Fig 2: The TNF‐α/TNFR2 signaling pathway promotes the proliferation and enhances the immunosuppressive phenotype and function of Tregs. (A) The proportions of Foxp3+ Tregs in CD4+ T cells and the expression levels of TNFR2, CTLA‐4 and ICOS in control Tregs and activated Tregs were compared by flow cytometry analysis. Three biological duplicates of independent experiments were carried out. (B) Tregs were stimulated by either TNF‐α or TNF‐α plus Mab726 for 72 hours in the absence of anti‐CD3/CD28 beads. The proportion of Foxp3+ Tregs in CD4+CD25+ T cells was determined by flow cytometry. Representatives of three independent experiments with biological duplicates are shown. (C) Tregs were stimulated with either TNF‐α or TNF‐α plus Mab726 for 72 hours in the absence of anti‐CD3/CD28 beads. The level of LAP expression in activated Tregs was determined by flow cytometry. (D) Proliferation suppression assay. CFSE‐labeled CD8+ T cells were cultured alone or coincubated with activated Tregs and stimulated with either TNF‐α or TNF‐α plus Mab726 for 72 hours to quantify the proliferation percentage by flow cytometry. (E) Purified CD8+ T cells were cultured alone or coincubated with activated Tregs and stimulated by either TNF‐α or TNF‐α plus Mab726 for 72 hours. The IFN‐γ production in CD8+ T cells was detected by ELISA. Statistical analyses were performed using GraphPad Prism 8.0.1. The statistical tests used are indicated in the figure legends: *P < .05; **P < .01; ***P < .001; ****P < .0001 [Color figure can be viewed at wileyonlinelibrary.com]

Fig 3: PPP improves autophagy-dependent anticancer chemotherapy in a T lymphocyte-dependent manner. (A, B) Human osteosarcoma cells were treated with PPP (10 μM) in the presence of a low dose of the ICD inducers mitoxantrone (MTX, 1 μM) for 6 hours. High-dose MTX (5 μM) was used as positive controls. Quinacrine staining was used to monitor intracellular ATP content. Representative images are shown in A and quinacrine dots were quantified in B. Scale bar equals 10 μm. Data are means±SD of three replicates (***p<0.001 vs untreated control; ###P<0.001 vs PPP; Tukey’s multiple comparisons test). (C–F) Human neuroglioma H4 cells wild-type or Atg5 knockout, human osteosarcoma U2OS wild type, PERK knockout or PEIF2α S51A knockin cells, murine fibrosarcoma MCA205 wild-type or AKTT308D/S473D knockin cells were treated with PPP (10 μM) alone or in combination with low doses of the ICD inducers MTX (1 μM) for 6 H as described above. High-dose MTX (5 μM) was used as positive controls. the extracellular ATP levels were measured in C–F. Data are means ± SD of three replicates (**P<0.01, ***P<0.001 vs untreated control; #P<0.05, ##P<0.01, ###P<0.001 vs WT; Tukey’s multiple comparisons test). (G–O) In vivo treatment of implanted murine MCA205 fibrosarcoma with oxaliplatin (OXA), anti-PD-1 antibody and PPP, alone or in combination (schematic view in G). (H, I) Growth kinetic of murine fibrosarcoma MCA205 cells evolving in immunocompetent C57BL/6 mice or athymic nu/nu mice (J) or MCA205 fibrosarcoma expressing constitutive active AKTT308D/S473D (M), Atg5 deficient MCA205 ATG5-/- (N) or MCA205 expressing the ectoATPase CD39 (O) evolving in immunocompetent C57BL/6 mice were treated as indicated in (G). When tumors became palpable, mice received systemic intraperitoneal (i.p.) injections of ppp alone or together with OXA or PD-1 blocker. Data are depicted as growth curves (mean±SD) (H, L–O) and tumor size distributions at day 24 (I). Individual tumor growth curves of mice treated with OXA and PPP, combined or not with PD-1 blockade are shown (K). The generation of immunological memory was assessed in cured animals by rechallenge with MCA205 and TC-1. Individual tumor growth curves are depicted (L). Data were analyzed with TumGrowth. n≥6 for mice in each group. (*P< 0.05 or ns, not statistically significant vs OXA; #P<0.05 or ns, not statistically significant vs OXA+PD-1 blockade, Student’s t-test, survival plots in online supplemental figure S7). (P) Schematic overview of the treatment of implanted murine MCA205 fibrosarcoma with OXA and PPP, alone or in combination. (P–T) Cytofluorometric analysis of tumor-infiltrating lymphocytes (TIL): CD3+CD8+ cytotoxic T lymphocytes (Q), CD8+PD-1+ T lymphocytes (R), CD4+FOXP3+CD25+ regulatory T cells (Treg) (S), and the ratio of CD3+CD8+ T cells over Treg (T). Data are means±SD (n≥5) (*p<0.05 or ns, not statistically significant vs control; #P<0.05 or ns, not statistically significant vs OXA, Student’s t-test). PPP, picropodophyllin.

Fig 4: Regulatory T cells (Tregs) represents the majority of CD4+ tumor infiltrating Puro+ cells.(A) Schematic diagram for the experiment. (B) Representative plots (left) and statistical analysis (mean ± SEM) of gated CD44+ CD4+ tumor-infiltrating lymphocytes (TILs) analyzed for puromycin incorporation. Quantitation shows an enrichment for the expression of CD44. Percentages of positive cells in each gate are shown. Data from three experiments pooled together (n = 6 mice per experiment) **p < 0.01. (C) Statistical analysis (mean ± SEM) for the indicated markers within Puro+ and Puro- CD4+ TILs shows that Puro+ translating CD4+ TILs are enriched for CTLA4, CD39, CD27, ICOS, CCR5, and CD103 expression. Data from two experiments pooled together (n = 5 mice per experiment) *p < 0.05; **p < 0.01; ****p < 0.0001. (D) Representative plots (left) and statistical analysis (mean ± SEM) of gated CD25+ Foxp3+ CD4+ lymphocytes show that the number of Puro+ Tregs is higher in tumors versus spleen. Percentages of positive cells in each gate are shown. Data from two experiments pooled together (n = 4–5 mice per experiment) ***p < 0.001. (E) Immunohistochemical analysis of puromycin in Foxp3+ TILs shows that clusters of highly translating Foxp3+ cells are found. Scale bar, 20 μm. (F) Representative plots (left) and statistical analysis (mean ± SEM) for pS6(S235/236) within Puro+ and Puro- Tregs showing that, in vivo, most of translating Tregs have the mTORC1-S6K pathway active. Percentages of positive cells in each gate are shown. Data from one experiment representative of two (n = 7) ***p < 0.001. Figure 5—source data 1.Source data for Figure 5B, C, D, and F.

Fig 5: The most prominent CD4+ T cell subsets in SS tissues are Th1 cells and Tfh cells. (a) Representative multi-color immunofluorescence image of CD3 (red), CD19 (green) and DAPI (blue) staining in a pSS lesion (patient number 2). (b) Absolute numbers of CD3+ T cells, CD19+ B cells in pSS (n = 10). Paired t test used to calculate p-value. (c) Representative multi-color staining showing each CD4+ T cell subset. [Th1: CD4+ (red) T-bet+ (light blue)] [Th2: CD4+ (red) GATA3+ (yellow)] [Th17: CD4+ (red) RORγ+ (white)] [Tfh: CD4+ (red) CXCR5+ (green)] [Treg: CD4+ (red) Foxp3+ (orange)] [CD4+ CTLs: CD4+ (red) GZMA+ (green)] (d) Relative proportions of Th1, Th2, Th17, Tfh, Treg and CD4+ CTL subsets in tissues from pSS (n = 13), secondary SS (n = 7) and IgG4-RD patients (n = 6). Error bars represent mean ± SEM. *p < 0.05.