Fig 2: (A–C) Decreased frequencies and numbers of T-bet+CD11chi B cells in Cd38−/− cGVHD mice. (D–F) Decreased anti-ssDNA autoantibody serum levels in Cd38−/− cGVHD mice. Correlation with the frequencies of T-bet+CD11chi B cells. (A) Representative flow cytometry plot showing T-bet+CD11chi cells and T-bet−/loCD11c− on gated CD19+ B cells from spleen. (B) Percentages of CD11chiT-bet+ cells (of CD19+ B cells). Mean ± SEM. P-values are shown for Welch’s t-test. (C) Total number of CD11chiT-bet+ cells. Mean ± SEM. P-values are shown for Welch’s t-test. (D) Anti-ssDNA serum levels. Median values. P-values are shown for Mann–Whitney test. (E) Total IgG serum levels. Median values. P-values are shown for Mann–Whitney test. (F) Positive correlation between anti-ssDNA serum levels from cGVHD mice and the frequencies of CD11chiT-bet+ B cells (simple linear regression). The anti-ssDNA levels and frequencies are shown in log scale. The results are cumulative data from two to four different experiments per time point and mouse type, each with three to five mice per experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, *****P < 0.00001.

Fig 3: Expression of T-cell activation/exhaustion markers on the CD8+ TIL. a Expression of Nrp-1, PD-1, LAG-3, CTLA-4 and Tim-3 on CD8+ T cells from B16F10 TIL isolated at day 15. Right: percentages of Nrp-1 among CD8+ T cells expressing or not PD-1 (n = 26), Tim-3 (n = 19), LAG-3 (n = 19) or CTLA-4 (n = 16). b Expression of Nrp-1 on CD8+ T cells correlated with that of PD-1 (n = 38). c Expression of Nrp-1 on CD8+ TIL is restricted to a PD-1hi T-cell population. Left, Expression of Nrp-1 and PD-1 on CD8+ TIL; identification of three T-cell subsets: Nrp-1+PD-1hi, Nrp-1−PD-1+and Nrp-1−PD-1−. Right: expression of exhaustion/activation markers, functional proteins and transcription factors in Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− CD8+ T-cell subsets. Heat maps including percentages of cells positive for expression of PD-1, Nrp-1, Tim-3, CTLA-4, LAG-3, granzyme B (GrzB) and Ki-67, and gMFI for NFATc1, IRF-4, Helios, Blimp-1 and T-bet (n = 5). d gMFI of PD-1 on Nrp-1+PD-1hi, Nrp-1−PD-1+ and Nrp-1−PD-1− TIL subsets (n = 16). Results are representative of three to five independent experiments. Means ± SEM two-tailed Student’s unpaired t test a or one-way ANOVA test with Bonferroni correction d. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file c

Fig 4: IL-6 secreted by cancer cells affects moDC-induced Th1 differentiation.a MoDCs were differentiated and stimulated (LPS, 100 ng/ml, 48 h) in the presence or absence of 20% of NCI-H226 supernatant (SN). Stimulation of moDCs was additionally carried out in the presence or absence of tocilizumab (5 µg/ml, anti-IL-6R mAb). IL-12p70 secretion was analyzed by CBA assay. Shown are 2 independent experiments. b Same as in a, but moDCs were differentiated and stimulated (LPS, 100 ng/ml, 48 h) in presence of IL-6 depleted- or control- supernatant (SN) of NCI-H226. The relative IL12-p70 secretion is presented (mean fold change ± SD; n = 6). c A MLR was performed with allogeneic CD4+ T cells and moDCs differentiated and stimulated (LPS, 100 ng/ml, 48 h) in the presence or absence of IL-6 (20 ng/ml) or of 20% of NCI-H226 supernatant (SN). T cell proliferation was determined via 3H-thymidine incorporation. Shown is a representative experiment. Error bars show the standard deviation of three technical replicates. d The induced T cell proliferation of IL-6-treated moDCs (20 ng/ml) (mean fold change ± SD; n = 4) and of e, moDCs treated with 20% of NCI-H226 supernatant (SN) was determined relative to the corresponding stimulated control (mean fold change ± SD; n = 4). f Naïve CD4+ T cells were expanded by stimulation with allogeneic moDCs, that have been differentiated and stimulated (LPS, 100 ng/ml, 48 h) in the presence or absence of IL-6 (20 ng/ml) or of 20% of NCI-H226 supernatant (SN). After restimulation of the CD4+ T cells with accordingly treated moDCs CD4+ T cells were stained for their T-bet expression. Shown is the percentage of the T-bet+ population (mean ± SD; n = 3) and the relative mean fluorescence of T-bet (mean fold change ± SD; n = 3).