Spontaneous ex vivo proliferation and IL-2 production of donor T cells was reduced in the absence of CD28. Splenocytes were obtained from control BDF1 mice without transplant and from BDF1 recipients of CD28^+/+ or CD28^−/− cells 2 days after transplantation. Three mice per group were analyzed separately, and the data represent the mean +/− 1 SD of three individual mice. (A) Splenocytes were cultured at 2.0 × 10⁵/well for 4 hours in medium, and triplicate cultures were pulsed with [³H]TdR at the beginning of the culture. Results show the proliferative capacity of splenocytes early in GVHD. (B) Splenocytes were cultured at 5.0 × 10⁶/mL for 24 hours in medium alone, and supernates were assayed for IL-2 by the proliferation of CTLL-2 cells in the presence of anti–IL-4 MoAb.