Role of CD28 in T helper cell responses. (A) Mice were transferred with naïve 4get.OT-II cells, treated with papain OVA as in Fig. 1 A, and administered α-CD28 blocking antibody or IgG isotype control intraperitoneally 24 h after immunization, and dLNs were harvested and assessed by flow cytometry at 48 h. Endogenous CD44⁺Ki67⁺BCL6⁻ CD4 T cells were assessed for Gata3 and IRF4 coexpression with the indicated treatment. (B) Naïve CD45.2 4get.OT-II cells were transferred to CD45.1 mice and injected with CpG OVA in the ear pinnae or front footpad. Mice were treated intraperitoneally with α-CD62L 6 h post immunization, treated intraperitoneally with α-CD28 blocking antibody or IgG isotype control 24 h post immunization, and dLNs were harvested and assessed by flow cytometry at 48 h. Quantification of CD44⁺ OT-II cell number, frequency of CXCR3⁺ cells, and Tbet gMFI on CD44⁺ OT-II cells is shown. (C) Naïve OT-II cells were cultured in vitro with the indicated concentrations of α-CD3 and α-CD28. IL-4 and α-IFNγ were added to the culture in some conditions (dashed lines). Cells were harvested for flow cytometry 48 h later and assessed for expression of Gata3 and IRF4. Data shown represents one independent experiment with at least n = 4 independently immunized lymph nodes from two mice per group or three wells per treatment group. Graphs show mean ± SD and were analyzed using unpaired Student’s t test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Figures are representative of four independent experiments.