Hybrid αβ-γδ T cells can be activated innately or via αβ or γδ TCRs. (A) Coexpression of IL-1R1 and IL-23R on Vγ4⁺ hybrid αβ-γδ, Vγ4⁺ γδ T cells, or αβ T cells. (B) Production of IFN-γ and IL-17 by purified Vγ4⁺TCRβ⁺ or Vγ4⁺TCRβ⁻ cells (3,000 cells/well) stimulated for 3 d with IL-1β and IL-23 in the presence or absence of plate-bound anti-CD3. (C) Expression of Sox13 and Rorc mRNA in purified Vγ4⁺TCRβ⁺ cells stimulated for 2 d with IL-1β and IL-23. (D) Production of IFN-γ by TCRδ⁺ cells isolated from OT-II or C57BL/6 mice and cultured for 2 d with DCs pulsed for 5 h with OVA peptide or KLH. (E) Gene expression in TCRδ⁺TCRβ⁺ cells isolated from OT-II mice and cultured for 3 d with DCs in the presence or absence of OVA peptide. (F) Cytokine production by γδ T cells isolated from OT-II mice stimulated for 3 d with or without OVA peptide in the presence or absence of IL-12p70 + IL-18 (Th1) or IL-4 + anti–IFN-γ + anti–IL-17 (Th2). (G) Gene expression in Vγ4⁺TCRβ⁺ cells isolated from MOG-immunized mice on day 7 and cultured for 3 d with DCs in the presence or absence of MOG and/or IL-1β and IL-23. (H) LN cells isolated from 7 d MOG + CFA immunized or naive mice incubated with MOG-tetramer-Pe or control tetramer-Pe, gated on CD3⁺CD4⁺CD44⁺ cells, examining TCRβ⁺TCRδ⁺ and TCRβ⁺TCRδ⁻ populations. (I) γδ T cells from OT-I mice incubated for 3 d with DCs ± OVA peptide ± IL-12p70 and IFN-γ quantified in supernatants by ELISA. (J) Flow cytometry analysis of NKG2D expression on naive CD3⁺ T cells gating on TCRβ⁺ and TCRδ⁺ populations. (K) CD3⁺ T cells were incubated with and without YAC-1 cells (10:1) for 48 h. Proliferation was measured through expression of Ki67 by Vγ4⁺TCRδ⁺TCRβ⁺ versus Vγ4⁺TCRδ⁺TCRβ⁻ cells, gated on live CD3⁺TCRδ⁺ cells. (L) Gene expression in TCRδ⁺TCRβ⁺ cells stimulated for 2 d with anti-TCRδ. (M) Gene expression in CD3⁺ cells cultured for 3 d with IL-1β and IL-23 in the presence or absence of anti-TCRδ. Data are representative of at least two independent experiments. Results are shown as mean ± SEM. P values were calculated using a one-way ANOVA with Tukey's test for multiple comparisons (B, D, F, G, I–K, and M) or an unpaired t test (C, E, and L). *, P < 0.05, **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001. ns, not significant.