CD28-mediated Lck signaling regulates GVHD development and iTreg generation in vivo. BALB/c mice were lethally irradiated (800 cGy) and transplanted with 5 × 10⁶ of TCD-BM cells alone or plus 1 × 10⁶ T cells (CD4⁺ or CD8⁺ CD25⁻) from CD28-WT or CD28-Lck mice. Recipient mice were monitored throughout the experimental period for survival (A) and weight change (B). Using the same BMT setting as in panels A and B, 0.5 × 10⁶ CD8⁺ cells from normal B6 donors alone or plus 0.5 × 10⁶ CD4⁺ cells from CD28-WT or CD28-Lck mice were transplanted. Data show recipient survival (C) and weight changes (D) at different days after BMT. (E) BALB/c mice were lethally irradiated (800 cGy) and transplanted with 5 × 10⁶ TCD-BM cells alone or plus 1 × 10⁶ T cells (CD4⁺CD25⁻) from CD28-WT and CD28-Lck mice. Two weeks after transplantation, recipient spleens were harvested and measured for expression of surface CD4, CD25, and intercellular Foxp3. Percentage of Tregs (CD25⁺Foxp3⁺) on gated donor T cells (H2K^b+CD4⁺) is shown in each mouse for a total of 3 mice per group, and the data represent 1 of 3 replicate experiments. In separate experiments, CFSE-labeled T cells (CD4⁺ or CD8⁺ CD25⁻) isolated from CD28-WT and CD28-Lck mice were transplanted into lethally irradiated (800 cGy) BALB/c mice at 2 × 10⁶ per mouse. Four days after transplantation, recipient spleens were harvested and measured for CFSE profile, expression of surface CD4 and H2K^b, and intracellular IFNγ. Expression of CD4 and H2K^b was shown on live spleen cells (F). CFSE profile (G) and intracellular IFNγ expression (H) were shown on gated donor CD4 (H2K^b+CD4⁺) or CD8 (H2K^b+CD4⁻) T cells. The data represent 1 of 2 replicate experiments.