In swine and nonhuman primates, kidney allografts can induce tolerance of heart allografts, leading to their long-term, immunosuppression-free survival. We refer to this phenomenon as kidney-induced cardiac allograft tolerance (KICAT). In this study, we have developed a murine model for KICAT to determine the underlining cellular/molecular mechanisms. Here, we show that spontaneously accepted DBA/2J kidneys in C57BL/6 recipients induce systemic tolerance that results in the long-term acceptance of DBA/2J heart allografts but not third-party cardiac allografts. The state of systemic tolerance of hearts was established 2 weeks after transplantation of the kidney, after which time, the kidney allograft is no longer required. Depletion of Foxp3+ T cells from these mice precipitated rejection of the heart allografts, indicating that KICAT is dependent on Treg function. Acceptance of kidney allografts and cotransplanted heart allografts did not require the thymus. In conclusion, these data show that kidney allografts induce systemic, donor-specific tolerance of cardiac allografts via Foxp3 cells, and that tolerance is independent of the thymus and continued presence of the kidney allograft. This experimental system should promote increased understanding of the tolerogenic mechanisms of the kidney.
Chao Yang, Jifu Ge, Ivy Rosales, Qing Yuan, Edward Szuter, Ellen Acheampong, Paul S. Russell, Joren C. Madsen, Robert B. Colvin, Alessandro Alessandrini
Acute rejection (AR) in renal transplantation is an established risk factor for reduced allograft survival. The incidence of AR is 10-20% despite standard of care immunosuppression, suggesting molecular pathways exist which are inadequately suppressed by current therapy. Molecules with regulatory control among these could serve as important targets for therapeutic manipulation to prevent rejection. Here, an integrative network-based computational strategy incorporating gene expression and genotype data of human renal allograft biopsy tissue was applied, to identify the master regulators- the key driver genes (KDGs)- within dyregulated AR pathways. A 982 meta-gene signature with differential expression in AR versus non-AR, was identified from a meta-analysis of microarray data from 735 human kidney allograft biopsy samples across seven data sets. Among the upregulated genes, enriched biologic processes include the immune response, leucocyte activation and antigen processing and presentation; where monocytes, macrophages and dendritic cells were identified as the major immune cell populations. Genomic key driver analysis of this signature predicted 14 KDGs. Expression of the KDGs provided risk stratification for subsequent graft loss with high prediction accuracy at 2 years post transplant (AUC=0.913) and 2 years post biopsy (AUC=0.889) in separate clinical cohorts. Interrogation of two drug-repositioning resources identified compounds with predicted efficacy against individual KDGs or a key driver-based gene set respectively, and therefore be repositioned for AR prevention. Minocycline, an FDA-approved tetracycline antibiotic, was chosen for experimental validation in a murine cardiac allograft model of AR. Minocycline alone attenuated the inflammatory profile of AR compared with controls, and when co-administered with immunosuppression prolonged graft survival. This study demonstrates the proof-of-concept that a network-based strategy, using gene expression and genotype data assists target prioritization for therapeutics in renal allograft rejection.
Zhengzi Yi, Karen L. Keung, Li Li, Min Hu, Bo Lu, Leigh Nicholson, Elvira Jimenez-Vera, Madhav C. Menon, Chengguo Wei, Stephen I. Alexander, Barbara Murphy, Philip J. O’Connell, Weijia Zhang
Acute graft versus host disease (aGvHD) remains a major impediment to successful allogeneic hematopoietic cell transplantation (allo-HCT). To solve this problem, a greater knowledge of factors which regulate the differentiation of donor T cells toward cytotoxic or regulatory cells is necessary. We report that the β2-adrenergic receptor (β2-AR) is critical for regulating this differentiation, and that its manipulation can control aGvHD without impairing the graft-versus-tumor (GvT) effect. Donor T cell β2-AR expression and signaling is associated with decreased aGvHD when compared to recipients of β2-AR–/– donor T cells. We determined that β2-AR activation skewed CD4+ T cell differentiation in vitro and in vivo toward regulatory T cells (Tregs) rather than the T helper 1 (Th1) phenotype. Treatment of allo-HCT recipients with a selective β2-agonist, (bambuterol) ameliorated aGvHD severity. This was associated with increased Tregs, decreased cytotoxic T cells, and increased donor bone marrow-derived myeloid derived suppressor cells (MDSCs) in allogeneic and humanized xenogeneic aGvHD models. β2-AR signaling resulted in increased Treg generation through glycogen synthase kinase-3 activation. Bambuterol preserved the GvT effect by inducing NKG2D+ effector cells and central memory T cells. These data reveal how β-AR signaling can be targeted to ameliorate GvHD severity while preserving GvT effect.
Hemn Mohammadpour, Joseph L. Sarow, Cameron R. MacDonald, George L. Chen, Jingxin Qiu, Umesh C. Sharma, Xuefang Cao, Megan M. Herr, Theresa E. Hahn, Bruce R. Blazar, Elizabeth A. Repasky, Philip L. McCarthy
Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop genetically-recipient T cells that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell depleted Lewis rats. The rats were then given Lewis x Dark Agouti (LWxDA) CTTI under the kidney capsule and vascularized DA heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naïve and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with (LWxDA) CTTI rejected the third-party BN hearts (mean survival time 10d; n=5). Controls did not (n=5). CTTI recipients produced antibody against third party BN donor but not against the DA thymus donor demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.
Jean Kwun, Jie Li, Clay Rouse, Jae Berm Park, Alton B. Farris III, Maragatha Kuchibhatla, Joseph W. Turek, Stuart Knechtle, Allan D. Kirk, M. Louise Markert
Vascularized composite allotransplantation (VCA) has become a valid therapeutic option to restore form and function after devastating tissue loss. However, the need for high-dose multidrug immunosuppression to maintain allograft survival is still hampering more widespread application of VCA. In this study, we investigated the immunoregulatory potential of costimulation blockade (CoB; CTLA4-Ig and anti-CD154 mAb) combined with nonmyeoablative total body irradiation (TBI) to promote allograft survival of VCA in a fully MHC-mismatched mouse model of orthotopic hind limb transplantation. Compared with untreated controls (median survival time [MST] 8 days) and CTLA4-Ig treatment alone (MST 17 days), CoB treatment increased graft survival (MST 82 days), and the addition of nonmyeloablative TBI led to indefinite graft survival (MST > 210 days). Our analysis suggests that VCA-derived BM induced mixed chimerism in animals treated with CoB and TBI + CoB, promoting gradual deletion of alloreactive T cells as the underlying mechanism of long-term allograft survival. Acceptance of donor-matched secondary skin grafts, decreased ex vivo T cell responsiveness, and increased graft-infiltrating Tregs further indicated donor-specific tolerance induced by TBI + CoB. In summary, our data suggest that vascularized BM-containing VCAs are immunologically favorable grafts promoting chimerism induction and long-term allograft survival in the context of CoB.
Byoung Chol Oh, Georg J. Furtmüller, Madeline L. Fryer, Yinan Guo, Franka Messner, Johanna Krapf, Stefan Schneeberger, Damon S. Cooney, W.P. Andrew Lee, Giorgio Raimondi, Gerald Brandacher
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
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