Human regulatory DCs induce T-cell anergy in alloreactive T cells.
(A) Human naive CD4+ T cells (105) were cultured with allogeneic normal or modified DCs (104), and the proliferative response was measured on day 5. In another experiment, human naive CD4+ T cells (5 × 106) were cultured with allogeneic normal or modified DCs (5 × 105) for 3 days. These CD4+ T cells were then rescued, and cells (105) were subsequently restimulated with allogeneic normal mDCs (104) generated from same donor or unrelated donor in the presence or absence of human IL-2 in a second coculture. The proliferative response was measured on day 5. Data were expressed as mean values ± SD of 5 individual experiments. *P < .01 compared with CD4+ T cells plus allogeneic normal mDCs under each set of experimental conditions by Student paired t test. (B) Human total CD4+ T cells (105) were cultured with the indicated types of allogeneic DCs (104), and the proliferative response was measured on day 5. In another experiment, human total CD4+T cells (5 × 106) were cultured with the indicated types of allogeneic DCs (5 × 105) for 3 days. These CD4+ T cells were then rescued, cells (105) were subsequently restimulated with allogeneic normal mDCs (104) generated from same donor in a second coculture, and the proliferative response was measured on day 5. Data were expressed as mean values ± SD of 5 individual experiments. *P < .01 compared with CD4+ T cells plus allogeneic normal mDCs under each set of experimental conditions by Student paired t test. (C) Human naive CD4+ T cells (105) were cultured with allogeneic normal or modified DCs (103-104), and the proliferative response was measured on day 5. In another experiment, human naive CD4+ T cells (5 × 106) were cultured with allogeneic normal or modified DCs (5 × 104 to 5 × 105) for 3 days. These CD4+ T cells were then rescued, and cells (105) were subsequently restimulated with allogeneic normal mDCs (104) generated from same donor in a second coculture. The proliferative response was measured on day 5. Data were expressed as mean values ± SD of 5 individual experiments. *P < .01 compared with CD4+ T cells plus allogeneic normal mDCs under each set of experimental conditions by Student paired t test. (D) Human naive CD4+ T cells (5 × 106) were cultured with allogeneic normal or modified DCs (5 × 105) for 3 days. These CD4+ T cells were then rescued, and cells (105) were subsequently restimulated with allogeneic normal mDCs (104) generated from same donor. The proliferative response was measured on the indicated days. Data were expressed as mean values ± SD of 5 individual experiments. *P < .01 compared with CD4+ T cells plus allogeneic normal mDCs by Student paired t test. (E) Human naive CD4+ T cells (105) were cultured with allogeneic normal mDCs (104), and the proliferative response was measured on day 5. In another experiment, human naive CD4+ T cells (5 × 106) were cultured with allogeneic normal mDCs (5 × 105) for 3 days. These CD4+ T cells were then rescued, and cells (105) were subsequently cultured in medium alone (none) or restimulated with allogeneic normal mDCs (104) in the presence or absence of allogeneic normal or modified DCs (103-104) in a second coculture. The proliferative response was measured on day 5. Data were expressed as mean values ± SD of 5 individual experiments. *P < .01 compared with Ag-primed CD4+ T cells plus allogeneic normal mDCs in second coculture by Student pairedt test. (F) Human allogeneic Ag-specific CD8+ T cells (5 × 106), which were obtained from the primed PBMCs with allogeneic fibroblasts (donor no. 1), were cocultured with or without allogeneic normal or modified DCs (5 × 103 to 5 × 105) derived from the indicated donors (no. 1 or 2) for 3 days. These CD8+ T cells were then rescued, and cells (5 × 105) were subsequently subjected to a cytotoxicity assay against the allogeneic fibroblasts (104) derived from the indicated donors (no. 1 or 2). The value of spontaneous release cpm was less than 10% of the total release cpm. Data were expressed as mean values ± SD of 5 individual experiments. *P < .01 compared with allogeneic Ag-specific CD8+ T cells alone by Student paired ttest.
