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.

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.

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.