Donor Toll-like receptor 4 contributes to ischemia and reperfusion injury following human kidney transplantation.

While studies in animal models have linked Toll-like receptor (TLR) 4 signaling to kidney injury induced by ischemia and reperfusion, the relevance of TLR4 activation to allograft injury in human kidney transplants is unknown. Here we show that TLR4 is constitutively expressed within all donor kidneys but is significantly higher in deceased-, compared with living-donor organs. Tubules from deceased- but not living-donor kidneys also stained positively for high-mobility group box-1 (HMGB1), a known endogenous TLR4 ligand. In vitro stimulation of human tubular cells with HMGB1, in a TLR4-dependent system, confirmed that HMGB1 can stimulate proinflammatory responses through TLR4. To assess the functional significance of TLR4 in human kidney transplantation, we determined whether TLR4 mutations that confer diminished affinity for HMGB1 influence intragraft gene-expression profiles and immediate graft function. Compared with kidneys expressing WT alleles, kidneys with a TLR4 loss-of-function allele contained less TNFalpha, MCP-1, and more heme oxygenase 1 (HO-1), and exhibited a higher rate of immediate graft function. These results represent previously undetected evidence that donor TLR4 contributes to graft inflammation and sterile injury following cold preservation and transplantation in humans. Targeting TLR4 signaling may have value in preventing or treating postischemic acute kidney injury after transplantation.

Reversal of type 1 diabetes by a new MHC II-peptide chimera: "Single-epitope-mediated suppression" to stabilize a polyclonal autoimmune T-cell process.

Polyclonality of self-reactive CD4(+) T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHC II-peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T-cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3: 383-391]. Since most of the glutamic acid decarboxylase 65 (GAD65)-specific CD4(+) T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T-regulatory cell population (GAD65(217-230)-specific CD4(+) T cells) using a soluble I-A(αß) (g7)/GAD65(217-230)/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T-cell process. Activated GAD65(217-230)-specific T cells and a fraction of the diabetogenic (B(9-23)-specific) T cells were polarized toward the IL-10-secreting T-regulatory type 1-like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post-therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the ß cells. These findings argue for the stabilization of a polyclonal self-reactive T-cell process by a single epitope-mediated bystander suppression. Dimeric MHC class II-peptide chimeras-like approach may provide rational grounds for the development of more efficient antigen-specific therapies in type 1 diabetes.

Inhibition of the alloimmune response through the generation of regulatory T cells by a MHC class II-derived peptide.

We have previously shown that HLA-DQA1, a peptide derived from a highly conserved region of MHC class II, prevents alloreactive T cell priming and effector function in vivo, although underlying mechanisms are obscure. In this study, we demonstrate that 28% of mice treated with HLA-DQA1 combined with low-dose rapamycin achieved permanent engraftment of fully MHC-disparate islet allografts and significantly prolonged survival in the remaining animals (log rank, p < 0.001). Immunohistologic examination of the grafts from HLA-DQA1/rapamycin-treated animals revealed up-regulated expression of TGF-ss and FoxP3. In vivo administration of blocking anti-TGF-ss or depleting anti-CD25 mAb augmented T cell alloimmunity and prevented the long-term engraft induced by HLA-DQA1. In vitro experiments further showed that HLA-DQA1 induced differentiation of CD4(+) T cells into CD4(+)CD25(+)FoxP3(+) regulatory T cells. Together, these data provide the first demonstration that HLA-DQA1, a MHC class II-derived peptide, can prolong allograft survival via a TGF-beta and regulatory T cell-dependent mechanisms.

A peptide-major histocompatibility complex II chimera favors survival of pancreatic beta-islets grafted in type 1 diabetic mice.

BACKGROUND: Transplantation of pancreatic islets showed a tremendous progress over the years as a promising, new therapeutic strategy in patients with type 1 diabetes. However, additional immunosuppressive drug therapy is required to prevent rejection of engrafted islets. The current immunosuppressive therapies showed limited success in maintaining long-term islet survival as required to achieve insulin independence in type 1 diabetes, and they induce severe adverse effects. Herein, we analyzed the effects of a soluble peptide-major histocompatibility complex (MHC) class II chimera aimed at devising an antigen-specific therapy for suppression of anti-islet T cell responses and to improve the survival of pancreatic islets transplants. METHODS: Pancreatic islets from transgenic mice expressing the hemagglutinin antigen in the beta islets under the rat insulin promoter (RIP-HA) were grafted under the kidney capsule of diabetic, double transgenic mice expressing hemagglutinin in the pancreas and T cells specific for hemagglutinin (RIP-HA, TCR-HA). The recipient double transgenic mice were treated or not with the soluble peptide-MHC II chimera, and the progression of diabetes, graft survival, and T cell responses to the grafted islets were analyzed. RESULTS: The peptide-MHC II chimera protected syngeneic pancreatic islet transplants against the islet-reactive CD4 T cells, and prolonged the survival of transplanted islets. Protection of transplanted islets occurred by polarization of antigen-specific memory CD4 T cells toward a Th2 anti-inflammatory response. CONCLUSIONS: The peptide-MHC II chimera approach is an efficient and specific therapeutic approach to suppress anti-islet T cell responses and provides a long survival of pancreatic grafted islets.

Effect of cholecalciferol supplementation on inflammation and cellular alloimmunity in hemodialysis patients: data from a randomized controlled pilot trial.

BACKGROUND: Memory T-cells are mediators of transplant injury, and no therapy is known to prevent the development of cross-reactive memory alloimmunity. Activated vitamin D is immunomodulatory, and vitamin D deficiency, common in hemodialysis patients awaiting transplantation, is associated with a heightened alloimmune response. Thus, we tested the hypothesis that vitamin D3 supplementation would prevent alloreactive T-cell memory formation in vitamin D-deficient hemodialysis patients. METHODS AND FINDINGS: We performed a 12-month single-center pilot randomized, controlled trial of 50,000 IU/week of cholecalciferol (D3) versus no supplementation in 96 hemodialysis patients with serum 25(OH)D<25 ng/mL, measuring effects on serum 25(OH)D and phenotypic and functional properties of T-cells. Participants were randomized 2:1 to active treatment versus control. D3 supplementation increased serum 25(OH)D at 6 weeks (13.5 [11.2] ng/mL to 42.5 [18.5] ng/mL, p<0.001) and for the duration of the study. No episodes of sustained hypercalcemia occurred in either group. Results of IFNγ ELISPOT-based panel of reactive T-cell assays (PRT), quantifying alloreactive memory, demonstrated greater increases in the controls over 1 year compared to the treatment group (delta PRT in treatment 104.8+/-330.8 vs 252.9+/-431.3 in control), but these changes in PRT between groups did not reach statistical significance (p = 0.25). CONCLUSIONS: D3 supplements are safe, effective at treating vitamin D deficiency, and may prevent time-dependent increases in T-cell alloimmunity in hemodialysis patients, but their effects on alloimmunity need to be confirmed in larger studies. These findings support the routine supplementation of vitamin D-deficient transplant candidates on hemodialysis and highlight the need for large-scale prospective studies of vitamin D supplementation in transplant candidates and recipients. TRIAL REGISTRATION: Clinicaltrials.gov NCT01175798.

Immune cell-derived c3 is required for autoimmune diabetes induced by multiple low doses of streptozotocin.

OBJECTIVE: The complement system contributes to autoimmune injury, but its involvement in promoting the development of autoimmune diabetes is unknown. In this study, our goal was to ascertain the role of complement C3 in autoimmune diabetes. RESEARCH DESIGN AND METHODS: Susceptibility to diabetes development after multiple low-dose streptozotocin treatment in wild-type (WT) and C3-deficient mice was analyzed. Bone marrow chimeras, luminex, and quantitative reverse transcription PCR assays were performed to evaluate the phenotypic and immunologic impact of C3 in the development of this diabetes model. RESULTS: Coincident with the induced elevations in blood glucose levels, we documented alternative pathway complement component gene expression within the islets of the diabetic WT mice. When we repeated the experiments with C3-deficient mice, we observed complete resistance to disease, as assessed by the absence of histologic insulitis and the absence of T-cell reactivity to islet antigens. Studies of WT chimeras bearing C3-deficient bone marrow cells showed that bone marrow cell-derived C3, and not serum C3, is involved in the induction of diabetes in this model. CONCLUSIONS: The data reveal a key role for immune cell-derived C3 in the pathogenesis of murine multiple low-dose streptozotocin-induced diabetes and support the concept that immune cell mediated diabetes is in part complement-dependent.

MHC Class II-mediated apoptosis by a nonpolymorphic MHC Class II peptide proceeds by activation of protein kinase C.

It was demonstrated previously that a peptide derived from a conserved region of MHC class II, HLA-DQA1, inhibits proliferation of allogeneic T cells in vitro. Administration of HLA-DQA1 in conjunction with allogeneic cells at the time of priming or at the time of rechallenge prevented the development of the delayed type hypersensitivity response in vivo. The immunomodulatory effects of HLA-DQA1 were associated with the induction of apoptosis in B cells, macrophages, and dendritic cells via a caspase-independent pathway. This study investigated the binding site and mechanism that mediates cell death induced by HLA-DQA1. It was demonstrated that HLA-DQA1 binds to MHC class II on the cell surface, causing MHC class II signaling, initiation of protein kinase C signaling, and mitochondrial membrane depolarization with resultant apoptosis. The data indicate that HLA-DQA1 binds to MHC class II outside the groove, in a manner similar to superantigen. These results suggest that HLA-DQA1 is a novel immunotherapy that may provide an effective means of targeting professional antigen-presenting cells, in particular B cells.

Soluble, dimeric HLA DR4-peptide chimeras: an approach for detection and immunoregulation of human type-1 diabetes.

Still there are no effective methods to predict or cure type 1 diabetes (T1D) in humans. Soluble, dimeric MHC class II-peptide (DEF) chimeras have potential for both early diagnosis and immunospecific therapy. DEF chimeras prevent and reverse diabetes in mice by stimulating antigen-specific type 1 T regulatory cell (Tr1)-like cells. We also showed that diabetes could be predicted by changes in the phenotype of autoreactive CD4 T cells in peripheral blood. Herein, we demonstrated that human DEF (HLA-DR*0401/Fcgamma1) chimeras expressing peptides of beta-cell antigens stimulate Tr1-like cells in blood of patients with T1D, non-diabetic relatives, and controls. Furthermore, the specific and stable binding of DEF chimeras to cognate TCR and CD4 coreceptor allowed quantification and phenotyping of autoreactive CD4 T cells in non-stimulated blood by FACS. Our results indicate that (1) autoreactive CD4 T cells to GAD65 autoantigen are commonly present in humans expressing diabetes-susceptible HLA-DR*0401 molecules; (2) these autoreactive T cells undergo avidity maturation upon encountering the self antigen early in life; (3) the disease is associated with an imbalance between autoreactive CD4+CD25+ and CD4+CD69+ T cells specific for GAD65. Based on this, we propose a model to explain the kinetics of autoreactive CD4 T cells in blood during the natural history of T1D.

A novel mechanism for the immunomodulatory functions of class II MHC-derived peptides.

There is now extensive evidence that synthetic peptides corresponding to linear sequences of MHC molecules are effective immunoregulators, targeting the immune response at many different sites. It has been previously shown that peptides derived from a highly conserved region of MHC class II inhibit proliferation to autoantigen and to both the direct and indirect pathways of allorecognition. This study demonstrates that inhibition of lymphocyte proliferation by nonpolymorphic MHC class II peptides, specifically HLA-DQA1, is sequence-specific and that the inhibitory effect is mediated through the induction of apoptosis in antigen-presenting cells via a caspase-independent mechanism. In addition, T lymphocytes stimulated in the presence of HLA-DQA1 are rendered hyporesponsive to subsequent stimuli. Immunomodulation by HLA-DQA1 is effective in vivo because it prevents both the priming and the effector function of primed allogeneic T cells in a murine DTH model. These observations have important implications for the development of a novel therapy for immune-mediated diseases.

The impact of costimulatory molecule gene polymorphisms on clinical outcomes in liver transplantation.

CTLA-4 and CD28 deliver opposing signals for T-cell proliferation. We examined single nucleotide polymorphisms (SNPs) CTLA-4 -318C/T and CD28 IVS3 +17T/C for associations with acute rejection in liver transplant recipients. These and two other polymorphisms in CTLA-4 [microsatellite polymorphism +642(AT)n and SNP +49 A/G] were also analyzed for influence on graft survival. Two hundred and eleven liver transplant recipient genotypes were determined by direct sequencing or restriction fragment length polymorphism analysis of PCR-amplified genomic DNA. Mean graft survival for patients with the GG genotype of CTLA-4 +49 A/G was 58.5 +/- 6.0 months compared with 70.3 +/- 4.0 months and 73.8 +/- 2.8 months for the AA and AG genotypes, respectively (p = 0.0055). This is in support of previous studies suggesting decreased CTLA-4 function and increased incidence of autoimmune disease for this genotype. The 92-, 94-, and 100-bp alleles of CTLA-4 +642(AT)n occurred more often in African-American transplant recipients and were associated with decreased graft survival (p = 0.0001 and 0.007, respectively) but the independence of these variables could not be established. No associations with acute rejection or graft survival were found for CTLA-4 -318C/T or CD28 IVS3 +17T/C. The described associations between CTLA-4 gene polymorphisms and transplant outcomes provide the foundation for further investigations leading to genetic risk stratification for transplant recipients.

The impact of polymorphisms in chemokine and chemokine receptors on outcomes in liver transplantation.

Chemokines and their corresponding receptors likely play a central role in directing mononuclear cells to the graft sites during rejection. Genes for the chemokine stromal derived factor-1 (SDF1) and CC chemokine receptors CCR2 and CCR5 are characterized by polymorphisms which alter their function. We genotyped DNA of 207 liver transplant recipients by PCR or PCR-RFLP for CCR2-641, CCR5delta32, and SDF1-3'A polymorphisms, and examined their association on outcomes in liver allograft recipients. Due to the low number of patients homozygous for CCR2-641 and CCR5delta32, only the effects of their heterozygous variants were addressed in this study. None of the investigated polymorphisms showed a significant shift in gene frequency in acute rejection and rejection-free groups, or for graft survival. The gene frequency of the SDF1-3'A allele was significantly (p = 0.034) higher in patients who died (29.0%, n = 31) compared to recipients still alive (17.1%, n = 172). The mean patient survival time post transplant was 134 months in patients with SDF1 wild-type, significantly (log rank p = 0.014) longer than 98 months in patients with at least one SDF1-3'A allele. The CCR2 and CCR5 polymorphisms were not associated with significant differences in mortality rate. In conclusion, CCR2-641, CCR5delta32, and SDF1-3'A genotypes did not influence the risk for acute rejection or graft survival. However, in liver allograft recipients SDF1-3'A is significantly associated with higher mortality.

Analysis of gene polymorphisms in the regulatory region of MCP-1, RANTES, and CCR5 in liver transplant recipients.

Chemokines and their receptors play a major role in the inflammatory and immune responses that mediate allograft outcome. The production of some chemokines varies among individuals and these variations may be determined by genetic polymorphisms, most commonly within the regulatory region of the gene. We investigated whether the functional polymorphisms of the chemokines RANTES, MCP-1 and chemokine receptor CCR5 are associated with the incidence of acute rejection and long-term liver graft survival. Two hundred nine liver transplant recipients were genotyped using polymerase chain reaction sequence-specific primers for the following polymorphisms: RANTES-28, MCP-1 -2518, and CCR5-59029. There was no association with any of the three genotypes and the incidence of acute rejection episodes. In addition, no association of RANTES-28, MCP-1 -2518, or CCR5 -59029 variants with long-term liver graft survival was found. In conclusion, variants of RANTES-28, MCP-1 -2518, and CCR5-59029 neither influenced the incidence of acute rejection nor affected long-term allograft survival upon liver transplantation in the context of this analysis.