Fig 2: TNF signaling promotes Tfh-B cell interactions(A and B) PBMC from young adults were freshly isolated and sorted for coculture of T cell subsets (naive, gated as CD4+CD45RA+CD27+; CXCR5- memory, non-naive CD4+CXCR5-; cTfh, non-naive CD4+CXCR5+PD-1+) with autologous naive B cells (CD3-CD19+CD27loIgD+). Supernatant IgM (A) and IgG1 (B) were measured after 7 days, as shown for the following conditions: unstimulated (gray), SEB alone (0.5 µg/mL, green), SEB with recombinant human TNF (125 ng/mL, tan), or SEB with a-TNF antibodies (2 µg/mL, sienna) (1-way repeated-measures ANOVA with Holm-Sidak’s test; n = 12 per group).(C and D) Correlation shown for the TNFRSF1A in ICOS+CD38+ cTfh at day 7 compared with the H1N1-specific (C) hemagglutinin inhibitory (HAI) titer (n = 14) or H3N2-specific (D) HAI titer (n = 14) in young (orange) and elderly (purple) adults.(E) Correlation of apoptosis and NF-?B gene set GSVA scores for ICOS+CD38+ cTfh at day 7 for young (orange, n = 6) and elderly (purple, n = 8) adults.(F) Pearson coefficients for BCL family member genes compared to GSVA scores for TNF-NF-?B signaling in ICOS+CD38+ cTfh at day 7 for young (x axis) and elderly (y axis) adults. Genes classified as either pro-survival (dark blue) or apoptotic (sea green).

Fig 3: Generation and basal characterization of CD4-P2Y10-KOs.a Expression of selected GPCRs for chemokines or lipids and metabolites in naïve and spinal cord-infiltrating CD4 T cells was determined by Nanostring mRNA quantification (naïve CD4: splenic CD4 T cells from two healthy mice; sc-inf. CD4: spinal cord-infiltrating CD4 T cells pooled from four individual mice at EAE day 14). b Design of the floxed P2Y10 allele. c, d Reduction of P2Y10 expression in CD4 T cells from control and CD4-P2Y10-KO mice was judged by immunoblotting (c, GAPDH as loading control) and RNA sequencing in isolated splenic CD4 T cells (d, n = 3; **p = 0.0021). Total leukocyte numbers (e) and proportion of lymphocyte populations in thymus (f), inguinal lymph nodes (g), or spleen (h) (n = 6–12). Cytokine production (i, n = 5–6) and proliferation (j, n = 7) in CD3/CD28-activated CD4 T cells. Percentage of IFN?/ IL-17A/FoxP3-expressing cells (k, n = 7–9) and proliferating cells (l, n = 4–5) after Th1 and Th17 differentiation, respectively. Data are means ± SEM (in (a) only means); comparisons between genotypes were performed using two-tailed unpaired Student’s t-test. n, number of mice per group.

Fig 4: Description of biomarker levels and neoantigen identification in a single human patient (ID 9567). (a) Longitudinal serum mesothelin levels. Indicated by triangles are the time points for first-line chemotherapy (red -cisplatin and pemetrexed); second line (blue – carboplatin and pemetrexed) and third line (green – vinorelbine). (b) PBMC stimulated for 1 h with mutant peptide pool, then expanded in the presence of IL2 and CD3 for 14 days. T cell reactivity was assessed by ELISpot using the indicated mutant peptide exposed HLA expressing single antigen (SAL) lines. Controls show results for indicated HLA expressing SAL without peptides. Controls for HLA expressing SAL with wild-type peptides were performed for each mutant peptide of interest (Additional File 7 and data not shown). Difference between groups determined using non-parametric tests Kruskal-Wallis with Dunn’s multiple correction or Mann-Whitney as appropriate. *p < .05. SFU, spot forming units. (c) PBMC (200,000 cells per well) stimulated for 1 h with mutant ROBO3 peptide, then expanded in the presence of IL2 and CD3 for 14 days. T-cell reactivity was assessed by ELISpot assay. Negative control was T cells and B49:01SAL without peptide. Positive control was T cells plus aCD3 antibody. (d) CD137 cell surface expression measured using flow cytometry after stimulation of T cells with ROBO3 mutant peptide Wild-type controls are included in additional file 6.

Fig 5: Reduced migration and polarization in P2Y10-deficient CD4 T cells.a Transwell migration of naïve CD4 T cells (nCD4) under basal conditions and after addition of 100 ng/ml of the indicated chemokines to the lower well (6–12 mice per group; *p = 0.044 (CCL21), 0.048 (SDF-1a), 0.013 (CCL5); **p = 0.007 (CCL4); ***p < 0.001 (CCL19); ***p < 0.001 (CXCL10). b Live-cell imaging analysis of migratory behaviour in CCL19-stimulated naïve CD4 T cells (homogenous CCL19 application): Quantification of immobile cells (cells from three mice per group were analyzed in five experiments, and per experiment 64–200 cells were evaluated; **p = 0.007). Live-cell imaging analysis of migratory behaviour in CCL19-stimulated nCD4 T cells (CCL19 gradient): Exemplary tracking result (c) and statistical evaluation of accumulated distance (d; *p = 0.037), velocity (e; *p = 0.021), and directness (f) (3–5 mice per group were analyzed in 4–8 tracking experiments, and per mouse 937–7969 cells were evaluated). Expression of chemokine receptors (g) or other genes related to T cell migration (h) in nCD4 (3 mice per group). Transwell migration of CD4 cells that had been unspecifically activated by CD3/CD28 stimulation (aCD4) (i, 7–8 experiments with cells from 7 to 8 mice per group; *p = 0.019 (bas), 0.038 (CCL19), 0.011 (SDF-1a) or differentiated towards Th1 (j, 3–7 experiments with cells from 3 to 4 mice per group; *p = 0.027; **p = 0.005) or Th17 (k, 7 experiments with cells from four mice per group). l Percentage of P2ry10-expressing cells among Th1- and Th17-differentiated lymph node CD4 T cells (reanalysis of single-cell expression data from ref. 10). m T ranswell migration of naïve human CD4 T cells isolated from healthy donors (left) or MS patients (right) after treatment with scramble siRNA or siRNA directed against human P2Y10. Migration was assessed under basal conditions and after addition of 100 ng/ml SDF-1a to the lower well (three patients per group; *p = 0.025 (healthy), 0.042 (MS). Data are means ± SEM; comparisons between genotypes were performed using two-tailed unpaired Student’s t-test. ns not significant.