However, the effect of
human DN T cells on resting CD4+ and CD8+ T cells, their potential immunomodulatory RGFP966 role, and the mechanism of suppression are still rather unclear. In the present study, we demonstrate that human DN T cells can strongly suppress proliferation of CD4+ and CD8+ T cells. Moreover, DN T cells are also able to downregulate proliferation and cytokine production of highly activated effector T cells. In contrast to their murine counterparts, human DN T cells do not eliminate effector T cells by Fas/FasL-mediated apoptosis but suppress by an active cell contact-dependent mechanism. Together, these data suggest that human DN T cells might regulate proliferation and effector function of T cells and thereby contribute to peripheral tolerance. To determine the role of human DN T cells in suppressing immune responses, DN T cells were isolated and stimulated with allogeneic
mature DC as described in Materials and methods. In contrast to freshly isolated DN T cells, DC-stimulated DN T cells expressed activation markers and revealed an effector-memory phenotype (Fig. 1A). However, both resting and stimulated DN T cells lacked expression FK506 of Foxp3 or the cytotoxic T lymphocyte antigen 4 (CTLA-4). First, we asked whether prestimulated DN T cells are able to inhibit proliferation of CD4+ and CD8+ T cells that are autologous to the DN T cells. To address this question, CFSE-labeled CD4+
or CD8+ T cells were cocultured with allogeneic DC in the presence or next absence of DN T cells and proliferation of CD4+ and CD8+ T cells was measured by flow cytometry. After 5 days, CD4+ and CD8+ T cells revealed a strong proliferation, which was completely abrogated by addition of DN T cells (Fig. 1A). The data obtained by CFSE staining were confirmed by [3H]thymidine incorporation demonstrating a strong suppressive activity of DN T cells (Supporting Information Fig. 1A). Of interest, DN T cells were able to suppress proliferation of both CD45RA+ naive as well as CD45RO+ memory T cells (Supporting Information Fig. 1B). We also examined the efficacy of DN T-cell-mediated suppression by titration of increasing numbers of suppressor to responder cells (Fig. 1C). Notably, DN T cells significantly suppressed proliferation of responder cells up to a ratio of 1:10. To exclude that the suppressive effect of DN T cells relates to in vitro expansion, we used expanded CD4+ or CD8+ T cells as suppressor cells in the MLR. Of importance, both expanded T-cell lines failed to suppress proliferation of responder cells (Supporting Information Fig. 1C). Since T-cell responses in autoimmune diseases and during allograft rejection are known to be very strong, we aimed to determine whether DN T cells are capable to suppress highly activated T-cell lines. Thus, CD4+ and CD8+ T cells were stimulated weekly with allogeneic DC.