CD45RC^low CD8 T cells inhibit the differentiation of alloreactive CD4 T cells into IFNγ-producing T cells. (A) Purified CD4 T cells from LEW rats (5 × 10⁵ cells/well) were stimulated with irradiated allogeneic, T-cell-depleted, BN splenocytes (2.5 × 10⁵) either without (▪) or with variable numbers of LEW CD45RC^high (•) or CD45RC^low (○) CD8 T cells. Tissue culture supernatants were collected on day 5 after allogeneic stimulation and assayed for IFNγ (left panel) and IL-10 (right panel) titer using capture ELISA. Similar results were obtained in 8 different experiments. (B) Results were expressed as the percentage of inhibition of IFNγ (□) and IL-10 (▦) production at a CD4/CD8 CD45RC subsets ratio of 1:1. The cytokine production by CD4 T cells activated without CD8 T cells was set as 100% of the response. Bars represent the mean values of 7 individual experiments, and filled circles represent the results of each individual experiment. (C) Purified CD4 T cells from LEW rats were stimulated with irradiated allogeneic, T-cell-depleted BN splenocytes, either with or without LEW CD45RC^high or CD45RC^low CD8 T cells added at a 1:1 ratio. T-cell blasts were collected after 96 hours of culture and were stimulated with phorbol myristate acetate (PMA) and ionomycin for 4 hours. Brefeldin A was added for the last 2 hours of incubation and staining was performed using anti-CD4 mAb and FITC anti-IFNγ, PE anti-IL-4, or PE anti-IL-10, as indicated in “Materials and methods.” The percentage of IFNγ-producing cells was enumerated after gating on CD4 T cells and is indicated in each plot. The dotted lines represent the isotype control Ab labeling. These data are representative of 3 independent experiments.