CD40L⁺ CD8⁺ T cells functionally resemble CD4⁺ T helper cells. (A) CD8⁺ and CD4⁺ T cells were stimulated with P/I and CD40L⁺ and CD40L⁻ cells were separated. Next, the T-cell subsets were cocultured for 24 hours with immature moDCs in the presence (red) or absence (black) of blocking αCD40L antibodies. Gray filled lines represent CD83 expression on DCs without T cells. The median ± SEM of the CD83 expression is written beneath the histograms in matching colors. (B) Supernatants of experiments described in panel A were collected to determine cytokine concentrations. MoDC controls without T cells were also cultured in the presence of αCD40 antibody or αCD40L antibody (gray or white bars, respectively). (C) As in panel A, the same sorted T-cell populations were cocultured with B cells in the presence (red) or absence (black) of blocking αCD40L antibodies. The proliferation of cocultured B cells was assessed after 8 days based on CFSE dilution, and the supernatant from the same cocultures was analyzed by ELISA for secretion of immunoglobulin M and immunoglobulin G. The percentages represent the frequencies of proliferated CFSE^low B cells. (D-E) CD40L^−/− mice received 10⁶ OT-1 and OT-1xCD40L^−/− CD8⁺ T cells (intravenously) and were challenged 1 day later with OVA-peptide (SIINFEKL) (subcutaneously). (D) The level of IL-12 in the blood serum was quantified by ELISA 36 hours postimmunization, and (E) the mean fluorescence intensity (MFI) of CD40 and CD80 from draining LN CD11c⁺ MHC-II⁺ cells was determined. (D-E) For statistical analysis, analysis of variance with the Bonferroni multiple comparison posttest was used (*P < .05; **P < .01; ***P < .001). (A-E) One representative experiment out of 2 or 3 is shown.