Rates of graft rejection are high among recipients of heart transplants. The onset and progression of clinically significant heart transplant rejection are currently monitored by serial biopsy, but this approach is highly invasive and lacks sensitivity. Here, we have developed what we believe to be a new technique to measure organ rejection noninvasively that involves the exploration of tissue-infiltrating leukocytes as biomarker sources for diagnostic imaging. Specifically, we profiled the myeloid response in a murine model of heart transplantation with the aim of defining and validating an imaging signature of graft rejection. Ly-6Chi monocytes, which promote inflammation, accumulated progressively in allografts but only transiently in isografts. Ly-6Clo monocytes, which help resolve inflammation, did not accumulate, although they composed the majority of the few remaining monocytes in isografts. The persistence of Ly-6Chi monocytes in allografts prompted us to screen for a Ly-6Chi monocyte–associated imaging marker. Low-density array data revealed that Ly-6Chi monocytes express 10-fold higher levels of myeloperoxidase (MPO) than Ly-6Clo monocytes. Noninvasive magnetic resonance imaging of MPO with an MPO-activatable Gd-chelate revealed a spatially defined T1-weighted signal in rejected allografts but not in isografts or MPO-deficient allograft recipients. Flow cytometry, enzymography, and histology validated the approach by mapping MPO activity to Ly-6Chi monocytes and neutrophils. Thus, MPO imaging represents a potential alternative to the current invasive clinical standard by which transplants are monitored.
Filip K. Swirski, Moritz Wildgruber, Takuya Ueno, Jose-Luiz Figueiredo, Peter Panizzi, Yoshiko Iwamoto, Elizabeth Zhang, James R. Stone, Elisenda Rodriguez, John W. Chen, Mikael J. Pittet, Ralph Weissleder, Matthias Nahrendorf
One of the main unresolved questions in solid organ transplantation is how to establish indefinite graft survival that is free from long-term treatment with immunosuppressive drugs and chronic rejection (i.e., the establishment of tolerance). The failure to achieve this goal may be related to the difficulty in identifying the phenotype and function of the cell subsets that participate in the induction of tolerance. To address this issue, we investigated the suppressive roles of recipient myeloid cells that may be manipulated to induce tolerance to transplanted hearts in mice. Using depleting mAbs, clodronate-loaded liposomes, and transgenic mice specific for depletion of CD11c+, CD11b+, or CD115+ cells, we identified a tolerogenic role for CD11b+CD115+Gr1+ monocytes during the induction of tolerance by costimulatory blockade with CD40L-specific mAb. Early after transplantation, Gr1+ monocytes migrated from the bone marrow into the transplanted organ, where they prevented the initiation of adaptive immune responses that lead to allograft rejection and participated in the development of Tregs. Our results suggest that mobilization of bone marrow CD11b+CD115+Gr1+ monocytes under sterile inflammatory conditions mediates the induction of indefinite allograft survival. We propose that manipulating the common bone marrow monocyte progenitor could be a useful clinical therapeutic approach for inducing transplantation tolerance.
Mercedes Rodriguez Garcia, Levi Ledgerwood, Yu Yang, Jiangnan Xu, Girdhari Lal, Bryna Burrell, Ge Ma, Daigo Hashimoto, Yansui Li, Peter Boros, Marcos Grisotto, Nico van Rooijen, Rafael Matesanz, Frank Tacke, Florent Ginhoux, Yaozhong Ding, Shu-Hsia Chen, Gwendalyn Randolph, Miriam Merad, Jonathan S. Bromberg, Jordi C. Ochando
Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to α-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell–related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.
Pervinder Sagoo, Esperanza Perucha, Birgit Sawitzki, Stefan Tomiuk, David A. Stephens, Patrick Miqueu, Stephanie Chapman, Ligia Craciun, Ruhena Sergeant, Sophie Brouard, Flavia Rovis, Elvira Jimenez, Amany Ballow, Magali Giral, Irene Rebollo-Mesa, Alain Le Moine, Cecile Braudeau, Rachel Hilton, Bernhard Gerstmayer, Katarzyna Bourcier, Adnan Sharif, Magdalena Krajewska, Graham M. Lord, Ian Roberts, Michel Goldman, Kathryn J. Wood, Kenneth Newell, Vicki Seyfert-Margolis, Anthony N. Warrens, Uwe Janssen, Hans-Dieter Volk, Jean-Paul Soulillou, Maria P. Hernandez-Fuentes, Robert I. Lechler
Chronic rejection currently limits the long-term efficacy of clinical transplantation. Although B cells have recently been shown to play a pivotal role in the induction of alloimmunity and are being targeted in other transplant contexts, the efficacy of preemptive B cell depletion to modulate alloimmunity or attenuate cardiac allograft vasculopathy (CAV) (classic chronic rejection lesions found in transplanted hearts) in a translational model has not previously been described. We report here that the CD20-specific antibody (αCD20) rituximab depleted CD20+ B cells in peripheral blood, secondary lymphoid organs, and the graft in cynomolgus monkey recipients of heterotopic cardiac allografts. Furthermore, CD20+ B cell depletion therapy combined with the calcineurin inhibitor cyclosporine A (CsA) prolonged median primary graft survival relative to treatment with αCD20 or CsA alone. In animals treated with both αCD20 and CsA that achieved efficient B cell depletion, alloantibody production was substantially inhibited and the CAV severity score was markedly reduced. We conclude therefore that efficient preemptive depletion of CD20+ B cells is effective in a preclinical model to modulate pathogenic alloimmunity and to attenuate chronic rejection when used in conjunction with a conventional clinical immunosuppressant. This study suggests that use of this treatment combination may improve the efficacy of transplantation in the clinic.
Shahrooz S. Kelishadi, Agnes M. Azimzadeh, Tianshu Zhang, Tiffany Stoddard, Emily Welty, Christopher Avon, Mitch Higuchi, Amal Laaris, Xiang-Fei Cheng, Christine McMahon, Richard N. Pierson III
Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell–dependent IFN-γ production by Gr-1+CD11b+ cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-γ production by NKT cells and Gr-1+CD11b+ cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell–dependent augmented IFN-γ production by Gr-1+CD11b+ cells. Thus, treatment with either IL-12– or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.
Nobuhide Matsuoka, Takeshi Itoh, Hiroshi Watarai, Etsuko Sekine-Kondo, Naoki Nagata, Kohji Okamoto, Toshiyuki Mera, Hiroshi Yamamoto, Shingo Yamada, Ikuro Maruyama, Masaru Taniguchi, Yohichi Yasunami
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
A fraction of liver transplant recipients are able to discontinue all immunosuppressive therapies without rejecting their grafts and are said to be operationally tolerant to the transplant. However, accurate identification of these recipients remains a challenge. To design a clinically applicable molecular test of operational tolerance in liver transplantation, we studied transcriptional patterns in the peripheral blood of 80 liver transplant recipients and 16 nontransplanted healthy individuals by employing oligonucleotide microarrays and quantitative real-time PCR. This resulted in the discovery and validation of several gene signatures comprising a modest number of genes capable of identifying tolerant and nontolerant recipients with high accuracy. Multiple peripheral blood lymphocyte subsets contributed to the tolerance-associated transcriptional patterns, although NK and γδTCR+ T cells exerted the predominant influence. These data suggest that transcriptional profiling of peripheral blood can be employed to identify liver transplant recipients who can discontinue immunosuppressive therapy and that innate immune cells are likely to play a major role in the maintenance of operational tolerance in liver transplantation.
Marc Martínez-Llordella, Juan José Lozano, Isabel Puig-Pey, Giuseppe Orlando, Giuseppe Tisone, Jan Lerut, Carlos Benítez, Jose Antonio Pons, Pascual Parrilla, Pablo Ramírez, Miquel Bruguera, Antoni Rimola, Alberto Sánchez-Fueyo
Successful transplantation requires the prevention of allograft rejection and, in the case of transplantation to treat autoimmune disease, the suppression of autoimmune responses. The standard immunosuppressive treatment regimen given to patients with autoimmune type 1 diabetes who have received an islet transplant results in the loss of T cells. In many other situations, the immune system responds to T cell loss through cytokine-dependant homeostatic proliferation of any remaining T cells. Here we show that T cell loss after islet transplantation in patients with autoimmune type 1 diabetes was associated with both increased serum concentrations of IL-7 and IL-15 and in vivo proliferation of memory CD45RO+ T cells, highly enriched in autoreactive glutamic acid decarboxylase 65–specific T cell clones. Immunosuppression with FK506 and rapamycin after transplantation resulted in a chronic homeostatic expansion of T cells, which acquired effector function after immunosuppression was removed. In contrast, the cytostatic drug mycophenolate mofetil efficiently blocked homeostatic T cell expansion. We propose that the increased production of cytokines that induce homeostatic expansion could contribute to recurrent autoimmunity in transplanted patients with autoimmune disease and that therapy that prevents the expansion of autoreactive T cells will improve the outcome of islet transplantation.
Paolo Monti, Miriam Scirpoli, Paola Maffi, Nadia Ghidoli, Francesca De Taddeo, Federico Bertuzzi, Lorenzo Piemonti, Marika Falcone, Antonio Secchi, Ezio Bonifacio
T cell Ig mucin 1 (TIM-1) plays an important role in regulating immune responses in autoimmune and asthma models, and it is expressed on both Th1 and Th2 cells. Using an antagonistic TIM-1–specific antibody, we studied the role of TIM-1 in alloimmunity. A short course of TIM-1–specific antibody monotherapy prolonged survival of fully MHC-mismatched vascularized mouse cardiac allografts. This prolongation was associated with inhibition of alloreactive Th1 responses and preservation of Th2 responses. TIM-1–specific antibody treatment was more effective in Th1-type cytokine–deficient Stat4–/– recipients as compared with Th2-type cytokine–deficient Stat6–/– recipients. Subtherapeutic doses of rapamycin plus TIM-1–specific antibody resulted in allograft acceptance and prevented the development of chronic allograft vasculopathy. Allograft survival via this treatment was accompanied by a Th1- to Th2-type cytokine switch. Depletion of natural Tregs abrogated the graft-protecting effect of the TIM-1–specific antibody. Importantly, CD4+CD25+ Tregs obtained from long-term survivors had enhanced regulatory activity as compared with naive CD4+CD25+ Tregs. Consistent with this, TIM-1–specific antibody treatment both preserved Tregs and prevented the expansion of alloreactive effector Th1 cells in an alloreactive TCR transgenic adoptive transfer model. These studies define previously unknown functions of TIM-1 in regulating alloimmune responses in vivo and may provide a novel approach to promoting transplantation tolerance.
Takuya Ueno, Antje Habicht, Michael R. Clarkson, Monica J. Albin, Kazuhiro Yamaura, Olaf Boenisch, Joyce Popoola, Ying Wang, Hideo Yagita, Hisaya Akiba, M. Javeed Ansari, Jaeseok Yang, Laurence A. Turka, David M. Rothstein, Robert F. Padera, Nader Najafian, Mohamed H. Sayegh
T cell Ig mucin (Tim) molecules modulate CD4+ T cell responses. In keeping with the view that Tim-1 generates a stimulatory signal for CD4+ T cell activation, we hypothesized that an agonist Tim-1–specific mAb would intensify the CD4+ T cell–dependant allograft response. Unexpectedly, we determined that a particular Tim-1–specific mAb exerted reciprocal effects upon the commitment of alloactivated T cells to regulatory and effector phenotypes. Commitment to the Th1 and Th17 phenotypes was fostered, whereas commitment to the Treg phenotype was hindered. Moreover, ligation of Tim-1 in vitro effectively deprogrammed Tregs and thus produced Tregs unable to control T cell responses. Overall, the effects of the agonist Tim-1–specific mAb on the allograft response stemmed from enhanced expansion and survival of T effector cells; a capacity to deprogram natural Tregs; and inhibition of the conversion of naive CD4+ T cells into Tregs. The reciprocal effects of agonist Tim-1–specific mAbs upon effector T cells and Tregs serve to prevent allogeneic transplant tolerance.
Nicolas Degauque, Christophe Mariat, James Kenny, Dong Zhang, Wenda Gao, Minh Diem Vu, Sophoclis Alexopoulos, Mohammed Oukka, Dale T. Umetsu, Rosemarie H. DeKruyff, Vijay Kuchroo, Xin Xiao Zheng, Terry B. Strom
Small airway fibrosis (bronchiolitis obliterans syndrome) is the primary obstacle to long-term survival following lung transplantation. Here, we show the importance of functional microvasculature in the prevention of epithelial loss and fibrosis due to rejection and for the first time, relate allograft microvascular injury and loss of tissue perfusion to immunotherapy-resistant rejection. To explore the role of alloimmune rejection and airway ischemia in the development of fibroproliferation, we used a murine orthotopic tracheal transplant model. We determined that transplants were reperfused by connection of recipient vessels to donor vessels at the surgical anastomosis site. Microcirculation through the newly formed vascular anastomoses appeared partially dependent on VEGFR2 and CXCR2 pathways. In the absence of immunosuppression, the microvasculature in rejecting allografts exhibited vascular complement deposition, diminished endothelial CD31 expression, and absent perfusion prior to the onset of fibroproliferation. Rejecting grafts with extensive endothelial cell injury were refractory to immunotherapy. After early microvascular loss, neovascularization was eventually observed in the membranous trachea, indicating a reestablishment of graft perfusion in established fibrosis. One implication of this study is that bronchial artery revascularization at the time of lung transplantation may decrease the risk of subsequent airway fibrosis.
Ashok N. Babu, Tomohiro Murakawa, Joshua M. Thurman, Edmund J. Miller, Peter M. Henson, Martin R. Zamora, Norbert F. Voelkel, Mark R. Nicolls
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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