Posttransplant IL-7 administration increases the proliferation of nonalloreactive donor T cells in recipients of allogeneic BMT. (a) Lethally irradiated CBA/J recipients were transplanted with B10.BR TCD-BM (5 × 10⁶) and received 10 μg/day IL-7 or PBS (control) on days 21–27. Animals were sacrificed on day 28 and splenocytes were stained with cell surface antibodies (anti-CD4, -CD8, and -Ly9.1), followed by fixation, permeabilization, and intracellular staining with 7-AAD for cell cycle analysis. The values shown represent the mean percentage ± SEM of donor CD4⁺ or CD8⁺ T cells in S/G2/M phase with P values comparing IL-7–treated with PBS-treated groups (four mice per group). *P < 0.05. (b) Mice were transplanted as described in a, and BrdU was added to their drinking water at a concentration of 0.8 mg/ml on days 15–33 after allogeneic BMT. The mice also received 1 μg/day IL-7 or PBS (control) on days 15–33. Splenocytes were harvested at day 33 and stained with anti-CD4, CD8, -CD44, and -BrdU antibodies. The values shown represent the mean percentage ± SEM of donor naive or memory CD8⁺ T cells that are BrdU⁺, with P values comparing IL-7–treated with PBS-treated groups (four mice per group). (c and d) Sublethally irradiated B6 (Ly5.1⁺) (syngeneic) or C3FeB6F1 (allogeneic) recipients were infused with CFSE-labeled B6 purified T cells (2 × 10⁷). The recipients received 10 μg/day IL-7 or PBS (control) on days 0–3. On day 3, splenocytes were harvested and stained with anti-CD4 and anti-CD8 antibodies for flow cytometric analysis. Results shown are representative of three duplicate experiments. (e) Splenocytes were harvested from C3FeB6F1 (allogeneic) recipients as in d, and incubated with PMA (10 ng/ml) and ionomycin (2 μM) for 5 hours. Brefeldin A was added during the last 3 hours to inhibit IFN-γ secretion. Cells were harvested and stained with anti-CD4 and anti-CD8 antibodies, then washed, fixed, permeabilized, and stained intracellularly with anti–IFN-γ antibody.