Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation.

Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, ß4galNT2, SLA-I α chain, and ß-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.

Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses.

Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, ß4galNT2, SLA-I α chain, and ß2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.

Analysis of anti-HLA antibodies in sensitized kidney transplant candidates subjected to desensitization with intravenous immunoglobulin and rituximab.

BACKGROUND: Preexisting donor-specific antibodies against human leukocyte antigens are major risk factors for acute antibody-mediated and chronic rejection of kidney transplant grafts. Immunomodulation (desensitization) protocols may reduce antibody concentration and improve the success of transplant. We investigated the effect of desensitization with intravenous immunoglobulin and rituximab on the antibody profile in highly sensitized kidney transplant candidates. METHODS: In 31 transplant candidates (calculated panel-reactive antibody [cPRA], 34%-99%), desensitization included intravenous immunoglobulin on days 0 and 30 and a single dose of rituximab on day 15. Anti-human leukocyte antigen antibodies were analyzed before and after desensitization. RESULTS: Reduction of cPRA from 25% to 50% was noted for anti-class I (5 patients, within 20-60 days) and anti-class II (3 patients, within 10-20 days) antibodies. After initial reduction of cPRA, the cPRA increased within 120 days. In 24 patients, decrease in mean fluorescence intensity of antibodies by more than 50% was noted at follow-up, but there was no reduction of cPRA. Rebound occurred in 65% patients for anti-class I antibodies at 350 days and anti-class II antibodies at 101 to 200 days. Probability of rebound effect was higher in patients with mean fluorescence intensity of more than 10,700 before desensitization, anti-class II antibodies, and history of previous transplant. CONCLUSIONS: The desensitization protocol had limited efficacy in highly sensitized kidney transplant candidate because of the short period with antibody reduction and high frequency of rebound effect.