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: Stability of mature T-bethigh, T-betlow, and T-bet- MP cells and their IL-12-independence. (a and b) Blockade of IL-12B p40 does not hamper the T-bethigh MP subpopulation. WT mice received anti-IL-12B p40 mAb or control IgG every other day for 1 week and were analyzed for MP CD4+ T cells. a The bar graph shows the frequency (mean ± SD) of MP cells among CD4+ T lymphocytes (Control n = 5 mice; Anti-IL-12B n = 3 mice). b The representative dot plots display T-bet and CXCR3 expression in MP cells while the bar graph indicates the frequency (mean ± SD) of T-bethigh subpopulation among MP cells from each group (Control n = 5 mice; Anti-IL-12B n = 3 mice). (c and d) Batf3 KO MP cells have lower levels of T-bethigh subpopulation before and after transfer into congenic WT mice. Sorted MP cells from CD45.2+ WT and Batf3 KO mice were transferred into CD45.1+ WT recipients and the donor as well as recipient MP cells analyzed 10 days later. The representative dot plots show T-bet and CXCR3 expression in donor MP cells (c) before and (d) after transfer while the bar graph indicates the frequency (mean ± SD) of the T-bethigh subpopulation among transferred MP cells (n = 5 mice). (e) MP cells largely maintain their T-bet expression levels after generation. T-bethigh, T-betlow, or T-bet- MP cells were sorted from CD45.2+ T-bet-ZsGreen reporter mice, transferred into CD45.1+ WT recipient animals, and analyzed for their T-bet and CXCR3 expression 10 days later. Representative dot plots display T-bet and CXCR3 expression by donor as well as recipient MP cells from three independent host mice per group. A two-sided t-test was performed to assess statistical significance. Source data are provided as a Source Data file.

Fig 4: CHD4 Is Essential for Early B Cell Development(A) Mature B cells are absent in spleens of Mb1cre/wtChd4fl/fl mice. Splenocytes were stained with B220 and CD19 and analyzed using flow cytometry (top panel). Absolute numbers of total splenocytes and CD19+ B220+ B cells from each mouse strain are shown (n = 3; **p < 0.005) (bottom left). The spleen from a representative 10-week-old Mb1cre/wtChd4fl/fl mouse was compared with littermate control (Chd4fl/fl) (bottom right).(B) CHD4 deletion impairs early B cell development in the bone marrow. Flow cytometric analysis shows expression of IgM and B220 in the bone marrow. Cellularity of bone marrow is shown on the right side of representative flow plots. BM cells were stained with c-Kit and CD25 and analyzed for pre-B (IgM- B220+ CD25+ c-Kit-) and pro-B (IgM- B220+ CD25- c-Kit+) cells. The red box represents the parent gate for analysis of pre-B and pro-B cells. n = 5; **p < 0.005. Furthermost right panel shows PCR-based genotyping and loss of the fl cel Chd4 allele in total bone marrow cells and sorted pro-B cells. Genotyping PCR from two independent experiments is shown.(C) Loss of p53 does not restore B cell developmental defects in Mb1cre/wtChd4fl/fl mice. Absolute numbers of bone marrow from each mouse strain are shown (n = 4; **p < 0.005) (left). Flow cytometric analysis shows expression of IgM and B220 expression or CD25 and c-Kit in bone marrow (BM) (right).(D) Transgenic expression of Bcl2 does not rescue B cell developmental defects in Mb1cre/wtChd4fl/fl mice. Absolute numbers of total bone marrow from each mouse strain are shown (left) (n = 4; **p < 0.005). Flow cytometric analysis shows IgM and B220 expression or CD25 and c-Kit expression in total bone marrow (BM) (right).

Fig 5: Representative FACS contour plots showing Live CD45+CD3+TCRß+ cells and the consequences of excessive concentration of Dispase used for gut digestion on the CD4 markerIdentical protocols were used to isolate intestinal immune cells from mouse #1 and #2 except for the concentration of Dispase as indicated. Lung and mesenteric lymph nodes were used as controls. Anti-CD45 BV786 (30-F11, BD Bioscience, CAT# 564225, 1/1000), anti-KLRG1 BUV395 (2F1, BD Bioscience, CAT# 740279, 1/200), anti-CD3 APC-eF780 (17A2, eBioscience, CAT# 47-0032-82, 1/200), anti-TCRß PerCP-Cy5.5 (H57-597, BioLegend, CAT# 109228, 1/200), and anti-CD4 APC (GK1.5, eBioscience, CAT# 17-0041-83, 1/200) antibodies together with the fixable viability dye eF506 (eBioscience, CAT# 65-0866-14, 1/800) were used.