Figure 3.

Loss of both DGKα and ζ enhances T_H1 and T_H17 differentiation in vivo. Thy1.1⁺Thy1.2⁺ congenic mice in vivo injected with 1.5 × 10⁶ Thy1.1⁻Thy1.2⁺TCRVα2⁺CD4⁺ WT or DKO naïve OT2 T cells on day −1 were immunized with OVA_323−339 peptide in CFA on day 0. Spleens and dLNs were harvested on the seventh day after immunization. (A) Representative dot plots of dLN cells and splenocytes. Top panels: CD4 and TCRVα2 staining. Bottom panels: Thy1.1 vs. Thy1.2 staining of the gated TCRVα2⁺CD4⁺ population. (B,C) Mean ± SEM of percentages (B) and number (C) of donor-derived OT2 T cells in dLNs and spleens (n = 4). (D–H) dLN cells and splenocytes were stimulated with PMA and ionomycin for 4–5 h in the presence of GolgiPlug, followed by cell surface and intracellular staining. (D) Representative dot plots of indicated cytokines in gated donor-derived OT2 cells. (E–H) Mean ± SEM of percentages of IFN-γ-producing cells (E) and IL-17-producing cells (F) as well as total numbers of donor-derived IFN-γ-producing (G) and IL17-producing (H) OT2 T cells. (I–K) Splenocytes and dLN cells were stimulated with (OVA+) or without (OVA–) OVA_323−339 for 2 days, with the addition of GolgiPlug in the last 5 h, and then were cell surfaced and intracellular stained for OT2 T cells and cytokine expression. (I) Representative dot plots of indicated ctyokine-producing cells in gated donor-derived OT2 cells. (J) Percentages of donor-derived cytokine-producing OT2 T cells (n = 4). (K,L) IFN-γ (K) and IL-17A (L) concentrations in culture supernatant harvested before adding GolgiPlug (n = 3). Data shown are representative of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 as determined by the Student t-test.