Figure 5. Interleukin (IL)-10-producing CD4⁺ T cells express T-bet and T-bet-inducible genes.

(A, B) 10BiT mice were infected with 3 × 10⁴ pfu of murine cytomegalovirus (MCMV). (A) Leukocytes were isolated from the salivary glands (SGs) on day 14 p.i. Representative histograms (left) and summary bar graph (right) show the expression of T-bet among Thy1.1⁺ CD4⁺ T cells (red) and Thy1.1⁻ CD4⁺ T cells (blue). Data are shown as mean ± standard error of the mean (SEM; n = 5–6 mice per group pooled from two independent experiments). MFI, median fluorescence intensity. **p < 0.01 (Mann–Whitney U test). (B) Leukocytes were isolated from the SGs on day 14 p.i. and sorted as CD4⁺ CD44⁺ CD62L⁻ CD90/90.1⁺ (Thy1.1⁺) or CD90/90.1⁻ (Thy1.1⁻) populations via fluorescence-activated cell sorting (FACS). ATAC-seq profiles show accessible chromatin regions in the Tbx21 gene for Thy1.1⁺ CD4⁺ T cells (red) and Thy1.1⁻ CD4⁺ T cells (blue). Data are shown as normalized values accounting for the total number of reads per lane. The black boxes indicate major differences in chromatin accessibility. Data are shown as pooled analyses from a minimum of n = 5 mice per group representing three independent experiments. (C) Venn diagram showing the overlap between genes enriched in Thy1.1⁺ CD4⁺ T cells (A, D) and genes enriched in T-bet⁺ CD4⁺ T cells (GSE38808). (D) Cd4^+/+Tbx21^flox/flox (Cd4^+/+) and Cd4^CreERT2/+Tbx21^flox/flox (Cd4^Cre/+) mice were infected with 3 × 10⁴ pfu of MCMV. Tamoxifen was administered (+Tam) or withheld (−Tam) from days 7 to 12 p.i. Leukocytes were isolated from the SGs on day 14 p.i. Representative flow cytometry plots show the expression of CD4 versus RFP (left) and CD44 versus T-bet for the gated populations (right). Data in the bar graph are shown as mean ± SEM (n = 4–5 mice per group representing four or five independent experiments). *p < 0.05, **p < 0.01 (Mann–Whitney U test).

Figure 5—figure supplement 1. Interleukin (IL)-10-producing CD4⁺ T cells are not selectively regulated by Eomes.

Representative flow cytometry plots (A) and histograms (B) showing the expression of Eomes versus Thy1.1. (C, D) Cd4^+/+Tbx21^flox/flox and Cd4^CreERT2/+Tbx21^flox/flox mice were infected with 3 × 10⁴ pfu of murine cytomegalovirus (MCMV). Tamoxifen was administered (+Tam) or withheld (−Tam) from days 7 to 12 p.i. (C) Leukocytes were isolated from the salivary glands (SGs) on day 14 p.i. The expression of T-bet was measured via flow cytometry. Data are shown as mean ± standard error of the mean (SEM; n = 4–5 mice per group representing four or five independent experiments). MFI, median fluorescence intensity. **p < 0.01 (Mann–Whitney U test). (D) MCMV replication in SG homogenates on day 28 p.i. measured via plaque assay. Data are shown as individual points with median values (n = 5 mice per group representing three independent experiments).