Fig 2: Tumors induce Cyp11a1 expression in T cells in vivo.a B16-F10 cells were injected subcutaneously into the Cyp11a1-reporter mice. After 12 days brachial lymph node (LN), blood, and tumor tissues were analyzed by flow cytometry. Gating strategy: Singlets>Live cells>CD45,Cyp11a1-mCherry. N = 5. CD45+Cyp11a1-mCherry+ cells were further gated to show T helper cell (CD4+CD3e+) expression of Cyp11a1. b Splenic cells were purified from tumor-bearing Cyp11a1-reporter mice, and restimulated in vitro using PMA/ionomycin and analyzed by flow cytometry. N = 9. c, d B16-F10 cells were injected subcutaneously into the Cyp11a1-mCherry reporter mice. After 5, 7, and 12 days tumor tissues were analyzed by flow cytometry to detect the CD4+ T cells (c), CD8+ T cells (d) N = 4. All cell>Singlets>Live cell>CD45+>CD4+CD3e+ or CD8+CD3e+. e–g EO771 cells were injected into the mammary fat pad of Cyp11a1-reporter mice. After 15 days, tumor tissues and tumor-draining LN were analyzed by flow cytometry to detect the Cyp11a1-mCherry expression in CD4+ T cells (e, f) CD8+ T cells (g). Gating: All cell>Singlets>Live cell>CD45+>CD4+TCRb+ or CD8+TCRb+. e Representative FACS profile of Cyp11a1+CD4+ T cells. f, g Representative graphical presentation of one experiment showing Cyp11a1-expressing CD4 and CD8 T cells. N = 4. h B16-F10 tumor-infiltrating leukocytes (TIL) and splenocytes were purified from tumor-bearing mice on post-inoculation day-12, cultured for 48 h, and the supernatant was analyzed by ELISA to measure pregnenolone. N = 12, pooled analysis of three independent experiments. i Metastasized lungs were harvested 10 days post-B16-F10 intravenous injection in C57BL/6 mice, dissociated into single-cell suspension, cultured for 48 h, and the supernatant was analyzed by ELISA. Naïve uninfected lungs (normal lung) were used as control. N = 6, pooled analysis of two independent experiments. j Hierarchical clustering of steroidogenic genes and IL4 mRNA expression across 44 melanoma patient samples (GEO: GSE19234). k Schematics showing glucocorticoid synthesis pathway. l Frequency distribution histogram showing CYP11A1 mRNA expression (normalized read counts, log10 scale) across 22 melanoma patients’ tumor-infiltrating CD4+ T cells (EGAD00001000325). Individual data points for CYP11A1 expression are shown in Supplementary Fig. 3i. All error bars in this figure represent mean with s.d. P-values were calculated by unpaired two-tailed t-test. N represents biologically independent animals.

Fig 3: Caspase-8 deficiency dramatically attenuates Th2 immune response in the allergic asthma model. (a) T cells from OVA–treated WT, Rip3-/-, and Rip3-/- Casp8-/- mice were analyzed for IL-4, IFN-?, and IL-17 expression by intracellular cytokine staining. (b) Percentage of T cells producing IL-4, IFN-?, and IL-17 relative to total CD4+ or CD8+ T cells in (a). (c) The total number of T cells producing IL-4, IFN-?, and IL-17 in (a). (d) RT-PCR analysis of Il4, Il5, Il13, and Ifng in OVA–treated WT, Rip3-/-, and Rip3-/-Casp8-/- mice. (e) ELISA analysis of IL-4, IL-5, IL-13 and IFN-? in OVA–treated WT, Rip3-/-, and Rip3-/-Casp8-/- mice. Each symbol indicates an individual mouse and mean ± SEM values are shown. Results are representative of 3 independent experiments. **P < 0.01; ***P < 0.001; **** P < 0.0001; ns, not significant.

Fig 4: T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFN? neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-µM 4µ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4µ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4µ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results (n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

Fig 5: The Early IL-4 Wave Is Localized at the Periphery of B Cell Follicles(A–C) Confocal microscopy analysis of (A and B) wild-type and CD1d-/- and (C) IL4-GFP mice on day 3 of influenza infection. Lymph nodes were labeled with CD1d tetramer (magenta) and anti-B220 antibody (green in A and B and white in C). The arrows in (C) indicate CD1d tetramer+ cells expressing IL-4 (IL-4 GFP, green). Scale bars, 300 µm (lymph node) and 60 µm (section).(D) Flow cytometry analysis of IL-4 GFP+ cells in mediastinal lymph nodes on day 3 of influenza infection, showing CD1d-tet- and CD1d-tet+ cells.(E) t-SNE plots of CD1d-tet- and CD1d-tet+ subsets. The CD1d-tet+ population separates into two clusters (1, dark purple; 2, light purple), as does the CD1d-tet- population (3, light green; 4, dark green).(F and G) Expression distribution (violin plots) in each population (horizontal axes) for (F) CD3e, Zbtb16, CXCR6, and CXCR3 and (G) Gata3, Tbx21, and Rorc. Expression levels are log2(tpm+1).(H) Flow cytometry analysis of CXCR3 levels in IL-4-GFP- (red) and GFP+ (blue) NKT cells on day 3 of influenza infection. Lines connect cells from the same mouse.(I) Flow cytometry analysis of IL-4 production by CXCR3- (red) and CXCR3+ (blue) NKT cells on day 3 of influenza infection. Lines connect cells from the same mouse.(J) ELISPOT analysis of IL-4-producing CXCR3- (red) and CXCR3+ (blue) NKT cells sorted on day 3 of influenza infection. Each dot represents pooled NKT cells from 6 mice.In graphs showing statistical analysis, data are representative of at least two independent experiments. Horizontal bars, mean; error bars, SEM. Student’s t test, *p < 0.05, **p < 0.01.