Model Of Allograft Rejection Research Articles

A Model of Acute Antibody-Mediated Renal Allograft Rejection in the Sensitized Rata.

Antibody-mediated rejection in transplant recipients with preexisting donor-specific antibodies is a challenging clinical situation. However, we lack suitable animal models to study this scenario. The aim of this study was to develop an animal model of acute antibody-mediated rejection of renal allografts in sensitized recipients. We used major histocompatibility complex class I and II incompatible rat strains (Dark Agouti RT1av1 and Lewis RT1l), which develop aggressive rejection. Recipient Lewis rats were immunized with donor strain spleen cells 5 days before surgery to induce donor-specific antibodies. Rats underwent bilateral nephrectomy and orthotopic transplant of the donor kidney. To minimize T-cell-mediated rejection while allowing the development of donor-specific antibodies, recipient animals were given tacrolimus starting the day before surgery. Hyperacute rejection was not seen, but acute graft dysfunction was evident on day 1 with a rapid deterioration of graft function by day 3. Histologic damage featured glomerulopathy, capillaritis, capillary thrombosis, and acute tubular injury. Recipients exhibited high serum levels of donor-specific antibodies and deposition of immunoglobulin G and C4d on graft endothelium. Immunostaining showed substantial endothelial damage, fibrin deposition in glomerular and peritubular capillaries, and infiltrates of macrophages, neutrophils, and natural killer cells. T-cell activation was efficiently suppressed by tacrolimus. We have developed a clinically relevant model of acute antibody-mediated rejection in recipients with preexisting donor-specific antibodies, which is suitable for testing novel therapies.

A model of acute renal allograft rejection in outbred Yorkshire piglets

Pigs represent a desirable animal model for the study of rejection in kidney transplantation with inbred Yucatan miniature swine (YMS) the most commonly studied strain due to well defined swine leukocyte antigen (SLA) genotypes. However, limitations to YMS may include cost and availability. Outbred Yorkshire pigs are widely available and significantly cheaper than YMS. Recent advances in SLA genotyping have allowed its application to outbred strains. On this basis, we theorized that Yorkshire pigs would be a viable alternative to YMS for the study of rejection in kidney transplantation. To address this question, we performed auto (Auto) and allotransplants (Allo) in 24 Yorkshire pigs, and assessed SLA genotypes and acute rejection after 72h. At sacrifice, and when compared to autotransplants, allotransplants had significant elevations in serum creatinine (8.4±1.3 vs 2.8±2.0mg/dL for Allo vs autotransplants, respectively) and BUN (61±9 vs 19.2±15mg/dL for Allo vs autotransplants, respectively). Warm ischemia times between the two groups did not differ (24±2.3 vs 26.4±1.4min for Auto vs Allo, respectively). There were 16 distinct SLA haplotypes identified from pigs undergoing allotransplantion, no matched donor-recipient pairs, and all allografts demonstrated rejection. Type IIA cellular rejection (Banff) was the most common. One allograft demonstrated hyperacute rejection due a blood group incompatibility. Histologically, the expression of regulatory Tcells and dendritic cells was increased in allografts. These data suggest that Yorkshire pigs may be a useful model for the study of acute rejection in experimental kidney transplantation.

Prevention of Donor Specific Antibody Production after Skin Allografting by Mobilization of Endogenous Stem Cells Using a Combination of AMD3100 and Low-dose FK506 in Rats

Background: We have developed a stem cell mobilizing strategy that enables long-term liver and kidney allograft survival without sustained immunosuppression in small and large animals using a safe combination of two FDA approved drugs AMD3100 and low-dose FK506 (AF). Furthermore, no increase in serum DSA levels was detected in animals displaying long-term survival even after donor skin grafts were rejected. Therefore, the purpose of this study is to determine if AF combination treatment prevents de novo DSA production in a rat model of skin allograft rejection. Methods: Split thickness skin allografts from Dark Agouti (DA, RT1a) rats were transplanted into adult Lewis (RT11) recipients. Recipient animals were randomly divided into AF treatment group (AMD3100 1 mg/kg and FK506 0.1 mg/kg, s.c., every other day for 14 days) and control group (same volume of saline, s.c.). Serum levels of DSA including both IgG and IgM were measured by flow cytometry at 14 days, 1 and 2 months after skin transplantation. The sensitization to donor antigens in Lewis rats at 2 months after skin allografting was further confirmed by transplanting donor DA kidneys. Full dose FK506 (1 mg/kg/day, s.c) was given to prevent acute cellular rejection after kidney transplantation. Results: Skin allografts were rejected in 8–10 days after transplantation in both groups. Serum levels of DSA-IgG increased 6 to 10 folds in control animals (n = 10), but only 2 to 3 folds in animals treated with AF combination (n = 11) at 1 month and remained at similar levels at 2 months after skin transplantation. All control animals (n = 5) died within 7 days after kidney transplantation. DSA deposition and AMR in kidney allografts were confirmed with signs of IgG deposition in and around glomeruli with C4d deposition on peritubular capillaries. In contrast, about 64% (7/11) animals with AF treatment after skin allografting survived over three weeks without antibody mediated rejection (AMR) and no increase in serum levels of DSA at 14 days after kidney transplantation. Conclusion: Mobilizing endogenous stem cells with AF combination treatment does not prolong skin allograft survival but prevents de novo DSA production after allograft rejection. AF treatment may inhibit B cell sensitization, prevent de novo DSA production and improve long-term allograft function after organ transplantation.

Transient Anti-CD45RC mAb Treatment Induces Specific Transplant Tolerance Through Potentiation of Tregs

Introduction: The main goal in transplantation is to induce specific rather than general immunosuppression. The CD45 protein is an essential regulator of T and B cell antigen receptor signaling. We have previously shown that T cells expressing low levels of CD45RC have strong immunoregulatory properties while T cells expressing high levels of CD45RC are essentially effector cells. Thus, we investigated the effect of depleting CD45RC+ cells on graft survival. Methods: Tregs were analyzed for the expression of CD45 isoforms by flow cytometry. LEW-1A rats were grafted in a heterotopic position with a LEW-1 W heart. NSG mice were 2Gy-irradiated or grafted with human skin and injected with 5x106 or 1,5x107 human PBMCs respectively. Animals were treated with 0.8 mg/kg/2.5d for 20d of anti-CD45RC mAb (OX22 or MT2 clones) or isotype. Humoral responses were assessed after KLH + CFA or exogenous red blood cells immunization. FACS Aria-sorted Tregs were i.v. transferred in 4,5Gy-irradiated recipients. CD45RClow/- Tregs were analyzed by RNA sequencing. Results: First, we have shown that CD45RC is the only CD45-isoform not expressed by Foxp3+ Tregs both in human and rat. Interestingly, transient treatment with anti-CD45RC Mabs induced transplant tolerance in a fully mismatch cardiac allograft model in rat. Importantly, this treatment prevented specifically anti-donor humoral responses while preserved primary and memory responses against cognate antigens. As expected, anti-CD45RC Mabs induced rapid cell death of CD45RC+ T cells through intrinsic cell signaling while preserved CD45RClow/- Tregs. Furthermore, the short-term treatment induced a transient homeostatic proliferation and sustained transcriptional changes in long-term CD8+ and CD4+ CD45RClow/- Tregs that maintained an indefinite, dominant and donor-specific tolerance, as adoptive transfer of Tregs succeeded in this allograft combination only. Finally, we translated these findings to human, in humanized mice models of xenogeneic GVHD and human skin allograft rejection. We demonstrated that ex vivo depletion of CD45RC+ PBMCs prevented development of GVHD, and that transient treatment with anti-human CD45RC Mabs delayed GVHD development and allograft rejection. Conclusion: This is the first proof of concept that targeting CD45RC with short-term Mabs treatment is an efficient innovative strategy to induce a donor-specific tolerance in transplantation without any other treatment.

Complement component 3 deficiency prolongs MHC-II disparate skin allograft survival by increasing the CD4(+) CD25(+) regulatory T cells population.

Recent reports suggest that complement system contributes to allograft rejection. However, its underlying mechanism is poorly understood. Herein, we investigate the role of complement component 3 (C3) in a single MHC-II molecule mismatched murine model of allograft rejection using C3 deficient mice (C3−/−) as skin graft donors or recipients. Compared with C3+/+ B6 allografts, C3−/− B6 grafts dramatically prolonged survival in MHC-II molecule mismatched H-2bm12 B6 recipients, indicating that C3 plays a critical role in allograft rejection. Compared with C3+/+ allografts, both Th17 cell infiltration and Th1/Th17 associated cytokine mRNA levels were clearly reduced in C3−/− allografts. Moreover, C3−/− allografts caused attenuated Th1/Th17 responses, but increased CD4+CD25+Foxp3+ regulatory T (Treg) cell expression markedly in local intragraft and H-2bm12 recipients. Depletion of Treg cells by anti-CD25 monoclonal antibody (mAb) negated the survival advantages conferred by C3 deficiency. Our results indicate for the first time that C3 deficiency can prolong MHC-II molecule mismatched skin allograft survival, which is further confirmed to be associated with increased CD4+ CD25+ Treg cell population expansion and attenuated Th1/Th17 response.

A chronic renal rejection model with a fully MHC-mismatched rat strain combination under immunosuppressive therapy

BackgroundThe Fischer-to-Lewis (LEW) rat model of kidney transplantation is a widely accepted and well-characterized model of chronic rejection. In contrast to transplantation in a clinical setting, however, the absence of treatment with immunosuppressants and only minor mismatch of major histocompatibility complexes (MHCs) are critical discrepancies. Here, we established a rat model of chronic rejection using fully MHC-mismatched strains in which kidney disease progresses even under immunosuppressive therapy. MethodsLEW (RT1l) rats were used as donors and Brown Norway (BN, RT1n) rats as recipients. Intramuscular administration of 0.1mg/kg of tacrolimus was initiated on the day of transplantation. Post-transplantation, this dose was maintained until Day 9, suspended until Day 28 and then resumed from Day 29. Renal function, histopathology, and levels of donor-specific antibody (DSA) and several biomarkers of renal injury were assessed. ResultsOn Day 91 post-transplantation, recipients received tacrolimus treatment with short-term suspension exhibited reduced renal function and changes in histology. Those were characteristics of chronic rejection including glomerulosclerosis, interstitial fibrosis, and tubular atrophy in human transplantation recipients. Urinary protein excretion increased in a linear fashion, and elevated levels of several biomarkers of renal injury and DSA were observed even under administration of an immunosuppressant. ConclusionsWe established an allograft rejection model with impaired renal function and typical histopathological changes of chronic rejection in fully MHC-mismatched rats by controlling administration of an immunosuppressant. These findings suggest that this model more accurately reflects transplantation in a clinical setting than existing models and enables the evaluation of therapeutic agents.

Toll-Like Receptor 4 Deficiency Improves Short-term Renal Function but not Long-term Graft Survival in a Fully MHC-Mismatched Murine Model of Renal Allograft Transplantation.

We have previously demonstrated that absence of myeloid differentiation primary response gene 88 (MyD88) induced donor-specific kidney allograft tolerance. The upstream pathways of MyD88 that mediate this process, however, remain unclear. Toll-like receptor 4 (TLR4) is an innate immune receptor that is dependent upon MyD88 for activity of its dominant signaling pathway. Here, we investigated the role of TLR4 in kidney allograft rejection using a fully major histocompatibility complex-mismatched, life-sustaining, murine model of renal allograft rejection. Donor (BALB/c) and recipient (C57BL/6) mice either both deficient or sufficient for TLR4 underwent heterotopic renal allograft transplantation, with an additional group of mice receiving renal isografts as controls. Survival was assessed up to 100 days posttransplantation. Animals were also sacrificed 14 days posttransplantation for assessment of the acute allograft rejection response. Both wild-type (WT) and TLR4 allografts showed inferior survival compared to isografts, with no difference in survival between the allograft groups. Serum creatinine was lower in TLR4 allografts at day 14 posttransplantation compared with WT allografts, but this was not sustained by day 100. At day 14 posttransplant, increased CD11c dendritic cell accumulation, expression of IL-2 and indoleamine 2,3-dioxygenase were evident in TLR4 compared with WT allografts, whereas expression of inducible nitric oxide synthase was decreased. Acute kidney allograft rejection was modestly attenuated in TLR4 mice; however, long-term allograft survival and function were not affected in our model. Protection against acute rejection may involve increased accumulation of CD11c cells and indoleamine 2,3-dioxygenase expression.

Renal Contrast-Enhanced Sonography Findings in a Model of Acute Cellular Allograft Rejection.

Noninvasive methods to diagnose and differentiate acute cellular rejection from acute tubular necrosis or acute calcineurin inhibitor toxicity are still missing. Because T lymphocytes play a decisive role in early states of rejection, we investigated the suitability and feasibility of antibody-mediated contrast-enhanced ultrasound by using microbubbles targeted to CD3(+) , CD4(+) , or CD8(+) T cells in different models of renal disease. In an established rat renal transplantation model, CD3-mediated ultrasound allows the detection of acute rejection as early as on postoperative day 2. Ultrasound signal intensities increased with the severity of inflammation. Further, an early response to therapy could be monitored by using contrast-enhanced sonography. Notably, acute tubular necrosis occurring after ischemia-reperfusion injury as well as acute calcineurin inhibitor toxicity could easily be differentiated. Finally, the quantified ultrasound signal correlated significantly with the number of infiltrating T cells obtained by histology and with CD3 mRNA levels, as well as with chemokine CXCL9, CXCL11, and CCL19 mRNA but not with KIM-1 mRNA expression, thereby representing the severity of graft inflammation but not the degree of kidney injury. In summary, we demonstrate that antibody-mediated contrast-enhanced ultrasound targeting T lymphocytes could be a promising tool for an easy and reproducible assessment of acute rejection after renal transplantation.

Mechanisms of antibody-mediated acute and chronic rejection of kidney allografts.

Antibody-mediated rejection is responsible for up to half of acute rejection episodes in kidney transplant patients and more than half of late graft failures. Antibodies cause acute graft abnormalities that are distinct from T cell-mediated rejection and at later times posttransplant, a distinct pathologic lesion is associated with capillary basement membrane multilayering and glomerulopathy. Despite the importance of donor-reactive antibodies as the leading cause of kidney graft failure, mechanisms underlying antibody-mediated acute and chronic kidney graft injury are poorly understood. Here, we review recent insights provided from clinical studies as well as from animal models that may help to identify new targets for therapy. Studies of biopsies from kidney grafts in patients with donor-specific antibody versus those without have utilized analysis of pathologic lesions and gene expression to identify the distinct characteristics of antibody-mediated rejection. These analyses have indicated the presence of natural killer cells and their activation during antibody-mediated rejection. The impact of studies of antibody-mediated allograft injury in animal models have lagged behind these clinical studies, but have been useful in testing the activation of innate immune components within allografts in the presence of donor-specific antibodies. Most insights into processes of antibody-mediated rejection of kidney grafts have come from carefully designed clinical studies. However, several new mouse models of antibody-mediated kidney allograft rejection may replicate the abnormalities observed in clinical kidney grafts and may be useful in directly testing mechanisms that underlie acute and chronic antibody-mediated graft injury.

Diversity of the CD4 T Cell Alloresponse: The Short and the Long of It.

SummaryMHC alloantigen is recognized by two pathways: “directly,” intact on donor cells, or “indirectly,” as self-restricted allopeptide. The duration of each pathway, and its relative contribution to allograft vasculopathy, remain unclear. Using a murine model of chronic allograft rejection, we report that direct-pathway CD4 T cell alloresponses, as well as indirect-pathway responses against MHC class II alloantigen, are curtailed by rapid elimination of donor hematopoietic antigen-presenting cells. In contrast, persistent presentation of epitope resulted in continual division and less-profound contraction of the class I allopeptide-specific CD4 T cell population, with approximately 10,000-fold more cells persisting than following acute allograft rejection. This expanded population nevertheless displayed sub-optimal anamnestic responses and was unable to provide co-stimulation-independent help for generating alloantibody. Indirect-pathway CD4 T cell responses are heterogeneous. Appreciation that responses against particular alloantigens dominate at late time points will likely inform development of strategies aimed at improving transplant outcomes.

Effects of FTY720 on peripheral blood lymphocytes and graft infiltrating cells in a rat model of chronic renal allograft rejection

AimsChronic renal allograft loss is still an unsolved problem in kidney transplantation. We evaluated the impact of FTY720, a S1P receptor agonist, known to deplete lymphocytes from the peripheral blood by sequestering them into lymph nodes and Peyer's patches, on blood lymphocytes and graft infiltrating cells in a rat model of chronic real allograft rejection. MethodsLEW rats served as recipients for LEW.1U7B kidney grafts. All animals were treated with CsA (5mg/kg) for 10days after renal transplantation and monitored for kidney function, peripheral blood lymphocytes and graft infiltrating cells.In the intervention group (n=7) FTY720 therapy was started 7weeks post-KTx in a dose of 0.5mg/kg p. o. three times a week. ResultsIn the control group the survival of the rats was 9, 11, 18 and 4×24 weeks, in the intervention group 2×8, 9, 2×11, 18 and 20weeks. While in the intervention group the number of T- and B-lymphocytes in the peripheral blood was successfully reduced during FTY720 treatment, both groups showed significant amounts of T- and B-lymphocytes in the kidney grafts. Animals in both groups developed donor specific antibodies, extensive albuminuria and severe chronic changes in the grafts. ConclusionsFTY720 was highly effective to reduce T- and B-lymphocytes in the peripheral blood, but not effective in clearing their infiltration in the graft. Graft survival was not prolonged by FTY720 treatment starting late after kidney transplantation.

Deletion of Smad3 improves cardiac allograft rejection in mice.

T cells play a critical role in acute allograft rejection. TGF-β/Smad3 signaling is a key pathway in regulating T cell development. We report here that Smad3 is a key transcriptional factor of TGF-β signaling that differentially regulates T cell immune responses in a mouse model of cardiac allograft rejection in which donor hearts from BALB/c mice were transplanted into Smad3 knockout (KO) and wild type (WT) mice. Results showed that the cardiac allograft survival was prolonged in Smad3 KO recipients. This allograft protection was associated with a significant inhibition of proinflammatory cytokines (IL-1β, TNF-α, and MCP-1) and infiltration of neutrophils, CD3+ T cells, and F4/80+ macrophages. Importantly, deletion of Smad3 markedly suppressed T-bet and IFN-γ while enhancing GATA3 and IL-4 expression, resulting in a shift from the Th1 to Th2 immune responses. Furthermore, mice lacking Smad3 were also protected from the Th17-mediated cardiac injury, although the regulatory T cell (Treg) response was also suppressed. In conclusion, Smad3 is an immune regulator in T cell-mediated cardiac allograft rejection. Loss of Smad3 results in a shift from Th1 to Th2 but suppressing Th17 immune responses. Thus, modulation of TGF-β/Smad3 signaling may be a novel therapy for acute allograft rejection.

Effect of novel PKCθ selective inhibitor AS2521780 on acute rejection in rat and non-human primate models of transplantation

Selective inhibition of protein kinase Cθ (PKCθ) may be useful in inducing T cell-specific immunosuppression with a reduced rate of side effects. To our knowledge, however, no reports have been published regarding the selective inhibition of PKCθ by small-molecule compounds in animal models of allograft rejection. Here, we investigated the effect of the newly synthesized PKCθ selective inhibitor AS2521780 in mono- and combination therapies on acute rejection in ACI-to-Lewis rat cardiac and non-human primate (NHP) renal transplantation models. In the rat cardiac transplantation model, AS2521780 significantly prolonged graft survival to 14days at 10mg/kg twice daily (b.i.d.) and to 20days at 30mg/kg b.i.d. In contrast, acute rejection occurred in all recipients in the non-treated group by Days 5 or 6 post-transplantation. Significant improvements (P<0.001) in graft survival were observed following treatment with a combination of AS2521780 at 3mg/kg b.i.d. and a suboptimal dose of tacrolimus (0.02mg/kg) or mycophenolate mofetil (15mg/kg). In the NHP renal transplantation model, AS2521780 at 3mg/kg b.i.d. and tacrolimus at 1mg/kg (suboptimal dose) significantly improved graft survival compared to tacrolimus alone (P<0.05). The present study of AS2521780 in rat cardiac and NHP renal transplantation models demonstrates the potential of PKCθ as a novel drug target for organ transplantation. As AS2521780 was well tolerated and the dose of tacrolimus or mycophenolate mofetil can be reduced when used in combination with this drug, immunosuppressive regimens containing selective inhibitors of PKCθ might have good safety profiles.

IL-17 Deficiency Attenuates Allograft Injury and Prolongs Survival in a Murine Model of Fully MHC-Mismatched Renal Allograft Transplantation

IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of inflammatory and autoimmune diseases. However the role of IL-17 in renal allograft rejection has not been fully explored. Here, we investigate the impact of IL-17 in a fully MHC-mismatched, life-sustaining, murine model of kidney allograft rejection using IL-17 deficient donors and recipients (IL-17(-/-) allografts). IL-17(-/-) allografts exhibited prolonged survival which was associated with reduced expression of the Th1 cytokine IFN-γ and histological attenuation of acute and chronic allograft rejection, as compared to wild-type allograft recipients. Results were confirmed in WT allograft recipients treated with an IL-17 blocking antibody. Subsequent experiments using either donors or recipients deficient in IL-17 showed a trend towards prolongation of survival only when recipients were IL-17(-/-) . Administration of a depleting anti-CD25 antibody to IL-17(-/-) recipients abrogated the survival advantage conferred by IL-17 deficiency, suggesting the involvement of a CD4(+) CD25(+) T cell regulatory mechanism. Therefore, IL-17 deficiency or neutralization was protective against the development of kidney allograft rejection, which may be mediated by impairment of Th1 responses and/or enhanced protection by Tregs.

Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome.

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice.

A proteomic analysis of transplanted liver in a rat model of chronic rejection

Chronic rejection (CR) is an important cause of liver allograft failure. In the latter condition, re-transplantation of the liver (ReLT) is the only option for survival. Unfortunately, with the current state of knowledge, it is difficult to diagnose and treat early CR. To explore the biomarkers of the chronic rejection in orthotopic liver transplantation (OLT). A rat model of chronic liver allograft rejection was established, and the differential protein expression in chronic allograft rejection (CR) was analyzed by iTRAQ-MALDI-TOF/TOF. Expression of sixty-two proteins was found to be significantly changed in CR rats. In the present study, CLU, Lcn2 and Krt19 were identified and quantified as early and reliable biomarkers for chronic rejection. Analysis of differential protein expression by iTRAQ-MALDI-TOF/TOF is a potentially effective method to help understand the mechanism of CR in orthotopic liver transplantation. The proteins CLU, Lcn2 and Krt19 might be potential prognostic markers for predicting chronic rejection after liver transplantation.

Murine corneal transplantation: a model to study the most common form of solid organ transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

'Default' generated neonatal regulatory T cells are hypomethylated at conserved non-coding sequence 2 and promote long-term cardiac allograft survival.

Regulatory T (Treg) cells play an important role in the maintenance of immune self-tolerance and homeostasis. We previously reported that neonatal CD4(+) T cells have an intrinsic 'default' mechanism to become Treg (neoTreg) cells in response to T-cell receptor (TCR) stimulation. However, the underlying mechanisms are unclear and the effects of neoTreg cells on regulating immune responses remain unknown. Due to their involvement in Foxp3 regulation, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b during the induction of neoTreg cells in the Foxp3(gfp) mice. The function of neoTreg cells was assessed in an acute allograft rejection model established in RAG2(-/-) mice with allograft cardiac transplantation and transferred with syngeneic CD4(+) effector T cells. Following ex vivo TCR stimulation, the DNMT activity was increased threefold in adult CD4(+) T cells, but not significantly increased in neonatal cells. However, adoptively transferred neoTreg cells significantly prolonged cardiac allograft survival (mean survival time 47 days, P < 0.001) and maintained Foxp3 expression similar to natural Treg cells. The neoTreg cells were hypomethylated at the conserved non-coding DNA sequence 2 locus of Foxp3 compared with adult Treg cells. The DNMT antagonist 5-aza-2'-deoxycytidine (5-Aza) induced increased Foxp3 expression in mature CD4(+) T cells. 5-Aza-inducible Treg cells combined with continuous 5-Aza treatment prolonged graft survival. These results indicate that the 'default' pathway of neoTreg cell differentiation is associated with reduced DNMT1 and DNMT3b response to TCR stimulus. The neoTreg cells may be a strategy to alleviate acute allograft rejection.

Mouse kidney transplantation: models of allograft rejection.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Bone marrow and adipose tissue derived mesenchymal stromal cells have similar immunosuppressive capacities in vitro and in a humanized allograft rejection model

Bone marrow and adipose tissue derived mesenchymal stromal cells have similar immunosuppressive capacities in vitro and in a humanized allograft rejection model