Thymic graft-versus-host disease (tGVHD) can contribute to profound T cell deficiency and repertoire restriction after allogeneic BM transplantation (allo-BMT). However, the cellular mechanisms of tGVHD and interactions between donor alloreactive T cells and thymic tissues remain poorly defined. Using clinically relevant murine allo-BMT models, we show here that even minimal numbers of donor alloreactive T cells, which caused mild nonlethal systemic graft-versus-host disease, were sufficient to damage the thymus, delay T lineage reconstitution, and compromise donor peripheral T cell function. Furthermore, to mediate tGVHD, donor alloreactive T cells required trafficking molecules, including CCR9, L selectin, P selectin glycoprotein ligand-1, the integrin subunits αE and β7, CCR2, and CXCR3, and costimulatory/inhibitory molecules, including Ox40 and carcinoembryonic antigen-associated cell adhesion molecule 1. We found that radiation in BMT conditioning regimens upregulated expression of the death receptors Fas and death receptor 5 (DR5) on thymic stromal cells (especially epithelium), while decreasing expression of the antiapoptotic regulator cellular caspase-8–like inhibitory protein. Donor alloreactive T cells used the cognate proteins FasL and TNF-related apoptosis-inducing ligand (TRAIL) (but not TNF or perforin) to mediate tGVHD, thereby damaging thymic stromal cells, cytoarchitecture, and function. Strategies that interfere with Fas/FasL and TRAIL/DR5 interactions may therefore represent a means to attenuate tGVHD and improve T cell reconstitution in allo-BMT recipients.
Il-Kang Na, Sydney X. Lu, Nury L. Yim, Gabrielle L. Goldberg, Jennifer Tsai, Uttam Rao, Odette M. Smith, Christopher G. King, David Suh, Daniel Hirschhorn-Cymerman, Lia Palomba, Olaf Penack, Amanda M. Holland, Robert R. Jenq, Arnab Ghosh, Hien Tran, Taha Merghoub, Chen Liu, Gregory D. Sempowski, Melissa Ventevogel, Nicole Beauchemin, Marcel R.M. van den Brink
After liver transplantation in HCV-infected patients, the virus load inevitably exceeds pre-transplantation levels. This phenomenon reflects suppression of the host-effector immune responses that control HCV replication by the immunosuppressive drugs used to prevent rejection of the transplanted liver. Here, we describe an adoptive immunotherapy approach, using lymphocytes extracted from liver allograft perfusate (termed herein liver allograft–derived lymphocytes), which includes an abundance of NK/NKT cells that mounted an anti-HCV response in HCV-infected liver transplantation recipients, despite the immunosuppressive environment. This therapy involved intravenously injecting patients 3 days after liver transplantation with liver allograft–derived lymphocytes treated with IL-2 and the CD3-specific mAb OKT3. During the first month after liver transplantation, the HCV RNA titers in the sera of recipients who received immunotherapy were markedly lower than those in the sera of recipients who did not receive immunotherapy. We further explored these observations in human hepatocyte–chimeric mice, in which mouse hepatocytes were replaced by human hepatocytes. These mice unfailingly developed HCV infections after inoculation with HCV-infected human serum. However, injection of human liver–derived lymphocytes treated with IL-2/OKT3 completely prevented HCV infection. Furthermore, an in vitro study using genomic HCV replicon–containing hepatic cells revealed that IFN-γ–secreting cells played a pivotal role in such anti-HCV responses. Thus, our study presents what we believe to be a novel paradigm for the inhibition of HCV replication in HCV-infected liver transplantation recipients.
Masahiro Ohira, Kohei Ishiyama, Yuka Tanaka, Marlen Doskali, Yuka Igarashi, Hirotaka Tashiro, Nobuhiko Hiraga, Michio Imamura, Naoya Sakamoto, Toshimasa Asahara, Kazuaki Chayama, Hideki Ohdan
T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules — a process known as indirect recognition. As CD4+CD25+ Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4+CD25+ cells from C57BL/6 mice (H-2b) with allogeneic DCs from BALB/c mice (H-2d). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2Kd presented by H-2Ab MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.
Julia Yuen-Shan Tsang, Yakup Tanriver, Shuiping Jiang, Shao-An Xue, Kulachelvy Ratnasothy, Daxin Chen, Hans J. Stauss, R. Pat Bucy, Giovanna Lombardi, Robert Lechler
Bronchiolitis obliterans syndrome (BOS), a process of fibro-obliterative occlusion of the small airways in the transplanted lung, is the most common cause of lung transplant failure. We tested the role of cell-mediated immunity to collagen type V [col(V)] in this process. PBMC responses to col(II) and col(V) were monitored prospectively over a 7-year period. PBMCs from lung transplant recipients, but not from healthy controls or col(IV)-reactive Goodpasture’s syndrome patients after renal transplant, were frequently col(V) reactive. Col(V)-specific responses were dependent on both CD4+ T cells and monocytes and required both IL-17 and the monokines TNF-α and IL-1β. Strong col(V)-specific responses were associated with substantially increased incidence and severity of BOS. Incidences of acute rejection, HLA-DR mismatched transplants, and induction of HLA-specific antibodies in the transplant recipient were not as strongly associated with a risk of BOS. These data suggest that while alloimmunity initiates lung transplant rejection, de novo autoimmunity mediated by col(V)-specific Th17 cells and monocyte/macrophage accessory cells ultimately causes progressive airway obliteration.
William J. Burlingham, Robert B. Love, Ewa Jankowska-Gan, Lynn D. Haynes, Qingyong Xu, Joseph L. Bobadilla, Keith C. Meyer, Mary S. Hayney, Ruedi K. Braun, Daniel S. Greenspan, Bagavathi Gopalakrishnan, Junchao Cai, David D. Brand, Shigetoshi Yoshida, Oscar W. Cummings, David S. Wilkes
Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.
John D. Lang Jr., Xinjun Teng, Phillip Chumley, Jack H. Crawford, T. Scott Isbell, Balu K. Chacko, Yuliang Liu, Nirag Jhala, D. Ralph Crowe, Alvin B. Smith, Richard C. Cross, Luc Frenette, Eric E. Kelley, Diana W. Wilhite, Cheryl R. Hall, Grier P. Page, Michael B. Fallon, J. Steven Bynon, Devin E. Eckhoff, Rakesh P. Patel
Anergic T cells generated ex vivo are reported to have immunosuppressive effects in vitro and in vivo. Here, we tested this concept in nonhuman primates. Alloreactive T cells were rendered anergic ex vivo by coculture with donor alloantigen in the presence of anti-CD80/CD86 mAbs before adoptive transfer via renal allograft to rhesus monkey recipients. The recipients were briefly treated with cyclophosphamide and cyclosporine A during the preparation of the anergic cells. Thirteen days after renal transplantation, the anergic T cells were transferred to the recipient, after which no further immunosuppressive agents were administered. Rejection-free survival was prolonged in all treated recipients, and 3 of 6 animals survived long term (410–880 days at study’s end). In the long-surviving recipients, proliferative responses against alloantigen were inhibited in a donor-specific manner, and donor-type, but not third-party, skin allografts were also accepted, which demonstrated that antigen-specific tolerance had been induced. We conclude that anergic T cells generated ex vivo by blocking CD28/B7 costimulation can suppress renal allograft rejection after adoptive transfer in nonhuman primates. This strategy may be applicable to the design of safe clinical trials in humans.
Hisashi Bashuda, Masaaki Kimikawa, Kenichiro Seino, Yojiro Kato, Fumiko Ono, Akira Shimizu, Hideo Yagita, Satoshi Teraoka, Ko Okumura
Angiogenesis and vascular remodeling support fibroproliferative processes; however, no study has addressed the importance of angiogenesis during fibro-obliteration of the allograft airway during bronchiolitis obliterans syndrome (BOS) that occurs after lung transplantation. The ELR+ CXC chemokines both mediate neutrophil recruitment and promote angiogenesis. Their shared endothelial cell receptor is the G-coupled protein receptor CXC chemokine receptor 2 (CXCR2). We found that elevated levels of multiple ELR+ CXC chemokines correlated with the presence of BOS. Proof-of-concept studies using a murine model of BOS not only demonstrated an early neutrophil infiltration but also marked vascular remodeling in the tracheal allografts. In addition, tracheal allograft ELR+ CXC chemokines were persistently expressed even in the absence of significant neutrophil infiltration and were temporally associated with vascular remodeling during fibro-obliteration of the tracheal allograft. Furthermore, in neutralizing studies, treatment with anti-CXCR2 Abs inhibited early neutrophil infiltration and later vascular remodeling, which resulted in the attenuation of murine BOS. A more profound attenuation of fibro-obliteration was seen when CXCR2–/– mice received cyclosporin A. This supports the notion that the CXCR2/CXCR2 ligand biological axis has a bimodal function during the course of BOS: early, it is important for neutrophil recruitment and later, during fibro-obliteration, it is important for vascular remodeling independent of neutrophil recruitment.
John A. Belperio, Michael P. Keane, Marie D. Burdick, Brigitte Gomperts, Ying Ying Xue, Kurt Hong, Javier Mestas, Abbas Ardehali, Borna Mehrad, Rajan Saggar, Joseph P. Lynch III, David J. Ross, Robert M. Strieter
Homeostatic regulation of T cells involves an ongoing balance of new T cell generation, peripheral expansion, and turnover. The recovery of T cells when this balance is disrupted provides insight into the mechanisms that govern homeostasis. In a long-term, single cohort study, we assessed the role of thymic function after autologous transplant in adults, correlating serial computed tomography imaging of thymic size with concurrent measurements of peripheral CD4+ T cell populations. We established the age-dependent incidence, time course, and duration of thymic enlargement in adults and demonstrated that these changes were correlated with peripheral recovery of naive CD45RA+CD62L+ and signal-joint TCR rearrangement excision circle–bearing CD4+ populations with broad TCR diversity. Furthermore, we demonstrated that renewed thymopoiesis was critical for the restoration of peripheral CD4+ T cell populations. This recovery encompassed the recovery of normal CD4+ T cell numbers, a low ratio of effector to central memory cells, and a broad repertoire of TCR Vβ diversity among these memory cells. These data define the timeline and consequences of renewal of adult thymopoietic activity at levels able to quantitatively restore peripheral T cell populations. They further suggest that structural thymic regrowth serves as a basis for the regeneration of peripheral T cell populations.
Frances T. Hakim, Sarfraz A. Memon, Rosemarie Cepeda, Elizabeth C. Jones, Catherine K. Chow, Claude Kasten-Sportes, Jeanne Odom, Barbara A. Vance, Barbara L. Christensen, Crystal L. Mackall, Ronald E. Gress
Porcine endogenous retrovirus (PERV) is a potential pathogen in clinical xenotransplantation; transmission of PERV in vivo has been suggested in murine xenotransplantation models. We analyzed the transmission of PERV to human cells in vivo using a model in which immunodeficient NOD/SCID transgenic mice were transplanted with porcine and human lymphohematopoietic tissues. Our results demonstrate, we believe for the first time, that human and pig cells can coexist long-term (up to 25 weeks) without direct PERV infection of human cells. Despite the transplantation of porcine cells that did not produce human-tropic PERV, human cells from the chimeric mice were frequently found to contain PERV sequences. However, this transmission was due to the pseudotyping of PERV-C (a virus without human tropism) by xenotropic murine leukemia virus, rather than to de novo generation of human-tropic PERV. Thus, pseudotyping might account for the PERV transmission previously observed in mice. The absence of direct human cell infection following long-term in vivo coexistence with large numbers of porcine cells provides encouragement regarding the potential safety of using pigs that do not produce human-tropic PERV as source animals for transplantation to humans.
Yong-Guang Yang, James C. Wood, Ping Lan, Robert A. Wilkinson, Megan Sykes, Jay A. Fishman, Clive Patience
Extracellular nucleotides play an important role in thrombosis and inflammation, triggering a range of effects such as platelet activation and recruitment, endothelial cell activation, and vasoconstriction. CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase), converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inflammatory mediator adenosine. Deletion of CD39 renders mice exquisitely sensitive to vascular injury, and CD39-null cardiac xenografts show reduced survival. Conversely, upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inflammation. In this study we examined the consequences of transgenic expression of human CD39 (hCD39) in mice. Importantly, these mice displayed no overt spontaneous bleeding tendency under normal circumstances. The hCD39 transgenic mice did, however, exhibit impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism. Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived longer in a mouse cardiac transplant model of vascular rejection. These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates.
Karen M. Dwyer, Simon C. Robson, Harshal H. Nandurkar, Duncan J. Campbell, Hilton Gock, Lisa J. Murray-Segal, Nella Fisicaro, Tharun B. Mysore, Elzbieta Kaczmarek, Peter J. Cowan, Anthony J.F. d’Apice
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