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. 2020 Feb 27;217(5):e20190834. doi: 10.1084/jem.20190834

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© 2020 Edwards et al.

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Figure 3. — Hybrid αβ-γδ T cells can be activated innately or via αβ or γδ TCRs. (A) Purified Vγ4⁺ cells or Vγ4-depleted CD3⁺ cells were stimulated for 3 d with IL-1β and IL-23 or with medium alone. Data show cytokine production measured by ELISA. (B) Flow cytometric analysis of IL-1R1 and IL-23R expression on Vγ4⁺TCRδ⁺TCRβ⁺, Vγ4⁺TCRδ⁺TCRβ⁻, or Vγ4^–TCRδ⁻TCRβ⁺ cells, gated on live CD3⁺ cells. (C) Relative Ifng and Il17a expression in purified Vγ4⁺TCRβ⁺ or Vγ4⁺TCRβ⁻ cells stimulated for 3 d with IL-1β, IL-2, and IL-23 in the presence or absence of plate-bound anti-CD3. (D and E) IFN-γ, IL-17, and GM-CSF production by TCRδ⁺TCRβ⁺ or TCRδ⁺TCRβ⁻ cells (D), or TCRδ⁺TCRβ⁺ cells (E), isolated from OT-II mice and stimulated for 3 d with or without OVA-pulsed DCs in the presence or absence of IL-1β and IL-23 and/or anti-MHCII. Cytokines were measured by ELISA. (F and G) Vγ4⁺TCRβ⁺ cells were 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. Relative gene expression was measured by RT-PCR (F), and cytokines were measured by ELISA (G). PECs were isolated from naive mice or 3 h after i.p. challenge with S. aureus. (H) Cytokine levels in PEC culture supernatants measured by ELISA. (I) Flow cytometric quantification of TCRδ⁺ cells, gated on live CD3⁺ cells. (J) Flow cytometric quantification of Vγ4⁺TCRδ⁺TCRβ⁺ or Vγ4⁺TCRδ⁺TCRβ⁻ cells, gated on live CD3⁺TCRδ⁺ cells. (K) Flow cytometric quantification of IFN-γ and IL-17 production by Vγ4⁺TCRδ⁺TCRβ⁺ cells. (L) Bacterial loads in the peritoneal cavity and kidneys of WT mice 3 d after S. aureus infection or IL-17^−/− mice 3 d after adoptive transfer of Vγ4⁺TCRβ⁺ cells from WT mice or mock transfer (PBS). Data are representative of three independent experiments (n = 4–6 in A–K) or combined from two experiments (n = 8 or 12 in L). Flow cytometry plots in B are representative of six samples. Results are shown as mean ± SEM; *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, ^#,P < 0.05; ns, not significant; two-way ANOVA with Tukey's test for multiple comparisons (A, D, and E), one-way ANOVA with Tukey's test for multiple comparisons (B and L), three-way ANOVA with Tukey's test for multiple comparisons (C), or unpaired t test (F–K).