Long-term Graft Tolerance Research Articles

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5mg/kg) for 14days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1×106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500µL ip, POD 1, 5). Banff grade III or 120days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28days in CTRL (n=7), 34 in ASC (n=6), and 27.5 in ASC-CYP (n=4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

The mechanism of CD8+ CD45RClow regulatory T cell in rat liver transplantation nature tolerance model

Objective To explore the mechanism of CD8+ CD45RClow regulatory T cells and its related cytokines function during the tolerance process of orthotopic liver transplantation in rats. Methods The male Brown Norway rats were selected as the donor, the Lewis rats were the recipients, the allogenic orthotopic liver transplantation model was established by the double cuff method. Experiment animals were divided into early transplantation group (10 rats), tolerance group (10 rats), sham group. Liver function were detected. The tolerance recipients were detected by hematoxylin and eosin (HE) staining. The Luminex used to detect of cytokine and the flow cytometry were used to analyze the changes of subsets of lymphocytes in the peripheral blood/graft and draining lymph node. Results 70% of recipients in operation group could be induced natural immune tolerance. The levels of aspartate aminotransferase (AST) in tolerance group were [(456±54) U/ml] vs. those of sham group [(460±151) U/ml], (P>0.05); the levels of alanine aminotransferase (ALT) in tolerance group were [(122±35) U/ml] vs. those of sham group [(121±46) U/ml, P>0.05]. The expression of CD8+ CD45RClow T cells in tolerance group were increased than in short term group; in grafts it was up regulated in tolerance group than in sham group, and it was no significance between sham group and tolerance group in lymph node around graft. The expression of pDC in short term group are significantly increased compared with sham group. The interferon (IFN)-γ was increased in short term group, return to normal level in tolerance group. interleukin (IL)-10 in grafts milieu were dramatically increased, keep in high level in tolerance group. Conclusion In the tolerance recipients of rat orthotopic liver transplantation model, CD8+ CD45RClow regulatory T cells were significantly increased and aggregated in the peripheral blood and liver. The inhibitory cytokines IL-10 was important to maintain the long-term graft tolerance. Key words: Regulatory T cell; Liver transplantation; Immune tolerance; Interleukin-10; Dendritic cell

ATP and T-cell-mediated rejection.

Purine nucleosides and nucleotides are released in the extracellular space following cell injury and act as paracrine mediators through a number of dedicated membrane receptors. In particular, extracellular ATP (eATP) significantly influences T-lymphocyte activation and phenotype. The purpose of this review is to discuss the role of ATP signaling in the T-cell-mediated alloimmune response. In various animal models of solid transplantation, the purinergic axis has been targeted to prevent acute rejection and to promote long-term graft tolerance. The inhibition of ATP-gated P2X receptors has been shown to halt lymphocyte activation, to downregulate both Th1 and Th17 responses and to promote T-regulatory (Treg) cell differentiation. Similarly, the inhibition of ATP signaling attenuated graft-versus-host disease in mice undergoing hematopoietic cell transplantation. Significantly, different drugs targeting the purinergic system have been recently approved for human use and may be a viable therapeutic option for transplant patients. The inhibition of eATP signaling downregulates the alloimmune response, expands Treg cells and promotes graft survival. This robust preclinical evidence and the recent advances in pharmacological research may lead to intriguing clinical applications.

Immunosuppression in the pediatric transplant recipient

The field of pediatric solid-organ transplantation has significantly evolved since its beginnings in the early 20th century. As advancements have led to the development of innovative surgical techniques and novel medication regimens, transplantation has now become a routine practice leading to an increase in the rates of organ recipients worldwide.The care of pediatric solid-organ transplant recipients differs from adults in several areas not only due to technically challenging surgeries, but mostly due to the complexity of their immunosuppression management. Although there is large variation of pediatric immunosuppression regimens worldwide, the use of calcineurin inhibitors, either tacrolimus or cyclosporine, still forms the backbone of immunosuppression regimens after solid-organ transplantation. Both medications are relatively well tolerated but are known to have long-term side effects, especially nephrotoxicity and neurotoxicity. The goal of care in long-term pediatric survivors of solid-organ transplant now aims to safely minimize exposure to immunosuppression and to achieve long-term graft tolerance.

Editorial: Heme Oxygenases: Novel Regulators of Reproductive Processes.

Editorial: Heme Oxygenases: Novel Regulators of Reproductive Processes.

Protection of islet grafts through transforming growth factor-β-induced tolerogenic dendritic cells.

In type 1 diabetes, the insulin-producing β-cells are destroyed by the immune system. One way of restoring glucose control is to transplant β-cells from a donor. Although this procedure may restore endogenous insulin production, immunosuppressive treatment is needed to prevent the recipient from rejecting the donor-derived islets. We investigated the possibilities of transient expression of the immunosuppressive cytokine transforming growth factor (TGF)-β within islets to achieve long-term graft tolerance. We found that brief expression of TGF-β prevented rejection of syngeneic islets, that there was reduction of dendritic cell (DC) activation in the graft, and that there was reduced reactivation of T cells in the graft-draining lymph nodes. In vitro exposure of bone marrow–derived DCs to TGF-β reduced expression of costimulatory molecules CD80 and CD86, as well as production of proinflammatory cytokines such as interleukin-12 p70 in DCs, but did not alter levels of major histocompatibility complex classes I and II. Furthermore, the capacity of TGF-β–treated bone marrow–derived DCs to activate both CD4+ and CD8+ T cells was reduced. Adding TGF-β–conditioned tolerogenic DCs to the grafted islets led to long-term survival of the graft, demonstrating that TGF-β–induced tolerogenic DCs can provide an effective means to restore immune tolerance in an already established autoimmune disease.

Innate immunity in transplant tolerance and rejection

Historically, research on transplant rejection and tolerance has focused on cells of the adaptive immune system, especially T cells. Anti-graft effector T cells are necessary and sufficient for the rejection of most allografts, while regulatory T cells, either arising naturally or as a result of a specific treatment regimen, are crucial to long-term graft tolerance. Although the role of T cells in transplant rejection and tolerance is well-established, the role that the innate immune system plays in these processes is only recently being appreciated. Cells of the innate immune system, such as dendritic cells (DCs) and natural killer cells, can become activated by microbial products or endogenous pro-inflammatory ligands released during the mechanical and ischemia-reperfusion injury associated with transplantation surgery, promoting the initiation of T-cell responses against the graft. In addition, innate immune cells are required for acute and chronic rejection in certain animal transplant models and by extension perhaps in clinical transplantation. However, innate immune cells are also implicated in the establishment of transplant tolerance through mechanisms including elimination or inhibition of activated host effector T cells and killing of donor DCs. A deeper understanding of the functions of the innate immune system during transplantation may lead to more successful tolerance strategies.

α4β7± Regulatory T Cells (Tregs) at Engraftment Predict Long-Term Graft-Versus-Host Disease (GVHD) Outcomes.

Abstract 2237Poster Board II-214 Background:GVHD occurs unpredictably after allogeneic stem cell transplant (ASCT). GVHD is characterized by profound immune dysregulation, which suggests that abnormalities in suppressor lymphocytes known as Tregs may account for the clinical features of this disease. We have shown that the frequency of Tregs or α4β7± Tregs at neutrophil engraftment predict for recurrent grade II-IV GVHD or recurrent gut GVHD during the first 100 days of transplant, respectively. These data indicate that early lymphocyte populations may determine long-term immune-tolerance following ASCT. We hypothesized that Treg subsets present at engraftment would be associated with the occurrence, National Institutes of Health (NIH) phenotype, and severity of GVHD after day±100. Methods:Patients (pts) undergoing T cell replete ASCT were enrolled. GVHD after day±100 was classified prospectively as either: acute GVHD, overlap GVHD, or classic chronic GVHD using consensus criteria derived from the NIH. Clinical severity was determined by the modified Glucksberg criteria for acute GVHD and the NIH global assessment of severity for overlap/classic chronic GVHD. The frequency of CD45RO±CD25±Foxp3±CD127lo Tregs was quantified within the CD4± T-lymphocyte population at the time of neutrophil engraftment using polychromatic flow cytometry. Gut-homing Tregs were identified by the presence of α4β7±, and their frequency was expressed as a percentage of the total Treg population. Results:Treg analysis was performed at a median of 19 days post-transplant (range, 10-31) on 41 pts undergoing ASCT and surviving until day±100. Twenty-four (59%) pts received ablative conditioning and 17 (41%) pts received a reduced intensity regimen followed by matched related [N= 24 (59%)] or unrelated donor [N= 17 (41%)] ASCT. The stem cell source was peripheral blood, bone marrow, or cord blood for 30 (73%), 9 (22%), and 2 (5%) pts, respectively. GVHD prophylaxis consisted of calcineurin inhibitor and methotrexate [N=23 (56%)] or mycophenolate mofetil [N=18 (44%)]. The majority of patients had grade II-IV acute GVHD prior to day±100 [N=34 (83%)]. After a median follow-up of 15 months (range, 5-24) GVHD after day±100 occurred in 32 (78%) pts at a median of 152 days post ASCT (range, 103-533). GVHD phenotype was classified as: acute (N=19), overlap (N=9), or classic chronic (N=4). Grade III-IV acute and moderate to severe overlap/classic chronic GVHD occurred in 3 (7%) and 11 (27%) pts, respectively. Pts with GVHD after day±100 had lower frequencies of Tregs and α4β7± Tregs at engraftment (4.4% vs. 14.3%; P=0.145) and (9.9% vs. 20.7%; P=0.009), respectively. After excluding pts with gut GVHD, the association between α4β7± Tregs and GVHD after day±100 persisted (P=0.034). Median Treg percentages did not differ among GVHD phenotypes [acute (4.4%) vs. overlap (4.2%) vs. classic chronic (5%) vs. none (14.3%); P=0.464]. However, significant differences between α4β7± Treg frequencies and GVHD subtypes were noted [acute (11%) vs. overlap (9.6%) vs. classic chronic (11.6%) vs. none (20.7%); P=0.002]. α4β7± Treg differences were most apparent when pts with any acute GVHD (acute±overlap) were compared to pts without features of acute GVHD (none±classic chronic) (9.9% vs. 19.4%; P=0.009). Increased frequencies of Tregs and α4β7± Tregs at engraftment tended to be associated with lower grades of acute GVHD after day±100 [odds ratio (OR), 0.95; 95% confidence interval (CI), 0.87-1.04; P=0.255 (Tregs) and OR, 0.81; 95% CI, 0.70-0.92; P=0.002 (α4β7± Tregs)] and less severe overlap/classic chronic GVHD [OR, 0.91; 95% CI, 0.79-1.04; P=0.158 (Tregs) and OR, 0.93; 95% CI, 0.86-1.01; P=0.071 (α4β7± Tregs)]. Using Cox Proportional Hazard regression (adjusting for donor type and acute GVHD prior to day ±100), increased frequencies of α4β7± Tregs at engraftment continued to be associated with decreased risk of GVHD after day ±100 (hazard ratio, 0.93; 95%CI, 0.88-0.99; P=0.013). Conclusion:Frequency of α4β7± Tregs at engraftment is associated with the incidence and severity of GVHD after day ±100. These data suggest that early lymphocyte subsets and gut associated mucosal immunity are important in determining long-term graft tolerance. Treg subsets at engraftment could be used to risk stratify pts prior to development of GVHD or as an endpoint in future clinical trials examining interventions aimed at increasing Tregs and preventing GVHD. Disclosures:No relevant conflicts of interest to declare.

Calcineurin inhibitor minimization in pediatric liver allograft recipients

The use of CNI in pediatric LTx has dramatically improved the outcome for children with end-stage liver disease by significantly reducing the rate of acute and chronic rejection. Long-term concerns about CNI-induced nephrotoxicity and other adverse effects remain an issue, particularly as the emphasis moves from short-term survival to long-term quality of life. This review summarizes lessons learnt from pediatric and adult solid organ transplantation in minimizing CNI use in immunosuppression protocols in children following LTx. There are three models for CNI minimization: dose reduction, withdrawal or avoidance, supplemented by the use of IL-2 receptor blocking antibodies in the peri-transplant period, and early transition to alternate drugs such as MMF or SRL. Prospective studies evaluating reduction or withdrawal protocols in adult and pediatric LTx indicate that rejection rates are comparable with traditional CNI-based immunosuppression and that two and five yr patient and graft survival are similar, with recovery in renal function. There are few studies evaluating complete avoidance of CNI, apart from that in renal transplantation, although the benefits of long-term reduction in cardiovascular, metabolic, and possibly neoplastic side effects may justify this approach. It is not clear yet how CNI minimization will affect the development of tolerance but experimental and preliminary clinical studies indicate that CNI and steroid avoidance or minimization in the peri-operative period may favor the development of long-term graft tolerance. In summary, CNI minimization may be safe and effective in the short term but large-scale pediatric randomized studies are required to evaluate the long-term efficacy of these regimes in the development of chronic rejection, PTLD, and graft tolerance.

Donor bone marrow transplantation: Chimerism and tolerance

Infusion of donor bone marrow (DBM)-derived cells continue to be tested in clinical protocols intended to induce specific immunologic tolerance. Central clonal deletion of donor-specific alloreactive cells associated with mixed chimerism reliably produced long-term graft tolerance. In this setting, depletion of recipient T cells by antilymphocyte antibodies and subsequent repopulation by donor hematopoietic cells after donor bone marrow infusion (DBMI) are prerequisites for tolerance induction. Major advances have been made in animal models and in pilot clinical trials and the key questions with the future perspectives are presented in this article.

Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells.

Naturally arising CD25(+)CD4(+) regulatory T (T(R)) cells can be exploited to establish immunologic tolerance to non-self antigens. In vivo exposure of CD25(+)CD4(+) T cells from normal naive mice to alloantigen in a T cell-deficient environment elicited spontaneous expansion of alloantigen-specific CD25(+)CD4(+) T(R) cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. The expanded T(R) cells, which became CD25(low) in the absence of other T cells, stably sustained suppressive activity, maintained expression levels of other T(R) cell-associated molecules, including Foxp3, CTLA-4 and GITR, and could adoptively transfer tolerance to normal mice. Furthermore, specific removal of the T(R) cells derived from originally transferred CD25(+)CD4(+) T(R) cells evoked graft rejection in the long-term tolerant mice, indicating that any T(R) cells deriving from CD25(-)CD4(+) naive T cells minimally contribute to graft tolerance and that natural T(R) cells are unable to infectiously confer significant suppressive activity to other T cells. Similar antigen-specific expansion of T(R) cells can also be achieved in vitro by stimulating naturally present CD25(+)CD4(+) T cells with alloantigen in the presence of IL-2. The expanded CD25(+)CD4(+) T cells potently suppressed even secondary MLR in vitro and, by in vivo transfer, established antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of antigen-specific expansion of naturally arising Foxp3(+)CD25(+)CD4(+) T(R) cells can establish antigen-specific dominant tolerance to non-self antigens, and would also be instrumental in re-establishing self-tolerance in autoimmune disease and antigen-specific negative control of pathological immune responses.

Tolerance induction in clinical transplantation: the pending questions.

Despite the dramatic improvement of early graft survival due to the introduction of cyclosporin, late graft loss caused by chronic rejection and the lethal consequences of long-term immunosuppression, such as infection and cancer, remain major concerns for the transplantation community. Tolerance induction would avoid these complications. The ways to go are controversial, reflecting the redundancy of rejection pathways. They include the induction of central tolerance by establishment of mixed chimerism through hematopoietic stem cell transplantation and the induction of "operational tolerance" through immunodeviation involving dendritic or regulatory T cells. Major advances have been made in animal models exploring these strategies and, some preliminary data are even now available in humans, allowing the initiation of pilot clinical trials. In this article, we discuss the key questions that these trials will have to address.

Microchimerism, GVHD, and tolerance in solid organ transplantation.

The phenomenon of microchimerism and its relationship to long-term graft tolerance is an area of active study. The ability to establish a tolerant state has been enhanced with current immunosuppressive drugs and emerging therapies such as donor HPC infusions. An undesirable outcome of host-donor WBC interaction is GVHD. GVHD is a rare complication reported most frequently in liver transplantation. Two cases of GVHD reported in recipients of organs from donors homozygous for a shared HLA haplotype would support a policy of avoiding the use of these donors. TA-GVHD is very rare in solid organ transplant recipients, with only four published cases; only two had convincing supportive evidence and one of these had an underlying hematologic abnormality. These few cases do not support a policy of routine irradiation of cellular blood components for all solid organ transplant recipients. The use of donor HPC infusions to enhance chimerism and graft tolerance has increased the number of GVHD cases observed (usually mild) and decreased the severity and number of rejection episodes. The long-term effects of donor HPC infusions on graft survival is under investigation.

Transplantation tolerance and mixed chimerism: at the frontier of clinical application

Although the persistence of donor-type hematopoietic cells in low numbers (microchimerism) is well established in some transplant recipients, its relevance for graft acceptance is still a matter of debate. On the other hand, clonal deletion of donor-specific alloreactive cells associated with mixed chimerism (macrochimerism) has reliably produced long-term graft tolerance in pre-clinical models. So far, the cytoablative conditioning regimens required to achieve mixed chimerism have hampered the clinical development of such protocols. Here, we discuss recent observations suggesting that the deliberate induction of hematopoietic cell chimerism might become a feasible strategy to achieve transplantation tolerance in clinics.

Induction of Long-Term Graft Tolerance and Donor/Recipient Chimerism

We compared the effect of different immunosuppressive drug regimens on both mean and long-term allograft survival and the histologic appearance of donor/recipient chimeras in the ACI to Lewis rat heterotopic cardiac transplant model. Intraabdominal cardiac transplantation was performed in standard fashion and microchimerism was assessed using immunohistochemical staining of cut sections of recipient spleen and lymph nodes. All of the drug regimens studied resulted in excellent mean allograft survival. Furthermore, long-term (>120 day) allograft survival was consistently observed in each group. In fact, many of the recipients continue to have functioning heterotopic grafts even as they approach the end of the natural life span of a Lewis rat. Mixed allogeneic chimerism, however, was not observed with 100% frequency despite the consistent induction of graft tolerance. This finding may be related to the immunosuppressive agents used or the sensitivity of the immunohistochemical assay. Therefore, the exact role, if any, of chimerism in the induction of graft tolerance remains unanswered.