Time requirements of T cell help and the role of T_regs cells.

(A) 2×10⁴ CD8⁺ T cells were transferred into CD3ε^−/− hosts. At 8 or 29 days after groups of hosts received CD4 T cells (2×10⁴ cells) and the mice were sacrificed 7 weeks after the first cell transfer. The mean CD8⁺ T cell recovery is shown for the three groups of hosts (***p≤0.001). It should be pointed out that the late transfer of CD4 T cells still induced the preferential accumulation of CD62L^lowCD8⁺ T_EM cells (not shown) (B) 2×10⁴ CD8⁺ T cells were transferred alone or with CD4⁺ T cells from Lat-DTR mice into a CD3ε^−/− host. A fraction of the mice were treated with diphtheria toxin (DT) for 10 days at either one or 4 weeks after cell transfer, and the mice were analyzed 7 weeks after cell transfer. The number of CD8⁺ T cells recovered 7 weeks after cell transfer is shown. CD4⁺ T cell depletion at week 1 or at week 4 reduced the number of CD8⁺ T cells recovered (**p≤0.01). Similar findings were obtained in two independent experiments. Dot plots show representative examples of the CD62L and CD44 expression by the CD8⁺ T cells recovered in the different groups of mice. (C) 10⁴ CD8⁺ T cells were transferred into CD3ε^−/− hosts alone or with 5×10⁴ CD4 T_reg cells. The number of CD8⁺ T cells recovered in individual mice is given as the mean value for each group. (D) 10⁴ CD8⁺ T cells were transferred into CD3ε^−/− immune-deficient hosts alone, co-transferred with 10⁴ CD4^naive T cells or co-transferred with 10⁴ CD4^naive T cells and 5×10⁴ CD4 T_reg cells in three independent groups of hosts. The number of CD8⁺ T cells recovered in individual mice is given as the mean value for each group. The increased expansion of CD8⁺ T cells seen upon co-transfer with CD4^naive T cells was abolished when the T_reg cells were added in the transferred mix. (**p≤0.01) (E) 10⁵ total CD8⁺ T cells were transferred into CD3ε^−/− hosts alone, co-transferred with 10⁴ CD4^naive T cells or co-transferred with 10⁴ CD4 T cells and then given 5×10⁴ T_reg cells four weeks latter. Mice were culled 8 weeks after the initial CD8⁺ T cell transfer. The data indicate the number of CD8⁺ T cells recovered in each host. Statistically significant differences are shown (**p<0.01). Similar findings were obtained in two independent experiments.