Fig 2: MP lymphocytes differentiate into the T-bethigh subset in the presence of DC1-derived IL-12.(a and b) IL-12 p40 and p35 are essential for optimal MP differentiation. a The frequency (mean ± SD) of MP cells among CD4+ T lymphocytes obtained from WT, Il12b KO, and Il12a KO mice is shown (WT n = 8 mice; Il12b KO n = 8 mice; Il12a KO n = 7 mice). b The representative plots show T-bet and CXCR3 expression in MP CD4+ T cells from each group while the bar graph indicates the frequency (mean ± SD) of T-bethighCXCR3+ fraction among the same CD4+ T cell population (WT n = 8 mice; Il12b KO n = 8 mice; Il12a KO n = 7 mice). Data are pooled from three independent experiments performed. (c) IL-23A p19 is dispensable for T-bethigh MP differentiation. The bar graph shows the frequency (mean ± SD) of T-bethighCXCR3+ cells among MP cells from WT and Il23a KO mice (n = 5 mice). Data are representative of two independent experiments performed. (d and e) DC1 tonically produce IL-12B p40. The representative contour plots in (d) show p40 expression by the indicated DC subsets from WT animals while the histograms in (e) depict YFP levels expressed by the same DC subsets from p40-YFP reporter mice. The filled histograms show negative control staining using non-reporter mice. The bar graphs indicate the p40+ and YFP+ fractions (mean ± SD) among indicated cell populations (n = 5 mice). Data are representative of three independent experiments. (f and g) Activated DC1 produce tonic IL-12B p40. The representative plots in (f) depict p40 expression in MHCIIvery highCD86highCD40high and MHCIIhighCD86lowCD40lowCD8a+ DCs from WT mice while the histograms in (g) show YFP expression in the same DC subsets from p40-YFP reporter mice. The bar graphs in (f) and (g) show the p40+ and YFP+ fractions (mean ± SD), respectively, among indicated CD8a+ DC subpopulations (n = 3 mice). Data are representative of two independent experiments performed. (h and i) p40+ DC1 are localized in the splenic T-cell zone. Different cell types in spleen of p40-YFP reporter mice were analyzed by multicolor tissue imaging. B220: yellow; CD4: purple; CD8: red; CD11c: blue; YFP: green. Scale bars show (h) 150 µm and (i) 15 µm. Images are representative of four sections from two mice. A two-sided t-test was performed to assess statistical significance. Source data are provided as a Source Data file.

Fig 3: TLR-MyD88 signaling homeostatically induces IL-12 and supports MP differentiation. (a and b) MyD88 is essential for homeostatic IL-12B p40 production and optimal T-bethigh MP differentiation. a Representative contour plots indicating p40 expression in CD8a+ DCs from WT and Myd88 KO animals and a bar graph showing the fraction (mean ± SD) of p40+ among CD8a+ DCs from each group (n = 4 mice). b The dot plots show T-bet and CXCR3 expression in MP CD4+ T lymphocytes from each group while the bar graph indicates the frequency (mean ± SD) of T-bethighCXCR3+ cells among the total MP population (WT n = 5 mice; Myd88 KO n = 4 mice). Data are representative of two independent experiments performed. (c and d) T cell-extrinsic MyD88 supports T-bethigh MP differentiation from naïve precursors. Sorted naïve CD4+ T lymphocytes were transferred into congenic WT and Myd88 KO animals and analyzed 3 weeks later. The representative dot plots indicate expression of (c) CD44 and CD62L in the donor cell population and (d) T-bet and CXCR3 in MP donor cells from each group. The bar graphs show the frequency (mean ± SD) of (c) MP cells among donor cell population and (d) T-bethighCXCR3+ subset among MP donor cells (WT n = 5 mice; Myd88 KO n = 4 mice). Data are pooled from two independent experiments. (e) MyD88 tonically activates DC1. The bar graph indicates the frequency (mean ± SD) of MHCIIvery highCD86highCD40high cells among CD8a+ DCs (n = 3 mice). (f and g) IL-1/18 signaling is dispensable for tonic IL-12B p40 production and T-bethigh MP differentiation. The bar graphs show the frequency (mean ± SD) of (f) p40+ cells among CD8a+ DCs and (g) T-bethighCXCR3+ cells within MP cells from the indicated KO animals (n = 5 mice). (h–j) Contribution of multiple TLRs to tonic IL-12B p40 production and MP differentiation. The bar graphs indicate the fraction (mean ± SD) of (h) p40+ cells among CD8a+ DCs, (i) MHCIIvery highCD86highCD40high cells among CD8a+ DCs, and (j) T-bethighCXCR3+ cells among MP lymphocytes obtained from indicated animals (WT n = 7 mice; Tlr2 KO n = 5 mice; Tlr4 KO n = 4 mice; 3D n = 3 mice). Data are pooled from two independent experiments. A two-sided t-test was performed to assess statistical significance. Source data are provided as a Source Data file.

Fig 4: DC1 are essential for optimal T-bethigh MP cell differentiation.(a–c) BatF3-sensitive DCs are essential for the optimal differentiation of MP cells. a The bar graph shows the frequency (mean ± SD) of MP cells among CD4+ T lymphocytes from WT and Batf3 KO mice (n = 5 mice). b The representative dot plots depict expression of T-bet and CXCR3 in MP CD4+ T cells from each group while the bar graph indicates the frequency (mean ± SD) of the T-bethighCXCR3+ subpopulation among the same MP population (n = 5 mice). c Total cell numbers (mean ± SD) of each of the MP subsets in (b) (n = 5 mice). Data are representative of two independent experiments. (d) CD4+ T cell-extrinsic BatF3 and IL-12 support differentiation of naïve CD4+ T lymphocytes toward the T-bethigh MP subset. Sorted naïve CD4+ T cells were transferred into congenic WT and the indicated KO mice and analyzed 3 weeks later. The representative dot plots depict T-bet and CXCR3 expression by donor- and recipient-derived MP cells while the bar graph shows the frequency (mean ± SD) of T-bethighCXCR3+ subpopulation among MP donor cells (WT n = 6 mice; Il12b KO n = 5 mice; Il12a KO n = 3 mice; Batf3 KO n = 5 mice). Data are pooled from two independent experiments performed. A two-sided t-test was performed to assess statistical significance. Source data are provided as a Source Data file.

Fig 5: Microbial products are dispensable for tonic p40 expression and T-bethigh MP differentiation.(a and b) Commensal bacteria are not required for IL-12B p40 expression or MP differentiation. a Representative contour plots comparing p40 expression in splenic CD8a+ DCs from SPF and GF mice together with a bar graph showing the frequency of p40+ cells (mean ± SD) among CD8a+ DCs from each group (n = 4 mice). b Representative dot plots depicting T-bet and CXCR3 expression in MP cells together with a summary graph indicating the frequency (mean ± SD) of T-bethighCXCR3+ lymphocytes among the same CD4+ T-cell subpopulations from each group (n = 3 mice). Data are representative of two independent experiments performed. (c–e) AF mice display homeostatic IL-12B p40 expression and MP differentiation indistinguishable from both SPF and GF mice. The bar graphs show the frequency (mean ± SD) of (c) p40+ cells among CD8a+ DCs and (d) T-bethighCXCR3+ cells among MP CD4+ T lymphocytes as well as (e) ?MFI of CD40L expression in the indicated cell populations from SPF, GF, and AF mice (n = 4 mice). A two-sided t-test was performed to assess statistical significance. Source data are provided as a Source Data file.