of whether the biopsy and urine were from the same or different patients. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of immune cell types in urinary cells compared with biopsies.CONCLUSIONS RNA-Seq of urinary cells and biopsies, in addition to identifying enriched gene signatures and pathways associated with TCMR or AMR, revealed genomic changes between TCMR and AMR, as well as between allograft biopsies and urinary cells.
Akanksha Verma, Thangamani Muthukumar, Hua Yang, Michelle Lubetzky, Michael F. Cassidy, John R. Lee, Darshana M. Dadhania, Catherine Snopkowski, Divya Shankaranarayanan, Steven P. Salvatore, Vijay K. Sharma, Jenny Z. Xiang, Iwijn De Vlaminck, Surya V. Seshan, Franco B. Mueller, Karsten Suhre, Olivier Elemento, Manikkam Suthanthiran
Neutrophils play critical roles during the initial phase of hepatic ischemia/reperfusion injury (HIRI). However, the regulation of neutrophil activation, infiltration, and proinflammatory cytokine secretion has not been fully elucidated. In this study, we revealed that OX40 was expressed by neutrophils, its expression in neutrophils was time-dependently upregulated following HIRI, and Ox40 knockout markedly alleviated liver injury. Compared with wild-type neutrophils, the adoptive transfer of Ox40–/– neutrophils decreased HIRI in neutrophil-depleted Rag2/Il2rg–/– or Ox40–/– mice. Moreover, consistently, the in vitro experiments showed that Ox40 not only prolonged neutrophil survival but also promoted proinflammatory cytokines, ROS production, and even neutrophil chemotaxis. Further investigation demonstrated that the knockout of Ox40 in neutrophils inhibited NF-κB signaling via the TRAF1/2/4 and IKKα/IKKβ/IκBα pathways. OX40L and OX86 stimulation could enhance neutrophil activation and survival in vitro and in vivo. In conclusion, our study provides a new understanding of OX40, which is expressed not only in adaptive immune cells but also in innate immune cells, i.e., neutrophils, contributing to the activation and survival of neutrophils. These findings provide a novel potential therapeutic target for the prevention of HIRI during liver transplantation or hepatic surgery.
Hua Jin, Chunpan Zhang, Chengyang Sun, Xinyan Zhao, Dan Tian, Wen Shi, Yue Tian, Kai Liu, Guangyong Sun, Hufeng Xu, Dong Zhang
Background: Innate immune activation impacts lung transplant outcomes. Dectin-1 is an innate receptor important for pathogen recognition. We hypothesized that genotypes reducing dectin-1 activity would be associated with infection, graft dysfunction, and death in lung transplant recipients. Methods: We assessed the rs16910526 CLEC7A gene polymorphism Y238X, which results in dectin-1 truncation, in 321 lung allograft recipients at a single institution and in 1,129 lung allograft recipients in the multi-center lung transplant outcomes group (LTOG) cohort. Differences in dectin-1 mRNA, cytokines, protein levels, immunophenotypes, and clinical factors were assessed. Results: Y238X carriers had decreased dectin-1 mRNA expression (P = 0.0001), decreased soluble dectin-1 protein concentrations in BAL (P = 0.008) and plasma (P = 0.04), and decreased monocyte surface dectin-1 (P = 0.01) compared to wild type subjects. Y238X carriers had an increased risk of fungal pathogens (HR 1.17, CI 1.0 – 1.4), an increased risk of graft dysfunction or death (HR 1.6, CI 1.0 – 2.6), as well increased mortality in the UCSF cohort (HR 1.8, CI 1.1 – 3.8) and in the LTOG cohort (HR 1.3, CI 1.1 – 1.6), compared to CLEC7A wildtype subjects. Conclusion: Increased rates of graft dysfunction and death associated with this dectin-1 polymorphism may be amplified by immunosuppression that drives higher fungal burden from compromised pathogen recognition. Funding: Project funding came from the UCSF Nina Ireland Program for Lung Health (NIPLH) Innovative Grant program, award number IK2CX001034 from the Clinical Sciences Research & Development Service of the VA Office of Research and Development, and the Joel D. Cooper Career Development Award from the International Society for Heart and Lung Transplantation.
Daniel R. Calabrese, Ping Wang, Tiffany Chong, Jonathan Hoover, Jonathan P. Singer, Dara Torgerson, Steven R. Hays, Jeffrey A. Golden, Jasleen Kukreja, Daniel Dugger, Jason D. Christie, LTOG investigators, John R. Greenland
Islet transplantation can restore lost glycemic control in type 1 diabetes subjects, but is restricted in its clinical application by limiting supplies of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signalling thresholds. Here we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for approximately 45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon regulatory T cells, was antigen-specific and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signalling, graft hyper-inflammation and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators with no impact on glucose stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signalling thresholds to favour immune tolerance and promotes islet allogeneic survival. Clinically this would allow for reduced immunosuppression and support the use of alternate islet sources.
Nathan W. Zammit, Stacey N. Walters, Karen L. Seeberger, Philip J. O’Connell, Gregory S. Korbutt, Shane T. Grey
B7-H4 is a negative regulatory B7 family member. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4–/– versus WT recipients markedly accelerated GVHD-induced lethality. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. Rapid mortality in B7-H4–/– recipients was associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased T effector function (proliferation, proinflammatory cytokines, cytolytic molecules), and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4–/– versus WT recipients required multiple metabolic pathways, increased extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs), and increased expression of fuel substrate transporters. During GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. B7-H4–/– donor T cells given to WT recipients increased GVHD mortality and had function and biological properties similar to WT T cells from allogeneic B7-H4–/– recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4–/– mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.
Asim Saha, Patricia A. Taylor, Christopher J. Lees, Angela Panoskaltsis-Mortari, Mark J. Osborn, Colby J. Feser, Govindarajan Thangavelu, Wolfgang Melchinger, Yosef Refaeli, Geoffrey R. Hill, David H. Munn, William J. Murphy, Jonathan S. Serody, Ivan Maillard, Katharina Kreymborg, Marcel van den Brink, Chen Dong, Shuyu Huang, Xingxing Zang, James P. Allison, Robert Zeiser, Bruce R. Blazar
Tissue engineering is a promising approach to address organ shortages currently limiting clinical transplantation. “Off-the-shelf” engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells basally express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. We report here that CRISPR/Cas9-mediated dual ablation of β2-microglobulin and CIITA in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.
Jonathan Merola, Melanie Reschke, Richard W. Pierce, Lingfeng Qin, Susann Spindler, Tania Baltazar, Thomas D. Manes, Francesc Lopez-Giraldez, Guangxin Li, Laura G. Bracaglia, Catherine Xie, Nancy Kirkiles-Smith, W. Mark Saltzman, Gregory T. Tietjen, George Tellides, Jordan S. Pober
Steroid-refractory intestinal acute graft-versus-host disease (aGVHD) is a frequently fatal condition with little known about mechanisms driving failed steroid responses in gut mucosa. To uncover novel molecular insights in steroid-refractory aGVHD, we compared gene expression profiles of rectosigmoid biopsies from patients at diagnosis of clinical stage 3-4 lower intestinal aGVHD (N=22), to repeat biopsies when the patients became steroid refractory (N=22), and normal controls (N=10). We also performed single gene analyses of factors associated with tolerance (programmed death ligand-1 [PDL1], indoleamine 2,3 dioxygenase [IDO1], and T cell immunoreceptor with Ig and ITIM domains [TIGIT]) and found that significantly higher expression levels of these aGVHD inhibitory genes (PDL1, IDO1, TIGIT) at aGVHD onset became decreased in the steroid-refractory state. We examined genes triggered by microbial ligands to stimulate gut repair, amphiregulin (AREG) and the aryl hydrocarbon receptor (AhR), and found that both AREG and AhR gene expression levels were increased at aGVHD onset and remained elevated in steroid-refractory aGVHD. We also identified higher expression levels of metallothioneines, metal-binding enzymes induced in stress responses, and M2 macrophage genes in steroid-refractory aGVHD. We observed no differences in T-cell subsets between onset and steroid-refractory aGVHD. Patients with a rapidly fatal course showed greater DNA damage and a distinct microbial signature at aGVHD onset, whereas patients with more prolonged survival exhibited a gene expression profile consistent with activation of Smoothened. Our results extend the paradigm beyond T cell-centric therapies for steroid-refractory GI aGVHD and highlight new mechanisms for therapeutic exploration.
Shernan G. Holtan, Ashraf Shabaneh, Brian C. Betts, Armin Rashidi, Margaret L. MacMillan, Celalettin Ustun, Khalid Amin, Byron P. Vaughn, Justin Howard, Alexander Khoruts, Mukta Arora, Todd E. DeFor, Darrell Johnson, Bruce R. Blazar, Daniel J. Weisdorf, Jinhua Wang
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