Phenotypic and functional characterization of human T cell clones indirectly activated against adult pig islet cells

Background: Xenotransplanted patients produce xenospecific IgG1 antibodies directed against epitopes other than Gal alpha 1,3Gal. IgG1 antibody production is believed to be dependent upon T cell help. Therefore, as a natural continuation of our work aimed at characterizing the xenoimmune antibody response against pig islet cells, we have also examined the T cell response. T cell reactivity against islet cells is believed to result from indirect antigen presentation, and our in vitro study was designed to mimic the response in vivo. The main purpose of this study was to characterize the phenotype, the immunological specificity and the functional capacity of indirectly activated T cell clones, reactive against pig islet cell antigens. Materials and methods: Human T cell clones, activated against pig islet cells in the presence of autologous antigen-presenting cells, were produced from limiting dilutions of bulk cultures. Clonality was investigated by T cell receptor V beta (TcRV beta) expression analysis. Clonal specificity was studied in proliferation assays using different pig cells as stimulators. ELISpot experiments were performed to detect cytokine production patterns. The cytotoxic capacity of the clones was assessed using standard cell-mediated lysis tests and different porcine and human target cells. Several long-term bulk cultures of human lymphocytes, indirectly activated against pig islet cells, maintained for up to 60 days, were used as a control for possible bias in the selection of the clones. Results: Nineteen CD4(+) TcRV alpha beta(+) T cell clones were recovered. No activation of natural killer T cells or gamma delta-T cells was recorded. There was no bias in the TcRV beta-usage. The immunological specificity differed between clones; some were specifically reactive against pig islet cell antigens, while others were reactive with antigens present on a variety of pig cells. All clones produced a broad spectrum of cytokines, e.g. interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)5, IL10 and IL13, with no evidence of bias for a particular phenotype. None of the T cell clones were cytotoxic against pig islet cells, but two clones were cytotoxic against pig phytohemagglutinin (PHA)-blasts. Conclusion: The analysis of several, indirectly activated, human CD4(+) T cell clones shows that the response against pig islet cells is heterogeneous both with regard to immunological specificity and functional characteristics. This heterogeneity was further confirmed by analysis of the long-term bulk cultures of human lymphocytes, indirectly activated against pig islet cells.