Codominant Role of Interferon-γ- and Interleukin-17-Producing T Cells During Rejection in Full Facial Transplant Recipients.

Facial transplantation is a life-changing procedure for patients with severe composite facial defects. However, skin is the most immunogenic of all transplants, and better understanding of the immunological processes after facial transplantation is of paramount importance. Here, we describe six patients who underwent full facial transplantation at our institution, with a mean follow-up of 2.7 years. Seum, peripheral blood mononuclear cells, and skin biopsy specimens were collected prospectively, and a detailed characterization of their immune response (51 time points) was performed, defining 47 immune cell subsets, 24 serum cytokines, anti-HLA antibodies, and donor alloreactivity on each sample, producing 4269 data points. In a nonrejecting state, patients had a predominant T helper 2 cell phenotype in the blood. All patients developed at least one episode of acute cellular rejection, which was characterized by increases in interferon-γ/interleukin-17-producing cells in peripheral blood and in the allograft's skin. Serum monocyte chemotactic protein-1 level was significantly increased during rejection compared with prerejection time points. None of the patients developed de novo donor-specific antibodies, despite a fourfold expansion in T follicular helper cells at 1 year posttransplantation. In sum, facial transplantation is frequently complicated by a codominant interferon-γ/interleukin-17-mediated acute cellular rejection process. Despite that, medium-term outcomes are promising with no evidence of de novo donor-specific antibody development.

TIM-1 signaling is required for maintenance and induction of regulatory B cells.

Apart from their role in humoral immunity, B cells can exhibit IL-10-dependent regulatory activity (Bregs). These regulatory subpopulations have been shown to inhibit inflammation and allograft rejection. However, our understanding of Bregs has been hampered by their rarity, lack of a specific marker, and poor insight into their induction and maintenance. We previously demonstrated that T cell immunoglobulin mucin domain-1 (TIM-1) identifies over 70% of IL-10-producing B cells, irrespective of other markers. We now show that TIM-1 is the primary receptor responsible for Breg induction by apoptotic cells (ACs). However, B cells that express a mutant form of TIM-1 lacking the mucin domain (TIM-1(Δmucin) ) exhibit decreased phosphatidylserine binding and are unable to produce IL-10 in response to ACs or by specific ligation with anti-TIM-1. TIM-1(Δmucin) mice also exhibit accelerated allograft rejection, which appears to be due in part to their defect in both baseline and induced IL-10(+) Bregs, since a single transfer of WT TIM-1(+) B cells can restore long-term graft survival. These data suggest that TIM-1 signaling plays a direct role in Breg maintenance and induction both under physiological conditions (in response to ACs) and in response to therapy through TIM-1 ligation. Moreover, they directly demonstrate that the mucin domain regulates TIM-1 signaling.

Standardization and cross validation of alloreactive IFNγ ELISPOT assays within the clinical trials in organ transplantation consortium.

Emerging evidence indicates memory donor-reactive T cells are detrimental to transplant outcome and that quantifying the frequency of IFNγ-producing, donor-reactive PBMCs by ELISPOT has potential utility as an immune monitoring tool. Nonetheless, differences in assay performance among laboratories limit the ability to compare results. In an effort to standardize assays, we prepared a panel of common cellular reagent standards, developed and cross validated a standard operating procedure (SOP) for alloreactive IFNγ ELISPOT assays in several research laboratories supported by the NIH-funded Clinical Trials in Organ Transplantation (CTOT) Consortium. We demonstrate that strict adherence to the SOP and centralized data analysis results in high reproducibility with a coefficient of variance (CV) of ≈ 30%. This standardization of IFNγ ELISPOT assay will facilitate interpretation of data from multicenter transplantation research studies and provide the foundation for developing clinical laboratory testing strategies to guide therapeutic decision-making in transplant patients.

Impact of calcineurin-inhibitor conversion to mTOR inhibitor on renal allograft function in a prednisone-free regimen.

Traditionally, chronic calcineurin inhibitor (CNI) nephrotoxicity has been considered to be one of the main nonimmune mechanisms causing chronic renal allograft dysfunction. CNI minimization and withdrawal strategies have yielded inconsistent results. Few studies address the feasibility of CNI elimination in a prednisone-free regimen. We report a prospective, randomized trial in 200 patients evaluating the impact on renal function and incidence of acute rejection after conversion from tacrolimus (Tac) to sirolimus (SRL). Patients with recent (<3 months) acute rejection episodes or with >0.5 g/day of proteinuria were excluded. All were induced with alemtuzumab, underwent rapid steroid elimination and were maintained on mycophenolate mofetil and Tac. At 12 months posttransplant, patients were randomized 2:1 to SRL (n = 123) or maintained on Tac (n = 64). Mean follow-up was 41.1 ± 15.8 months in the SRL group and 40.7 ± 14.4 months in the Tac group. Biopsy-proven acute rejection at 24 months postrandomization was similar between the groups. Patient survival, graft survival and estimated GFR were also not statistically different. Our study demonstrates that in a prednisone-free immunosuppressive regimen, conversion from Tac to SRL at 12 months posttransplantation is not associated with increased rates of acute rejection and graft loss. However, despite CNI elimination, renal allograft function is equally maintained in both groups.

The role of costimulatory molecules in directing the functional differentiation of alloreactive T helper cells.

Costimulatory molecules are a heterogenous group of cell surface molecules that act to amplify or counteract the initial activating signals provided to T cells from the T cell receptor following its interaction with an antigen/major histocompatibility complex, thereby influencing T cell differentiation and fate. Although costimulation was previously thought to be indispensable for T cell activation at all stages of development, it is now known that the requirements for costimulation, and the costimulatory molecules involved, vary according to the stage of T cell differentiation. The ability to influence T cell fate is of paramount interest in the field of transplantation as we seek therapeutic options that inhibit detrimental alloimmune responses whilst simultaneously promoting allograft tolerance. As with many immune mechanisms, there is a degree of functional overlap between certain costimulatory molecules, whereas some have diametrically opposite effects on different T cell subsets despite sharing common ligands. This is a critical point when considering these molecules as therapeutic targets in transplantation, as blockade of a costimulatory pathway, although desirable in itself, may prevent the ligation of an essential regulatory coinhibitory molecule. This review discusses the T helper cell lineages pertinent to transplantation and the costimulatory molecules involved in their differentiation.

Ex vivo expansion of human Tregs by rabbit ATG is dependent on intact STAT3-signaling in CD4⁺ T cells and requires the presence of monocytes.

The addition of low, nondepleting doses of rabbit antithymocyte globulin (ATG) to human peripheral blood mononuclear cells has been shown to expand functional CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) in vitro. This report is the first to elucidate the exact cellular mechanisms of ATG-mediated Treg expansion. CD4(+) T cells require monocytes, but not other antigen presenting cell subsets, to be present in coculture to expand Tregs. However, T cells do not require direct cell-cell contact with monocytes, suggesting the importance of soluble factors. Moreover, ATG initially "reprograms" CD4(+) T cells, but not monocytes, and induces STAT3 and STAT5 signaling in CD4(+) cells. These reprogrammed CD4(+) T cells subsequently secrete GM-CSF and IL-10 only in case of intact STAT3 signaling, which in turn promote the generation of tolerogenic CD14(+) CD11c(+) dendritic cells characterized by enhanced IL-10 and decreased IL-12 production. Treg expansion following ATG treatment is accompanied by enhanced gene expression of both GM-CSF and Bcl-2, but not TGF-ß, in peripheral blood mononuclear cells. These results demonstrate that ex vivo expansion of human Tregs by ATG is due to its ability to reprogram CD4(+) T cells in a STAT3-dependent but TGF-ß-independent manner, leading to the generation of monocyte-derived dendritic cells with a tolerogenic cytokine profile.

Overcoming memory T-cell responses for induction of delayed tolerance in nonhuman primates.

The presence of alloreactive memory T cells is a major barrier for induction of tolerance in primates. In theory, delaying conditioning for tolerance induction until after organ transplantation could further decrease the efficacy of the regimen, since preexisting alloreactive memory T cells might be stimulated by the transplanted organ. Here, we show that such "delayed tolerance" can be induced in nonhuman primates through the mixed chimerism approach, if specific modifications to overcome/avoid donor-specific memory T-cell responses are provided. These modifications include adequate depletion of CD8+ memory T cells and timing of donor bone marrow administration to minimize levels of proinflammatory cytokines. Using this modified approach, mixed chimerism was induced successfully in 11 of 13 recipients of previously placed renal allografts and long-term survival without immunosuppression could be achieved in at least 6 of these 11 animals.

The emerging role of the TIM molecules in transplantation.

Since their discovery in 2001, the T-cell immunoglobulin mucin (TIM) family members have been shown to play important roles in the regulation of immune responses. The TIM family comprises of eight genes in the mouse, three of which are conserved in humans (TIM-1, TIM-3 and TIM-4). Initially, TIM-1 and TIM-3 were thought to be expressed solely on T cells. However, emerging data suggest a much broader expression pattern where their presence on APCs confers differing functions, including the ability to mediate phagocytosis. In contrast, TIM-4 is exclusively expressed on APCs. Together, the TIM molecules provide a functional repertoire for determining the fate of T-cell activation and differentiation. To date, much of the knowledge about the TIM family members has been garnered from the models of asthma, allergy and autoimmunity. More recently, data from experimental models of transplantation demonstrate that TIM family members also have a key role in alloimmunity. This review will serve to highlight the emerging data regarding this unique family of molecules and to identify their potential in transplantation tolerance.

Essential role of PDL1 expression on nonhematopoietic donor cells in acquired tolerance to vascularized cardiac allografts.

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen-presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC-mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4-Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long-term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1-deficient endothelium hearts had significant higher frequency of IFN-γ-producing alloreactive cells as well as higher frequency of CD8(+) effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance.

In vivo function of immune inhibitory molecule B7-H4 in alloimmune responses.

B7 ligands deliver both costimulatory and coinhibitory signals to the CD28 family of receptors on T lymphocytes, the balance between which determines the ultimate immune response. Although B7-H4, a recently discovered member of the B7 family, is known to negatively regulate T cell immunity in autoimmunity and cancer, its role in solid organ allograft rejection and tolerance has not been established. Targeting the B7-H4 molecule by a blocking antibody or use of B7-H4(-/-) mice as recipients of fully MHC-mismatched cardiac allografts did not affect graft survival. However, B7-H4 blockade resulted in accelerated allograft rejection in CD28-deficient recipients. B7-1/B7-2-double-deficient recipients are truly independent of CD28/CTLA-4:B7 signals and usually accept MHC-mismatched heart allografts. Blockade of B7-H4 in these mice also precipitated rejection, demonstrating regulatory function of this molecule independent of an intact CD28/CTLA-4:B7 costimulatory pathway. Accelerated allograft rejection was always accompanied by increased frequencies of alloreactive IFN-γ-, IL-4- and Granzyme B-producing splenocytes. Finally, intact recipient, but not donor, B7-H4 is essential for prolongation of allograft survival by blocking CD28/CTLA4:B7 pathway using CTLA4-Ig. These data are the first to provide evidence of the regulatory effects of B7-H4 in alloimmune responses in a murine model of solid organ transplantation.

Differential requirement of CD27 costimulatory signaling for naïve versus alloantigen-primed effector/memory CD8+ T cells.

CD8(+) memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T-cell costimulatory pathway in alloreactive CD8(+)/CD4(+) T-cell activation. CD27-deficient (CD27(-/-)) and wild-type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4(+) or CD8(+) T cells, suggesting that CD27 is not essential during primary T-cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27(-/-) or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor-type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40- but not day 10-sensitized CD27(-/-) recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8(+) T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27(-/-) recipients of class I- but not class II-mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8(+) memory T cells in acute and chronic allograft rejection.

Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4(+) T cells secreting Th1 cytokines, while recovery from disease is associated with expression of Th2 cytokines. Investigations into the role of individual cytokines in disease induction have yielded contradictory results. Here we used animals with targeted deletion of the STAT4 or STAT6 genes to determine the role of these signaling molecules in EAE. The STAT4 pathway controls the differentiation of cells into a Th1 phenotype, while the STAT6 pathway controls the differentiation of cells into a Th2 phenotype. We found that mice deficient in STAT4 are resistant to the induction of EAE, with minimal inflammatory infiltrates in the central nervous system. In contrast, STAT6-deficient mice, which have a predominantly Th1 phenotype, experience a more severe clinical course of EAE as compared with wild-type or STAT4 knockout mice. In addition, adoptive transfer studies confirm the regulatory functions of a Th2 environment in vivo. These novel data indicate that STAT4 and STAT6 genes play a critical role in regulating the autoimmune response in EAE.

CD28-independent induction of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated disease initiated by antigen-specific CD4(+) T cells. Signaling through CD28 is a critical second signal for activation of T cells, and CD28 knockout (CD28KO) mice have been reported to be resistant to induction of EAE. We now report that CD28KO mice have no intrinsic defect in mediating disease, because they developed EAE after passive transfer of primed T cells. After immunization, peripheral T cells from CD28KO mice were primed and developed memory phenotype, but had decreased antigen-specific IFN-gamma production as compared with cells from wild-type (WT) animals. Reimmunization of CD28KO mice brought out clinical disease and increased IFN-gamma production in vitro. Pathologically, there were cellular infiltrates in the central nervous system, in contrast to single-immunized mice. We show furthermore that blocking B7-1 or CTLA4, but not B7-2, in CD28KO mice induces disease after a single immunization. Thus, EAE can be induced in animals lacking CD28-dependent costimulation, suggesting that alternative costimulatory pathways were used. Blocking the OX40-OX40L costimulatory pathway differentially affected disease induction in CD28KO mice as compared with WT controls. Our data show that EAE may develop in the absence of CD28 T-cell costimulation. These findings have implications for therapies aimed at blocking costimulatory signals in autoimmune diseases.

Regulatory functions of self-restricted MHC class II allopeptide-specific Th2 clones in vivo.

We studied T-cell clones generated from grafts of rejecting and tolerant animals and investigated the regulatory function of Th2 clones in vitro and in vivo. To prevent allograft rejection, we treated LEW strain recipient rats of WF strain kidney grafts with CTLA4Ig to block CD28-B7 costimulation. We then isolated epitope-specific T-cell clones from the engrafted tissue, using a donor-derived immunodominant class II MHC allopeptide presented by recipient antigen-presenting cells. Acutely rejected tissue from untreated animals yielded self-restricted, allopeptide-specific T-cell clones that produced IFN-gamma, whereas clones from tolerant animals produced IL-4 and IL-10. Adoptive transfer into naive recipients of Th1 clones, but not Th2 clones, induced alloantigen-specific delayed-type hypersensitivity (DTH) responses. In addition, Th2 clones suppressed DTH responses mediated by Th1 clones in vivo and blocked Th1 cell proliferation and IFN-gamma production in vitro. A pilot human study showed that HLA-DR allopeptide-specific T-cell clones generated from patients with chronic rejection secrete Th1 cytokines, whereas those from patients with stable graft function produce Th2 cytokines in response to donor-specific HLA-DR allopeptides. We suggest that self-restricted alloantigen-specific Th2 clones may regulate the alloimmune responses and promote long-term allograft survival and tolerance.

Ganciclovir Dosing Strategies and Development of Cytomegalovirus Resistance in a Kidney Transplant Recipient: A Case Report.

In renal transplant recipients, delayed graft function and accompanying renal impairment may lead to therapeutic underexposure of valganciclovir. We describe a case of a cytomegalovirus (CMV)-seronegative kidney transplant recipient from a CMV-seropositive donor, whose course was complicated during valganciclovir prophylaxis by CMV disease, ultimately progressing to ganciclovir, foscarnet, and cidofovir resistance. Assessments and adjustments for renal dysfunction, according to both Cockgroft-Gault and Modification of Diet in Renal Disease study equations, are described. Therapy was complicated by outpatient parenteral therapy with pump-administered antiviral therapy, which may have led to drug underexposure and the fostering of antiviral resistance. Suppression was ultimately achieved in conjunction with reduction in immunosuppressive therapy, CMV immunoglobulin, and initiation of leflunomide. At-risk recipients may benefit from 24 hour creatinine clearance assessments, direct creatinine clearance measurement, or therapeutic drug monitoring. Optimal dosing strategies in recipients with impaired kidney function remain undefined, with limited pharmacokinetic data to date.

CTLA4-Ig: a novel immunosuppressive agent.

Activation of naive T-cells requires two signals: one is antigen-specific and based on T-cell receptor (TCR) recognition of a peptide-MHC complex and the second is antigen-nonspecific and delivered by specific T-cell receptors after ligation with their ligands (costimulatory molecules) expressed by antigen-presenting cells (APCs). Engagement of the B7 family of molecules on APCs with their T-cell associated ligands, CD28 and CTLA-4 (CD152), provides a pivotal costimulatoty signal in T-cell activation. The lack of costimulation after engagement of the T-cell receptor by antigen, results in a state of antigen-specific unresponsiveness, termed anergy. Manipulation of CD28/B7 pathway has therefore been envisioned as a potential strategy for achieving therapeutically useful immunosuppression or tolerance. CTLA4-Ig has been initially developed by Bristol-Myers Squibb as a competitive inhibitor of CD28/B7 pathway (BMS-188667). Thereafter, CTLA4-Ig was produced by Repligen and also in some individual laboratories. In various animal models, discussed in this paper, CTLA4-Ig has been shown to inhibit T-cell-dependent antibody responses, significantly prolong transplanted organ survival, induce long-term donor-specific tolerance in some models, slow progression of autoimmune disease and to have immunomodulatory function in several other immunological disease models. Recently, CTLA4-Ig has entered Phase I clinical trials for the treatment of psoriasis, a T-cell mediated skin disease and treatment of graft-versus-host disease in allogeneic bone marrow transplantation. Large clinical randomised trials on the use of CTLA4-Ig are missing, nevertheless, its immunosuppressive effects coupled with features such as specificity of interaction and low toxicity, make CTLA4-Ig a promising new therapeutic agent for induction of donor-specific immunological tolerance, the ultimate goal of clinical immunosuppression.

T Helper Cells Fate Mapping by Co-stimulatory Molecules and its Functions in Allograft Rejection and Tolerance.

T cell differentiation is dictated by a combination of T cell receptor (TCR) interaction with an antigen-bound major histocompatibility complex (MHC), and co-stimulatory molecules signal. The co-stimulatory signal can be positive or negative, and amplifying or diminishing the initial signal. However, the secondary co-stimulatory signal is not obligatory and its necessity is dictated, in part, by the stage of T cell development. In the field of transplantation, directing the T cell differentiation process can lead to therapeutic possibilities that promote allograft tolerance, and hinder unfavorable alloimmune responses. Therefore, understanding the details of T cell differentiation process, including the influence of co-stimulatory signals, is of paramount importance. It is important to note there is functional overlap between co-stimulatory molecules. It has been observed that some co-stimulatory signals have different effects on different T cell subsets. Hence, blockade of a co-stimulatory signal pathway, as part of a therapeutic regimen in transplantation, may have far reaching effects beyond the initial therapeutic intent and inhibit co-stimulatory signals necessary for desirable regulatory responses. In this review, co-stimulatory molecules involved in the differentiation of naïve T cells into T helper 1 (Th1), T helper 2 (Th2), T helper 17 (Th17), inducible regulatory T cells (iTregs), and T helper 9 (Th9) cells and their overlap are discussed.