Reactivity of T cells from CD28-deficient scurfy mice. (A,B) In vivo activation of cd28^−/−scurfy mouse T cells. Lymph node cells from mice indicated above each panel were stained for CD4, CD8, CD3, CD44, and CD62L. Shown are (A) CD3 and (B) CD44 and CD62L staining on CD4⁺CD8⁻-gated cells. In panel A, numbers in each subpanel indicate the percentage of cells in each region against the total. In panel B, numbers in or above each quadrant indicate the percentage of cells in each region against the total. (C) Proliferative response of T cells from cd28^−/−scurfy mouse. Purified T cells from cd28^−/− and cd28^+/+ mice were stimulated with anti-CD3 (left panel) or anti-CD3+anti-CD28 (right panel) antibodies. Proliferation of T cells was measured in response to titrated doses of anti-CD3, respectively, by ³H-thymidine incorporation (mean ± SD, n = 3). (D) Cytokine production by T cells from cd28^−/−scurfy mice. Mo-Flo sorted TCR⁺CD4⁺DX5⁻ cells (2 × 10⁴ cells/well in flat-bottomed 96-well plate) were activated with 1 μg/mL anti-CD3-coated plates in the absence □, or presence ■, of anti-CD28 antibody (0.5 μg/mL). Forty-eight hours later, a portion of culture supernatant was harvested for analysis of cytokines as indicated on the top of the panels and culture supernatant from stimulated T cells were analyzed for cytokines denoted above each panel by enzyme-linked immunosorbent assay. CD4 T cells from wild-type (WT), cd28^−/− [28(-)sf)] and cd28^+/+scurfy (sf) mice were analyzed (mean ± SD, n = 2).