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 Vb 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 Vb diversity was detected and associated with the expansion of terminally differentiated effector memory (TEMRA; CD45RA + CCR7 2 CD27 2 CD28 2 ) 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-a and IFN-g. 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.

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.

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.

The in vitro purification of pancreatic islets offers an opportunity for their modification by ex vivo gene transfer. We investigated the efficiency and functional consequences of adenovirus-mediated gene transfer into adult murine pancreatic islets with a recombinant adenovirus encoding for the beta-galactosidase (beta-Gal) reporter gene. At 10(6) pfu/islet, almost all of the islets were transduced, but maximal transduction was obtained at 10(7) pfu/islet. Histochemical analysis of frozen islet sections showed that transduced cells were only located at the periphery of islets. Transduced islets showed normal insulin secretion in vitro, and were able to normalize in vivo the glycemia of streptozotocin-induced diabetes in syngeneic and allogeneic mice. beta-Gal expression in transduced islets was observed for at least 6 weeks in naive normal recipients and in immunodeficient mice, but was shortened in mice preimmunized to adenovirus. Nevertheless, islets maintained normal control of glycemia in all mice. An early leukocyte infiltrate was observed in syngeneic grafts of transduced islets, but no acceleration in rejection of fully MHC-incompatible islet grafts occurred. In summary, adenovirus-mediated gene transfer in adult mouse islets, although sparing most of the beta-cells, was highly efficient and did not impair insulin secretion by islets. The immune response to the adenovirus and/or to the transgene might be only partially responsible for the decreased expression over time of the transduced gene. Accordingly, adenovirus-mediated gene transfer might allow efficient expression of vectorized sequences with potential immunosuppressive effects in the islet microenvironment.