Alloantigen gene transfer to hepatocytes promotes tolerance to pancreatic islet graft by inducing CD8+ regulatory T cells.

BACKGROUND & AIMS: Induction of donor-specific immune tolerance is a good alternative to chronic life-long immunosuppression for transplant patients. Donor major histocompatibility complex (MHC) molecules represent the main targets of the allogeneic immune response of transplant recipients. Liver targeted gene transfer with viral vectors induces tolerance toward the encoded antigen. The aim of this work was to determine whether alloantigen gene transfer to hepatocytes induces tolerance and promotes graft acceptance. METHODS: C57BL/6 (H-2b) mice were treated with adeno-associated viral (AAV) vector targeting the expression of the MHC class I molecule H-2Kd to hepatocytes, before transplantation with fully allogeneic pancreatic islet from BALB/c mice (H-2d). RESULTS: AAV H-2Kd treated mice were tolerant to the alloantigen, as demonstrated by its long-term expression by the hepatocytes, even after a highly immunogenic challenge with an adenoviral vector. After chemical induction of diabetes, the AAV treated mice had significantly delayed rejection of fully allogeneic pancreatic islet grafts, with more than 40% of recipients tolerant (>100days). AAV-mediated expression of H-2Kd in the liver induced the local expansion of CD8+ T lymphocytes with allo-specific suppressive properties. The adoptive transfer of these liver-generated CD8+ Tregs into naive diabetic mice promoted the long-term survival of allogeneic pancreatic islet grafts. CONCLUSION: AAV-mediated long-term expression of a single MHC class I molecule in the liver induces the generation of a subset of allo-specific CD8+ Treg cells, which promote tolerance toward fully allogeneic graft. Liver gene transfer represents a promising strategy for in vivo induction of donor-specific tolerance. LAY SUMMARY: The liver has a special immune system, biased toward tolerance. In this study, we investigated the possibility of harnessing this property of the liver to induce tolerance to an allogeneic transplantation. We demonstrate for the first time that the in vivo gene transfer of an allogeneic antigen with an adeno-associated viral vector to mouse hepatocytes induces the expansion of a population of CD8+ regulatory T lymphocytes. These Tregs are then instrumental in preventing the rejection of allogeneic pancreatic islets transplanted in these animals. Allogeneic transplantation is the main treatment for the end-stage diseases of a number of organs. Life-long immunosuppressive treatments are still required to limit graft rejection, and these treatments exhibit serious side effects. Our present findings open a new avenue for promoting allo-specific tolerance via in vivo induction of CD8+ Treg expansion.

Expansion of highly differentiated cytotoxic terminally differentiated effector memory CD8+ T cells in a subset of clinically stable kidney transplant recipients: a potential marker for late graft dysfunction.

Despite the effectiveness of immunosuppressive drugs, kidney transplant recipients still face late graft dysfunction. Thus, it is necessary to identify biomarkers to detect the first pathologic events and guide therapeutic target development. Previously, we identified differences in the T-cell receptor Vß repertoire in patients with stable graft function. In this prospective study, we assessed the long-term effect of CD8(+) T-cell differentiation and function in 131 patients who had stable graft function. In 45 of 131 patients, a restriction of TCR Vß diversity was detected and associated with the expansion of terminally differentiated effector memory (TEMRA; CD45RA(+)CCR7(-)CD27(-)CD28(-)) CD8(+) T cells expressing high levels of perforin, granzyme B, and T-bet. This phenotype positively correlated with the level of CD57 and the ability of CD8(+) T cells to secrete TNF-α and IFN-γ. Finally, 47 of 131 patients experienced kidney dysfunction during the median 15-year follow-up period. Using a Cox regression model, we found a 2-fold higher risk (P=0.06) of long-term graft dysfunction in patients who had increased levels of differentiated TEMRA CD8(+) T cells at inclusion. Collectively, these results suggest that monitoring the phenotype and function of circulating CD8(+) T cells may improve the early identification of at-risk patients.

IL-7 receptor blockade following T cell depletion promotes long-term allograft survival.

T cell depletion is commonly used in organ transplantation for immunosuppression; however, a restoration of T cell homeostasis following depletion leads to increased memory T cells, which may promote transplant rejection. The cytokine IL-7 is important for controlling lymphopoiesis under both normal and lymphopenic conditions. Here, we investigated whether blocking IL-7 signaling with a mAb that targets IL-7 receptor α (IL-7Rα) alone or following T cell depletion confers an advantage for allograft survival in murine transplant models. We found that IL-7R blockade alone induced indefinite pancreatic islet allograft survival if anti-IL-7R treatment was started 3 weeks before graft. IL-7R blockade following anti-CD4- and anti-CD8-mediated T cell depletion markedly prolonged skin allograft survival. Furthermore, IL-7 inhibition in combination with T cell depletion synergized with either CTLA-4Ig administration or suboptimal doses of tacrolimus to induce long-term skin graft acceptance in this stringent transplant model. Together, these therapies inhibited T cell reconstitution, decreased memory T cell numbers, increased the relative frequency of Tregs, and abrogated both cellular and humoral alloimmune responses. Our data suggest that IL-7R blockade following T cell depletion has potential as a robust, immunosuppressive therapy in transplantation.

The blood of healthy individuals exhibits CD8 T cells with a highly altered TCR Vb repertoire but with an unmodified phenotype.

CD8 T cell clonal expansions (TCE) have been observed in elderly, healthy individuals as well in old mice, and have been associated with the ageing process. Both chronic latent and non-persistent viral infections have been proposed to drive the development of distinct non-functional and functional TCE respectively. Biases in TCR Vß repertoire diversity are also recurrently observed in patients that have undergone strong immune challenge, and are preferentially observed in the CD8 compartment. Healthy adults can also exhibit CD8 T cells with strong alterations of their CDR3 length distribution. Surprisingly, no specific investigations have been conducted to analyze the CD8 T cell repertoire in normal adults, to determine if such alterations in TCR Vß repertoire share the features of TCE. In this study, we characterized the phenotype and function of the CD8 population in healthy individuals of 25-52 years of age. All but one of the EBV-positive HLA-B8 healthy volunteers that were studied were CMV-negative. Using a specific unsupervised statistical method, we identified Vß families with altered CDR3 length distribution and increased TCR Vß/HPRT transcript ratios in all individuals tested. The increase in TCR Vß/HPRT transcript ratio was more frequently associated with an increase in the percentage of the corresponding Vß(+) T cells than with an absence of modification of their percentage. However, in contrast with the previously described TCE, these CD8(+) T cells were not preferentially found in the memory CD8 subset, they exhibited normal effector functions (cytokine secretion and cytotoxic molecule expression) and they were not reactive to a pool of EBV/CMV/Flu virus peptides. Taken together, the combined analysis of transcripts and proteins of the TCR Vß repertoire led to the identification of different types of CD8(+) T cell clone expansion or contraction in healthy individuals, a situation that appears more complex than previously described in aged individuals.

TNF blockade abrogates the induction of T cell-dependent humoral responses in an allotransplantation model.

TNF blockade modulates many aspects of the immune response and is commonly used in a wide array of immune-mediated inflammatory diseases. As anti-TNF induces anti-dsDNA IgM antibodies but not other antinuclear reactivities in human arthritis, we investigated here the effect of TNF blockade on the induction of TD humoral responses using cardiac allograft and xenograft models. A single injection of an anti-rat TNF antibody in LEW.1A recipients grafted with congenic LEW.1W hearts almost completely abrogated the induction of IgM and IgG alloantibodies. This was associated with decreased Ig deposition and leukocyte infiltration in the graft at Day 5. TNF blockade did not affect germinal-center formation in the spleen or expression of Th1/Th2 cytokines, costimulatory and regulatory molecules, and TLRs in spleen and graft of the recipient animals. Clinically, the abrogation of the induction of the alloantibodies was associated with a marked prolongation of graft survival. In contrast, anti-TNF did not alter acute xenograft rejection mediated by TI antibodies in a hamster-to-rat model. Taken together, these data indicate that TNF blockade abrogates the induction of TD humoral responses and accordingly, may have a beneficial effect in antibody-mediated inflammatory pathologies.

Mesenchymal stem cells induce a weak immune response in the rat striatum after allo or xenotransplantation.

Mesenchymal stem cells (MSCs) have attracted attention for their potential use in regenerative medicine such as brain transplantation. As MSCs are considered to be hypoimmunogenic, transplanted MSCs should not trigger a strong host inflammatory response. To verify this hypothesis, we studied the brain immune response after transplantation of human or rat MSCs into the rat striatum and MSC fate at days 5, 14, 21 and 63 after transplantation. Flow cytometry analysis indicated that both MSCs express CD90 and human leucocyte antigen (MHC) class I, but no MHC class II molecules. They do not express CD45 or CD34 antigens. However, MSC phenotype varies with passage number. Human MSCs have mRNAs for interleukin (IL)-6, IL-8, IL-12, tumour necrosis factor (TNF)-alpha and TGF-beta(1), whereas rat MSCs express IL-6-, IL-10-, IL-12- and TGF-beta(1)-mRNAs. The quantification shows higher levels of mRNAs for the anti-inflammatory molecules IL-6 and TGF-beta(1) than for pro-inflammatory cytokines IL-8 and IL-12; ELISA analysis showed no IL-12 whereas TGF-beta(1) and IL-6 were detected. Transplant size did not significantly vary between 14 and 63 days after transplantation, indicating an absence of immune rejection of the grafts. Very few mast cells and moderate macrophage and microglial infiltrations, observed at day 5 remained stable until day 63 after transplantation in both rat and human MSC grafts. The observations of very few dendritic cells, T alphabeta-cells, and no T gammadelta-lymphocytes, all three being associated with Tp rejection in the brain, support the contention that MSCs are hypoimmunogenic. Our results suggest that MSCs are of great interest in regenerative medicine in a (xeno)transplantation setting.

Transgenic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation.

The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.

Adenovirus-mediated CTLA4Ig or CD40Ig gene transfer delays pancreatic islet rejection in a rat-to-mouse xenotransplantation model after systemic but not local expression.

Transient costimulation signal blockade of either CD28/CD80-86 interactions and/or CD40/CD154 interactions can prevent islet rejection in some models of both allo- and xenotransplantation. We have used adenoviruses coding for CTLA4Ig or CD40Ig and compared the efficacy of genetic modification of islets to systemic production through either intramuscular (i.m.) or intravenous (i.v.) injection of these vectors in a rat-to-mouse islet transplantation model. When gene transfer was performed into islets, a high level of primary nonfunction was induced. Furthermore, transduced functional grafts were rejected with the same kinetics as nontransduced islets. In contrast, i.m. AdCTLA4Ig and i.v. AdCD40Ig significantly delayed rejection (mean survival time of 54 +/- 26.9 and 67.6 +/- 44.9 days, respectively, vs. 24.3 +/- 9.7 days for unmodified islets, p < 0.05). Combination of ex vivo AdCTLA4Ig islet transduction and i.v. AdCD40Ig did not improve graft survival further. In conclusion, islet graft transduction with adenoviruses coding for costimulation inhibitors resulted in local expression with low serum concentrations of CTLA4Ig or CD40Ig and was unable to protect islet xenografts from rejection. In contrast, i.m. or i.v. gene transfer resulted in high serum concentrations of these molecules and was highly efficient in prolonging xenograft survival. These results contrast with the efficacy of AdCTLA4Ig we observed in a rat islet allotransplantation model and suggest that islet xenograft rejection might be more difficult to control.

CTLA4Ig adenoviral gene transfer induces long-term islet rat allograft survival, without tolerance, after systemic but not local intragraft expression.

Genetic engineering using recombinant adenoviruses offers an opportunity to modify islet grafts in order to prevent allograft rejection. We have used an adenovirus coding for CTLA4Ig to compare its efficacy in preventing islet rejection depending on local or systemic production after gene transfer either into the islets or intramuscularly, respectively. Islet allograft survival was also evaluated using recombinant CTLA4Ig administered intraperitoneally or incubated ex vivo with islets prior to transplantation. Transduction of islets with 10(3) or 10(4) plaque-forming units (pfu) per islets of AdCTLA4Ig prolonged islet survival (mean +/- standard deviation [SD] days = 19.5 +/- 5.8 and 19.5 +/- 5.6, respectively, vs. 10.6 +/- 2.4 in control islets, p < 0.001), with low levels of circulating CTLA4Ig. In contrast, long-term survival (>60 days) was obtained after intramuscular injection of AdCTLA4Ig that resulted in sustained high levels of circulating CTLA4Ig. Islets incubated in vitro with CTLA4Ig did not show prolonged survival (10.3 +/- 2.5 days). Graft rejection was delayed after one injection of CTLA4Ig (23 +/- 7.6 days, p < 0.003 vs. control). Recipients of long-term surviving grafts after intramuscular AdCTLA4Ig gene transfer were not tolerant because second islet grafts of donor origin were rejected. These recipients also had a strong inhibition of humoral responses against nominal antigens, whereas animals receiving transduced islets showed normal responses. These data demonstrate that local production of CTLA4Ig after gene transfer was as efficient as a single injection of CTLA4Ig in preventing graft rejection. Furthermore, intramuscular gene transfer of CTLA4Ig was the most efficient way to induce long-term islet graft survival but no donor-specific tolerance was induced.

Rapid failure of pig islet transplantation in non human primates.

We have previously demonstrated that adult pig islets of Langerhans are not destroyed in vitro by primate sera. Whether these islets can function when placed into the liver of non-human primates is not known. We now report on the outcome of pig islet xenotransplantation into five non diabetic primates (four baboons and one macacus fascicularis) receiving intraportally purified adult pig islets. The average number of islet-equivalent per graft was 110,000 (60-180,000). All animals received associations of ATG, cyclosporine or LF 195 (a deoxyspergualin analog), mycophenolate mofetil and corticosteroids. A specific porcine C-peptide (C-pep) RIA test was used to monitor insulin secretion. Two hours after grafting, porcine C-peptide was positive (from 0.37 to 4.25 ng/ml) in all monkeys except one. Primate C-pep was normal in all cases. Only two monkeys had detectable levels of porcine C-pep on day 1 or 2 with undetectable levels thereafter, even after glucagon challenge between days 6 and 10. Several normal islets with moderate inflammatory infiltration were observed in one animal liver on day 2 (the time of necropsy) as well as islets with IgM and complement deposition. Among animals sacrificed on days 14, 16 and 38, some residual islet cells could be identified only in livers collected on day 14. Partial glycaemic control was achieved in some rats receiving islets from the same preparations. In conclusion, adult pig islets are not able to maintain insulin secretion for more than 24 h when injected intraportally into non diabetic immunosuppressed monkeys. suggesting immediate islet xenograft destruction.