Functional characterization of the CD28^+/−and CD28^−/− P1CTL.

(A) Proliferative response of transgenic spleen cells to varying concentrations of the P1A peptide was measured by pulsing the culture for 6 hours with ³H-TdR, starting at 42 hours of culture. A mixture of anti–B7-1 and anti–B7-2 (1 μg/mL) mAbs was added at the beginning of the culture. Data presented are means and SE of triplicates of cpm. (B, C) Role of CD28 in production of cytokines, IL-2 (B) and IFN-γ (C) in response to antigenic P1A peptide (0.1 μg/mL). Cytokines released into the supernatants at 48 hours after antigenic stimulation were measured by sandwich ELISA. (D) Cytotoxicity of activated CD28^+/− and CD28^−/− P1CTL. Spleen cells activated in vitro for 4 days were used as the effectors, whereas the macrophage cell line P388D1, pulsed with (+P) or without (−P) P1A peptide (1 μg/mL) was used as the target. (E) Anti-B7 mAbs inhibit IFN-γ production by CD28^−/− P1CTL, as detailed in panel C, except that anti–B7-1 and anti–B7-2 mAbs (10 μg/mL) were added into the culture. (F) Anti-B7 mAbs (added before the addition of effector T cells and present during CTL assay only) did not inhibit the cytolysis of P1A peptide–pulsed P388D1 target cells by P1CTL. Data shown are representative of at least 2 independent experiments.