Figure 3.

NK1.1⁺CD4⁺ T cells express Tbet and Bcl6 and produce effector cytokines. (A) Representative intracellular staining of NK1.1⁻ and NK1.1⁺ CD4⁺ T cells for the transcription factors Tbet, Bcl6, RORγt, Gata3, and Foxp3. Cells previously gated on live, CD44^hiCD62L^loCD4⁺TCRβ⁺ splenocytes. Gates based on FMO controls shown in Supplemental Figure 4. (B) Frequency of NK1.1⁺ and NK1.1⁻ CD4⁺ T cells expressing Tbet, Bcl6 or, Tbet and Bcl6 on days 8 and 11 p.i. An aligned rank transformation was performed on non-parametric data before determining significance by two-way ANOVA with a post hoc Holm-Sidak's multiple comparisons test. *p < 0.05, **p < 0.01. (C) Median fluorescence intensity (MFI) for Tbet and Bcl6 in NK1.1⁺ and NK1.1⁻ CD4⁺ T cells on day 8 and 11 p.i. An aligned rank transformation was performed on non-parametric data before determining significance by two-way ANOVA with a post hoc Holm-Sidak's multiple comparisons test. *p < 0.05. (D) Representative intracellular cytokine staining of NK1.1⁻ and NK1.1⁺ CD4⁺ T cells previously gated on live CD4⁺TCRβ⁺ T cells, on day 7 post-infection. Splenocytes were stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A before being stained for IFN-γ, IL-21, and TNFα. (E) The frequency of single, double, or triple cytokine-producing cells at day 7 post-infection. Significance assessed via an unpaired non-parametric Mann-Whitney test. *p < 0.05. Data are representative of three independent experiments (error bars, s.e.m.).