Extended Data Fig. 9: In vivo B cell derived IL-12 influences both B and T cells, and synergizes with IFNγ signals for maximal PB differentiation.
a, Representative FACS plots of B cells from mice treated with PBS or recombinant IL-12 as in Fig. 2b, and b, summary data of B cells expressing T-bet and producing IFNγ after IL-12 treatment. Data combined from two experiments, n = 10 per group, analyzed by two-tailed t-test. c-i, Additional data related to experiment described in Fig. 8c. Data combined from two experiments, n = 11 (WT/WT), n = 9 (WT/KO), n = 11 (KO/WT), n = 10 (KO/KO). For e and i, all symbols represent individual mice and bars the mean +/- SD. P values calculated by two-tailed t-tests.c, Gating scheme used to identify donor derived B cells and PB for Fig. 8d. Example shown is from the group IL12RKO T cells + WT B cells. d, Example FACS plots of donor derived B cells (TCRb-, CD19+ or CD138+ from either CD45.1 WT or CD45.2 KO gate), donor genotype is indicated above each chart. e, Additional summary from Fig. 8d showing the number of CD138+ donor B cells per spleen, and the fold change of IRF4 MFI on PB compared to ‘naïve’ (CD138− CD19+) B cells of the same mouse. f, Gating scheme used to analyze CD4 T cells from donor cells, example mouse received WT T cells and WT B cells. g,h, Representative FACS plots of donor derived T cells showing expression of T-bet+ IFNγ + Th1 cells in g, and CXCR5high Bcl6high TFH in h and i, Example FACS plots (h), and summary data (i), of the percentage of TFH among donor T cells per mouse. j, Additional analysis of mixed bone marrow chimera mice described in Fig. 8h. Fold advantage was calculated for GC B cells compared to resting cells from the same mouse as described in Extended Data Fig. 2. Data shown are combined from 2 experiments, (HK: n = 12 for IL-12R, n = 13 for IFNgR and IL-12R+IFNgR, Inf: n = 15 IL-12R, n = 16 IFNgR, n = 14 IL-12R+IFNgR), symbols represent individual mice and bars the mean +/− SD. Individual groups are tested against the hypothesis of no competitive difference by one sample t-test (two-tailed) compared to a hypothetical value of 1, and compared to each other by two-tailed t-tests. k, Visual representation of the proposed model. Early (4 hours) IL-12 initiates IFNγ production and autocrine IFNγ signals induce expression of genes that both establish negative feedback of IFNγ signaling, and an overlapping positive feedback loop between IL-12 and IFNγ signaling. IFNγ promotes IL-12 signaling by upregulating IL-12 signaling components (IL-12R and STAT4) and STAT1 which IFNγ signals through. At 24 hours, if both IL-12 and IFNγ are present, positive feedback overcomes negative regulation to sustain IFNγ signaling. Additionally, IL-12 and IFNγ synergize to induce genes which are not expressed under either condition alone. For all charts (b, e, i, j), NS = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
