Fig 2: CAR design and expression. (a) Schematic representations of the CAR constructs; parental STEAP1 CAR (JK11), control CD19 CAR, dominant negative mutant -STEAP1 CAR (Dnm) with the TGFß receptor (TGFßR) lacking the intracellular signaling domain, TGFß-switch receptor (SwR)-STEAP1 CAR (JK59) with the extracellular domain (ECD) of the TGFßRI and TGFßRII subunits fused to the transmembrane domain (TM) and intracellular domain (ICD) of the IL-2/IL-15 receptor ß and ? chains, respectively (IL-2Rß and IL-2R?). (b) Flow analysis of CAR expression in primary human T cells stained with CD3, CD4, and CD8 (left panel), and with the antibody recognizing the RQR8 sequence of JK11 and CD19 control CAR (middle panel) or the TGFßRII antibody recognizing Dnm and JK59 CARs (right panel). T cells were stained five days after viral transduction. (c) Graphs representing the expression of JK11 and CD19 CARs (right panel) and Dnm and JK59 CARs (left panel). Data represent the mean values ± SEM of 4 different T cell transductions from 4 different healthy donors (each transduction was performed in duplicate). (d) Flow analysis of the CAR expression in human T cells after CAR T cells freezing/thawing and two days activation with anti-CD3/CD28 antibodies. Contour plots of JK11 and CD19 CAR (left panel) and of Dnm and JK59 CAR expression (right panel). (e) Graphs representing the expression of JK11 and CD19 CARs (left panel) and Dnm and JK59 CARs (right panel) after freeze-thawing and activation for two days, from three to five healthy donors. Data represent the mean values ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. *** p < 0.001; **** p < 0.0001.

Fig 3: CD161 expression in CTLs indicates poor therapeutic responses and patient outcomes. Paraffin tumor sections of 535 patients in validation cohort 2 were analyzed for CD161+ CTL infiltration and its relation to tumor cell apoptosis and clinical outcomes. A, Representative immunofluorescent staining of CD3, CD8, CD161, CK, and TUNEL in breast cancer specimens. Arrows indicate CD161+ CTLs. Scale bar, 50 µm. B, The proportions of CD161+ CTLs in patients with different responses to chemotherapy (mean ± s.e.m.; CR, n = 88; PR, n = 237; SD, n = 180; PD, n = 30). C, Correlation between CD161 expression in CTLs and tumor cell apoptosis. D, Receiver operator characteristic (ROC) curves to predict neoadjuvant chemotherapeutic responses from intratumoral CD161+ CTL enrichment. AUC, area under the curve. E, Kaplan–Meier survival curves for disease-free survival of breast cancer patients with low or high CD161+ proportions of CD8+ T cells. HR, hazard ratio. F, Schematic diagram of our main findings. n = 535 (A–D) or 529 (E) patients. P values were calculated by two-sided one-way ANOVA with Tukey test (B), two-tailed Pearson correlation coefficient test (C) or two-sided log-rank test (E).

Fig 4: ZEB1 and JAK2 are potential targets for the treatment of MS(A) Dot plot showing linear correlation of ZEB1 gene expression with JAK2 gene expression in myelin-reactive Th17 cells from MS patients (Th17 MS) and healthy controls (Th17 Ctrl).(B) Contour plots showing sorting strategy for CD3+CD4+CD45RO+CCR6-CXCR3+ (Th1), CD3+CD4+CD45RO+CCR6+CXCR3+ (Th1/Th17), and CD3+CD4+CD45RO+CCR6+CXCR3- (Th17) cells from PBMC of MS patients (Silveira-Mattos et al., 2019).(C–H) Flow cytometry evaluating IFN-? and IL-17A expression in CCR6-CXCR3+ (Th1), CCR6+CXCR3+ (Th1/Th17), and CCR6+CXCR3- (Th17) transfected with siCTL, siJAK2, or siCTL. n = 3 different MS patients.(I) Schematic displaying the experimental design for treating EAE using fedratinib.(J) Clinical scores of C57BL/6 mice treated with vehicle or fedratinib (90 mg/kg) in EAE (n = 15 mice per group). Statistic difference is tested by nonparametric Mann-Whitney test.(K) Flow cytometry evaluating the number of CD4+ cells, CD4+IFN-?+ cells, CD4+IL-17A+ cells, CD4+GM-CSF+cells, CD4+IL4+ cells, CD4+CD25+FOXP3+ cells, CD8+ cells, and CD8+IFN-?+ cells in the CNS of mice treated with vehicle or fedratinib at 29 days post immunization.All statistical differences in (D), (F), (H), and (K) were tested using paired Student’s t test (two-tailed). NS, not significant; *p < 0.05, **p < 0.01.

Fig 5: The CD161+CD8+ T-cell subset is enriched in chemoresistant tumors. CD161 expression was examined in tumor-infiltrating CD8+ T cells of tumor suspensions or paraffin sections from pretreatment biopsies of breast cancer patients. A, Representative gating strategy for flow cytometry of CD161+ CTLs. Va7.2 was used to exclude MAIT cells and Va24 to exclude NKT cells. B, Representative flow cytometry of CD161 expression gated as A in paired tumor samples and peripheral blood (n = 24 different patients). See stratified quantification in Supplementary Fig. S2E. C and D, Representative immunofluorescence images (C) and quantification (D) for CD3, CD8, CD161, Va7.2, and Va24 in serial sections of breast cancer biopsies (n = 28 different patients). Arrows indicate CD161+ CTLs. Scale bar, 50 µm. E and F, Quantitative RT-PCR (E) and representative western blot (F) for CD161 expression in FACS-isolated conventional CD8+ T cells from tumor suspensions (n = 8 different patients for E and 3 for F). See quantification of F in Supplementary Fig. S2G. Results are mean ± SEM. ***, P < 0.001 by Student t test (B–E).