Anti-donor Immune Response Research Articles

MSCs Isolated From Patients With Ischemic Vascular Disease Have Normal Angiogenic Potential

MSCs Isolated From Patients With Ischemic Vascular Disease Have Normal Angiogenic Potential

B Cells and CD40/CD154 Interactions Are Required for CD8 T Cell Recovery Following Antibody-Mediated Lymphoablation.

Antibody-meditaed lymphoablation is commonly used as an induction therapy in transplant patients. However, recipients eventually reconstitute their T cell repertoire, develop anti-donor immune responses and reject transplanted organ. The goal of this study was to investigate the requirements for T cell recovery following lymphoablation in a mouse model of heterotopic cardiac transplantation. We previously reported that memory CD4 T cells are less susceptible to depletion by murine antithymocyte globulin (mATG) than CD8 T cells. Additional CD4 T cell depletion impaired CD8 T cell recovery after mATG treatment in B6 recipients of BALB/c heart allografts (0.2% vs 3-5% of spleen CD8 T cells recovered by d. 10 posttransplant) and in non-transplanted B6 mice. Analogous findings were made in recipients treated with anti-CD8 ± anti-CD4 mAbs demonstrating that the requirement for CD4 cell help is a common feature of antibody-mediated lymphoablation. In addition, memory CD8 T cell reconstitution depended on CD4 T cell help in non-transplanted B6 mice and in B6 isograft recipients treated with mATG. Treatment with blocking anti-CD154 mAb plus mATG inhibited memory CD8 T cell recovery in B6 heart allograft recipients similar to the levels observed after CD4 T cell depletion. Conversely, agonistic anti-CD40 mAb restored expansion of memory CD8 T cells following mATG treatment of CD4 T cell depleted heart allograft recipients. To test whether B cells are required for CD8 T cell recovery, we used B6.huCD20tg mice. Treatment with anti-human CD20 mAb depleted >90% of peripheral B cells and markedly inhibited CD8 T cell expansion in huCD20tg recipients compared to B cell-sufficient mice. Next, B cells were isolated from mATG treated recipients with or without CD4 depletion. In the absence of CD4 T cells, B cells expressed lower levels of MHC class I, CD80, CD86, and TNFa suggesting that both cognate interactions and cytokines promote CD8 T cell expansion. Our results show that CD4 T cell help mediated through B cells and CD40/CD154 interactions drives CD8 T cell reconstitution following antibody-mediated depletion. These findings may be used to improve the efficiency of lymphoablation in sensitized transplant recipients.

An Unanticipated Role for Survivin in Organ Transplant Damage

Ischemia/reperfusion (I/R) injury is a major determinant of graft survival in kidney transplantation. Survivin, an inhibitor of apoptosis that participates in the control of mitosis and cell cycle progression, has been implicated in renal protection and repair after I/R injury; however, no study has been performed in the transplant setting. We investigated the role of survivin in modulating posttransplant I/R injury in syngeneic and allogeneic kidney grafts, and studied whether protection from I/R injury impacted on the recipient immune system, on chronic allograft nephropathy and rejection. We used genetically engineered mice with survivin haploinsufficiency and WT mice in which survivin over-expression was induced by gene-delivery. Survivin haploinsufficiency in syngeneic grafts was associated with exuberant I/R tissue injury, which triggered inflammation eventually resulting in graft loss. Conversely, survivin over-expression in the grafts minimized I/R injury and dysfunction in syngeneic grafts and in a clinically relevant fully MHC-mismatched allogeneic combination. In the latter, survivin over-expression translated into limited anti-donor adaptive immune response and less long-term allograft injury with protection from renal parenchymal damage. Our data support survivin over-expression in the graft as a novel target for protocols aimed at limiting tissue damage at the time of transplant ultimately modulating the recipient immune system.

Attenuation of Portal Hypertension by Continuous Portal Infusion of PGE1 and Immunologic Impact in Adult-to-Adult Living-Donor Liver Transplantation

Small-for-size syndrome remains the greatest limiting factor of expanding segmental liver transplantation from living donors. Portal hyperperfusion is considered to substantially contribute to small-for-size syndrome. We investigated the impact of continuous portal infusion of prostaglandin E1 (PGE1) on small-for-size grafts (SFSGs) in adult-to-adult living-donor liver transplantation (LDLT). From July 2003 to December 2009, LDLT was performed in 122 patients. We introduced continuous portal infusion of PGE1 to five SFSG patients (PG group) from November 2007 to December 2009 and retrospectively compared them with a historical control group of eight relevant SFSG patients without PGE1 infusion (non-PG group) from July 2003 to October 2007 to determine the safety and efficacy of continuous PGE1 portal infusion for SFSGs. Splenectomy cases were excluded from analysis. The PG group demonstrated significantly lower postoperative portal pressure than the non-PG group. Moreover, the PG group demonstrated significantly improved liver function in the early posttransplantation period and significantly better recovery from hyperammonemia at 1 week after transplantation and from hyperbilirubinemia in the late posttransplantation period. Overall survival was significantly better in the PG group than in the non-PG group. Three patients in the non-PG group died of rejection-related reasons. Interestingly, immunomonitoring assay revealed that antidonor immune responses were significantly accelerated in the non-PG group compared with the PG group after LDLT. In contrast, the PG group showed well-suppressed antidonor immune responses. Continuous portal infusion of PGE1 for SFSG attenuated portal hypertension, improved graft function, and suppressed antidonor immune responses, resulting in better survival.

Pretransplant Antithymocyte Globulin Has Increased Efficacy in Controlling Donor-Reactive Memory T Cells in Mice

Antibody-mediated lymphocyte depletion is frequently used as induction therapy in sensitized transplant patients. Although T cells with an effector/memory phenotype remain detectable after lymphoablative therapies in human transplant recipients, the role of preexisting donor-reactive memory in reconstitution of the T cell repertoire and induction of alloimmune responses following lymphoablation is poorly understood. We show in a mouse cardiac transplantation model that antidonor immune responses following treatment with rabbit antimouse thymocyte globulin (mATG) were dominated by T cells derived from the preexisting memory compartment. Administration of mATG 1 week prior to transplantation (pre-TP) was more efficient in targeting preexisting donor-reactive memory T cells, inhibiting overall antidonor T cell responses, and prolonging heart allograft survival than the commonly used treatment at the time of transplantation (peri-TP). The failure of peri-TP mATG to control antidonor memory responses was due to faster recovery of preexisting memory T cells rather than their inefficient depletion. This rapid recovery did not depend on T cell specificity for donor alloantigens suggesting an important role for posttransplant inflammation in this process. Our findings provide insights into the components of the alloimmune response remaining after lymphoablation and may help guide the future use of ATG in sensitized transplant recipients.

Anti‐donor immune responses elicited by allogeneic mesenchymal stem cells: what have we learned so far?

Mesenchymal stem (stromal) cells (MSCs) have potent anti-inflammatory/immunosuppressive properties which underlie much of their therapeutic potential. This fact has led to the widely accepted belief that MSCs from genetically unrelated individuals (allogeneic (allo)-MSCs) can be used therapeutically with equal efficacy to autologous MSCs and without triggering the donor-specific immune responses that are typically associated with allo-transplants. In this article, we critically review available experimental data to determine whether good in vivo evidence exists in support of the 'immune privileged' status of allo-MSCs. We also examine published studies regarding the immunogenicity of allo-MSCs following activation ('licensing') by inflammatory stimuli or following differentiation. Among the identified studies which have addressed in vivo immunogenicity of allo-MSCs, there was substantial variability as regards experimental species, disease model, route of MSC administration, cell dose and stringency of the immunological assays employed. Nonetheless, the majority of these studies has documented specific cellular (T-cell) and humoral (B-cell/antibody) immune responses against donor antigens following administration of non-manipulated, interferon-γ-activated and differentiated allo-MSCs. The consequences of such anti-donor immune responses were also variable and ranged from reduced in vivo survival of allo-MSCs with accelerated rejection of subsequent allogeneic transplants to apparent promotion of donor-specific tolerance. On the basis of these findings and on existing knowledge of allo-antigen recognition from the field of transplant immunology, we propose that the concept of the immune privileged nature of allo-MSCs should be reconsidered and that the range and clinical implications of anti-donor immune responses elicited by allo-MSCs be more precisely studied in human and animal recipients.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Adaptive Immunity Rather Than Viral Cytopathology Mediates Polyomavirus-Associated Nephropathy in Mice

Nephropathy associated with BK polyomavirus causes kidney allograft dysfunction and failure. Understanding the pathogenesis of polyomavirus-associated allograft nephropathy (PVAN) is hampered by the species specificity of Polyomaviridae family members. Using a mouse polyomavirus (MPyV) kidney transplant model, we investigated clinically relevant variables that may contribute to PVAN. We found that the timing and source (i.e. donor vs. recipient) of MPyV infection and the titer of the viral inoculum have significant effects on the extent of allograft injury, with acute infection of the recipient by high-titer MPyV inoculums producing the most profound PVAN. In contrast, altering the degree of MHC matching or increasing ischemia/reperfusion injury by prolonging the cold ischemic time of the allograft did not affect the severity of PVAN. Survival correlated positively with serum creatinine levels, but not with viral loads in the kidney allograft. Using splenectomized alymphoplasia mice, which are unable to mount primary adaptive immune responses, we further demonstrate that persistent high viral loads in the kidney are not sufficient to cause advanced PVAN. These findings suggest that the mechanism of PVAN in mice is not a direct consequence of viral cytopathology, but rather involves interplay between viral infection and the recipient antidonor immune response.

Cell-Based Therapies in the Prevention of Solid Organ Transplant Rejection

Problem statement: Organ transplantation is a life-saving and increasi ngly common procedure, as it often serves as the only treatment available for end-stage organ disease. Although th e constant development of new and more effective immunosuppressive drugs has revolutionized the prevention and treatment of acute graft rejection, these drugs have significant toxicity, greatly incr ease patient susceptibility to neoplasms and infection a nd exert little impact on chronic rejection. Approach: The literature was reviewed to illuminate the mech anisms by which the anti-donor immune response is initiated and how cellular therapies im pact this response. Results: Data show that Donor Specific Transfusion, Apoptotic Cell therapies and Dendritic Cell therapies all function as a source o f alloantigen to suppress the anti-donor T cell respo nse. Conclusion: Cellular therapies hold promise in the prevention of solid organ allograft rejection, but require optimization and study in large animal mode ls before clinical implementation.

IL-21 Is an Antitolerogenic Cytokine of the Late-Phase Alloimmune Response

OBJECTIVEInterleukin-21 (IL-21) is a proinflammatory cytokine that has been shown to affect Treg/Teff balance. However, the mechanism by which IL-21 orchestrates alloimmune response and interplays with Tregs is still unclear.RESEARCH DESIGN AND METHODSThe interplay between IL-21/IL-21R signaling, FoxP3 expression, and Treg survival and function was evaluated in vitro in immunologically relevant assays and in vivo in allogenic and autoimmune models of islet transplantation.RESULTSIL-21R expression decreases on T cells and B cells in vitro and increases in the graft in vivo, while IL-21 levels increase in vitro and in vivo during anti-CD3/anti-CD28 stimulation/allostimulation in the late phase of the alloimmune response. In vitro, IL-21/IL-21R signaling (by using rmIL-21 or genetically modified CD4+ T cells [IL-21 pOrf plasmid–treated or hIL-21-Tg mice]) enhances the T-cell response during anti-CD3/anti-CD28 stimulation/allostimulation, prevents Treg generation, inhibits Treg function, induces Treg apoptosis, and reduces FoxP3 and FoxP3-dependent gene transcripts without affecting FoxP3 methylation status. In vivo targeting of IL-21/IL-21R expands intragraft and peripheral Tregs, promotes Treg neogenesis, and regulates the antidonor immune response, whereas IL-21/IL-21R signaling in Doxa-inducible ROSA-rtTA-IL-21-Tg mice expands Teffs and FoxP3− cells. Treatment with a combination of mIL-21R.Fc and CTLA4-Ig (an inhibitor of the early alloimmune response) leads to robust graft tolerance in a purely alloimmune setting and prolonged islet graft survival in NOD mice.CONCLUSIONSIL-21 interferes with different checkpoints of the FoxP3 Treg chain in the late phase of alloimmune response and, thus, acts as an antitolerogenic cytokine. Blockade of the IL-21/IL-21R pathway could be a precondition for tolerogenic protocols in transplantation.

Non-Engraftment Haploidentical Cellular Immunotherapy for Refractory Malignancies: Tumor Responses without Chimerism

Background: Allogeneic bone marrow transplantation (alloBMT) relies on immunosuppression to control graft-versus-host disease (GVHD) and allow successful engraftment; this happens at the expense of graft versus-tumor (GVT) activity. Advances in hematologic transplantation have prompted the development of effective, less toxic regimens attempting to balance graft-versus-host and GVT immunoreactions Conventional dogma holds that sustained chimerism/engraftment is necessary for prolonged responses. However, our previous study showed long-term response with transient chimerism, suggesting it was not necessary for therapeutic effect. In addition, Deyet al. showed anti-tumor responses with chimerism loss in 9/22 patients after non-myeloablative conditioning and alloBMT [Brit J Haem 128:2005]. In this phase I/II study, we determined recipient response rates with HLA-haploidentical donors, while infusing varying numbers of G-CSF primed cells.Methods: Forty-one patients with relapsed refractory cancers, significant co-morbidities, and ineligible for standard palliative or curative therapy, received 100-cGy total body irradiation (TBI) and infusion of G-CSF primed mobilized HLA-haploidentical cells. The product contained 1×106−2×108 CD3+ cells/kg. Twenty-nine patients received the highest dose.Results: The two highest CD3+ infusion cohorts caused a post-infusional cellular graft rejection syndrome. Symptoms resembled engraftment syndrome with cytokine elevations of IL-6, IL-10, IL-8, IFN-g, IL-5, IL-7, MCP-1 and MIP-1b. In the 26 patients with hematological malignancies there were 14-responses, 9-major. Two of six patients with lymphoma are presently free of disease at 74 and 80-months respectively; there were five durable complete-responses and four partial-responses in 13-patients with AML. All responses occurred without the presence of donor chimerism. Two patients who engrafted died; one due to GVHD and the other disease progression. We speculate that the cells responsible for the cytokine-storm, possibly tied to donor cell rejection, altered host tolerance to tumor and allowed for a host immune response against tumor. This theory is further supported in murine models showing that host anti-donor immune responses causing spontaneous or intentionally induced graft-rejection by recipient lymphocyte infusions, gave anti-tumor responses against recipient tumors [Blood 102:2003].Conclusion: Non-engrafting Haploidentical transplant with minimal myeloablation (100cGy) is a therapeutic strategy appropriate for older patients with significant comorbidities, and it provides a virtual universal donor pool. Side-effects include well tolerated myelotoxicity and an immediate post-infusional steroid responsive immunologic syndrome. TBI of 100cGy followed by HLA-haploidentical transplant is a biologically active therapy for refractory hematologic disease. The responses outside of chimerism are intriguing and may be related to marrow rejection and or a unique type of cytokinestorm. These data further indicate that persistent donor chimerism may not be necessary for durable anti-tumor response. The rejection of donor-derived cells, in concert with elevations of specific cytokines, may represent a novel therapeutic approach for hematological malignancies.

The Control of Anti-Donor Immune Responses by Regulatory T Cells in Organ Transplant Patients

The role of regulatory T cells in the induction and maintenance of transplant tolerance has been widely investigated in experimental animal models. Their involvement in the regulation of allogeneic immune reactivity in immunosuppressed organ transplant patients, however, remains unclear. Measurements of regulatory T cells after clinical organ transplantation may contribute to understanding their role in anti-donor immune responses. Several studies have investigated the frequency and/or immune regulatory function of peripheral regulatory T-cell populations, such as, the CD4 +CD25 + regulatory T cells or the CD8 +CD28 − suppressor T cells, in clinically stable organ transplant patients and patients with acute or chronic rejection. This review summarizes these studies and discusses the correlations found between the number and function of regulatory T cells and the immunological state of the patient. This knowledge is warranted for a better understanding of the control of allogeneic immune responses by regulatory T cells. Subsequently, monitoring regulatory T cells may help us to identify patients in whom the anti-donor reactivity is actively suppressed.

FOXP3 mRNA expression analysis in the peripheral blood and allograft of heart transplant patients

Previously, we demonstrated in heart transplant patients that FOXP3, a gene required for the development and function of regulatory T cells, was highly expressed in the graft during an acute cellular rejection. In this study, we analyzed whether the FOXP3 gene expression in the peripheral blood also reflects anti-donor immune responses, and therefore may provide clues for non-invasive detection of non-responsiveness or acute rejection. We examined the FOXP3 expression patterns of peripheral blood mononuclear cells (PBMC; n=69) of 19 heart transplant patients during quiescence and rejection in comparison with those of endomyocardial biopsies (EMB; n=75) of 24 heart transplant patients. While the FOXP3 mRNA levels were abundantly expressed in rejecting EMB (ISHLT rejection grade >1R) compared with EMB without histological evidence of myocardial damage (ISHLT rejection grade 0R–1R; p=0.003), no association with rejection or non-responsiveness was found for the FOXP3 mRNA levels in the peripheral blood. Thus, in contrast to intragraft FOXP3 gene expression, the peripheral FOXP3 mRNA levels lack correlation with anti-donor immune responses in the graft, and, consequently, FOXP3 does not appear to be a potential candidate gene for non-invasive diagnosis of non-responsiveness or rejection.

The pig‐to‐primate immune response: relevance for xenotransplantation

The allotransplantation of some solid organs can be associated with a graft-vs.-host (GVH) response from the activity of donor B or T cells. We have investigated whether there is a risk of a GVH response following pig-to-primate organ xenotransplantation. The responses of 16 pigs (six farm-housed wild-type and five wild-type housed under high herd health conditions [all designated WT], and 5 alpha1,3-galactosyltransferase gene-knockout [GT-KO] housed under high herd health conditions) to human (n = 6) and baboon (n = 6) peripheral blood mononuclear cells (PBMC) were determined. Assays included flow cytometry, complement-dependent cytotoxicity, and mixed lymphocyte reaction. Anti-primate cytotoxic IgM antibodies were detected in the sera of all pigs, but anti-primate IgG antibodies were minimal. All pigs demonstrated a cellular proliferative response to primate PBMC that was equivalent to, or greater than, the allo response. The strength of the pig-to-primate GVH responses was proportional to the health status of the pigs, those from a high health status herd, particularly from a specific pathogen-free herd maintained under clean husbandry conditions, where colonization of the gastrointestinal tract may be reduced, having lower responses. After pig organ transplantation in a primate, if the organ is from an early-weaned, early-segregated GT-KO pig, the strength of a GVH response is likely to be relatively weak. Although not investigated here, any GVH response is likely to be suppressed by the immunosuppressive therapy administered to the recipient to suppress the anti-donor immune response.

Donor Lymphoid Organs Are a Major Site of Alloreactive T-Cell Priming Following Intestinal Transplantation

We hypothesized that lymphoid organs within intestinal allografts contribute to their immunogenicity. Consistent with this hypothesis recipient T cells rapidly migrated to the lymph nodes and Peyer's patches of syngeneic and allogeneic intestinal grafts such that at 24 h approximately 50% of the lymphocytes isolated from donor lymphoid organs were of recipient origin. However, only in the lymphoid organs of allografts did recipient T cells display an activated phenotype, proliferate and produce IFNgamma. Rejection of allogeneic intestines lacking lymphoid organs was dramatically impaired in splenectomized, lymph node-deficient recipients compared to lymph node bearing, wild-type allogeneic intestines. This demonstrates the important role of donor lymphoid organs in the rejection process. Furthermore, recipient T cells proliferated more extensively and produced more IFNgamma in donor lymphoid organs than in recipient lymphoid organs, indicating that donor lymphoid organs play a dominant role in initiating the recipient anti-donor immune response following intestinal transplantation.

Immunological Unresponsiveness in Chimeric Miniature Swine following MHC-Mismatched Spleen Transplantation

In rodents, spleen allotransplantation (SpTx) induces tolerance. We investigated the induction of chimerism and donor-specific unresponsiveness following pig SpTx. Thirteen pigs underwent splenectomy (day 0); all received a blood transfusion. In 11/13 pigs, SpTx was performed across a MHC class I (n=1) or full (n=10) barrier; two control pigs received no SpTx. All pigs were monitored for chimerism, and anti-donor immune responses, including suppressor assays. Four pigs (two asplenic controls and two with SpTx) underwent delayed donor-matched kidney transplantation without immunosuppression. Six of the 11 spleen grafts were lost from rejection (n=5) or splenic vein thrombosis (n=1), and five remained viable. All 11 SpTx recipients developed multilineage chimerism, but chimerism was rapidly lost if the graft failed. Two control pigs showed <6% blood chimerism for 4 and 11 days only. Pigs with functioning spleen grafts had multilineage chimerism in blood, thymus and bone marrow for at least 2-6 months, without graft-versus-host disease. These pigs developed in vitro donor-specific hyporesponsiveness and suppression. In 2 pigs tolerant to the spleen graft, donor MHC-matched kidney grafts survived for >4 and >7 months in the absence of exogenous immunosuppression; in two asplenic pigs, kidney grafts were rejected on days 4 and 15. Successful SpTx can result in hematopoietic cell engraftment and in vitro donor-specific unresponsiveness, enabling prolonged survival of subsequent donor-matched kidney grafts without immunosuppression.

Induction of transplantation tolerance in non-human primate preclinical models

Short-term outcomes following organ transplantation have improved considerably since the availability of cyclosporine ushered in the modern era of immunosuppression. In spite of this, many of the current limitations to progress in the field are directly related to the existing practice of relatively non-specific immunosuppression. These include increased risks of opportunistic infection and cancer, and toxicity associated with long-term immunosuppressive drug exposure. In addition, long-term graft loss continues to result in part from a failure to adequately control the anti-donor immune response. The development of a safe and reliable means of inducing tolerance would ameliorate these issues and improve the lives of transplant recipients, yet given the improving clinical standard of care, the translation of new therapies has become appropriately more cautious and dependent on increasingly predictive preclinical models. While convenient and easy to use, rodent tolerance models have not to date been reliably capable of predicting a therapy's potential efficacy in humans. Non-human primates possess an immune system that more closely approximates that found in humans, and have served as a more rigorous preclinical testing ground for novel therapies. Prior to clinical adaptation therefore, tolerance regimens should be vetted in non-human primates to ensure that there is sufficient potential for efficacy to justify the risk of its application.

Host thymectomy and cyclosporine lead to unstable skin graft tolerance after class I mismatched allogeneic neonatal thymic transplantation in mice

Background Our laboratory has demonstrated that xenogeneic porcine thymus tissue grafted in thymectomized (ATX) and T cell-depleted mice induces donor-specific tolerance. Recipient thymectomy is essential for the success of tolerance induction. In contrast, studies in pigs grafted with non-vascularized allogeneic class I mismatched thymus tissue under the cover of CyA have shown that removal of host thymus is detrimental to thymic graft survival. To determine the requirements for nonvascularized allogeneic class I-mismatched thymic engraftment in mice, we performed thymic allotransplantation under the cover of CyA. Materials and methods Euthymic and ATX B10.MBR mice received class I mismatched B10.AKM neonatal mouse thymus (NMTHY) tissue under the kidney capsule with or without a short course of CyA. The grafts were allowed to engraft for two and a half months before exploratory laparotomy was performed to evaluate them. Three months after the thymic transplant, mice were challenged with donor-specific skin grafts to assess tolerance. One month after donor-specific skin grafting, they received third party B10.BR skin grafts. Cellular anti-donor immune responses were studied at the time of euthanasia. Results CyA-treated ATX and euthymic control mice showed good engraftment of the allogeneic thymic tissue at the time of exploratory laparotomy, whereas non-CyA-treated ATX and euthymic controls had rejected the grafts. The CyA-treated ATX B10.MBR mice accepted donor-specific skin grafts, but rejected them following a challenge with third party B10.BR skin grafts. Untreated ATX and euthymic mice and 6 of 7 CyA-treated euthymic mice rejected donor skin within 15 days. Mixed lymphocyte reactions did not show an increased anti-donor response, but CML clearly showed sensitization and increased killing activity against donor-type targets in these mice. Conclusion Allogeneic thymic transplantation across a class I MHC barrier under the cover of CyA induces a metastable state of tolerance in mice. To achieve this state, ATX of the recipient is required.

Early biological and immune response to semi-identical liver or kidney allograft in miniature swine

In inbred miniature swine, semi-identical liver allograft recipients survive up to 3 months without immunosuppression, whereas similarly mismatched kidney allografts are uniformly rejected within 2 weeks. The early biological and immunological events were assessed in this unique model. SLA(d/d) pigs (MGH, Harvard Medical School, Boston, MA, USA) received liver or kidney allograft from heterozygous SLA(c/d) miniature swine. Survival, graft function, histology, intragraft cytokines, peripheral lymphocyte and platelet count, plasma cortisol level and cellular/humoral anti-donor immune response were assessed. Kidney allografts were uniformly rejected within 2 weeks, whereas liver allografts survived for up to 87 days. After both liver and kidney transplantation, the peripheral lymphocyte count decreased during the first week concomitantly to a significant elevation of plasma cortisol level. Early decrease of peripheral platelet count was observed after liver but not renal transplantation. Up-regulation of transforming growth factor beta1 (TGF-beta1) and interferon-gamma (IFN-gamma) was observed during the first postoperative week in semi-identical liver allografts and IFN-gamma as well as IL-10 in kidney allografts. In liver recipients, labelled autologous lymphocytes accumulated in the liver graft and native spleen, whereas after renal allograft, lymphocytes accumulated in the native spleen and liver but never in the kidney allograft. Specific cellular anti-donor unresponsiveness was observed from the first post-transplant day in both liver and kidney recipients, while the humoral anti-donor response remained intact. In semi-identical liver allograft, recipient rejection is milder and slower than in similarly matched kidney allograft. The intragraft up-regulation of TGF-beta1 in semi-identical liver allograft might be one mediator to explain the modulation of rejection after liver transplant. The rapid, nonspecific accumulation of recipient lymphocytes in the liver allograft but not in kidney allograft might also play a role in the different survival time in this model.

Intravenous Infusion of Apoptotic Cells Simultaneously with Allogeneic Hematopoietic Grafts Alters Anti-Donor Humoral Immune Responses

Intravenous infusion of apoptotic donor or third-party leukocytes simultaneously with an allogeneic donor bone marrow (BM) graft favors engraftment across major histocompatibility barriers. While verifying that such apoptotic cell infusion might not also be associated with antibody (Ab)-mediated allo-immune responses, we found, rather strikingly, that apoptotic cell infusion could in fact successfully prevent a humoral allo-immunization against a BM graft in mice. Indeed, among recipients having rejected their BM graft, prior apoptotic cell infusion was associated with a near absence of Ab-mediated allo-responses, while such an immunization was frequently observed in the absence of apoptotic cell infusion. This was also observed when infusing host apoptotic cells, thus showing that the prevention of immunization was linked to the apoptotic state of the cells rather than mediated by residual anti-recipient activity. In vivo anti-transforming growth factor-β (TGF-β) treatment resulted in the loss of this apoptotic cell infusion-associated protective effect on humoral allo-responses. Further studies will determine whether apoptotic cell infusion, in addition to hematopoietic graft facilitation might also contribute to preventing deleterious Ab-mediated allo-responses in various transplantation settings.