Fig 2: Show accumulation of HBV viral Ag-specific iPSC-CTLs in the liver with HBV replicationEuthanize mice at day 8 after the adoptive transfer of T cells. Harvest the spleens or livers and isolate the intra-hepatic lymphocytes by enzymatic digestion.(A) Analyze accumulation of HBV-specific T cells by flow cytometry. Make single cell suspension from the intra-hepatic lymphocytes and stain cells with fluorochrome-conjugated antibodies. Gate on CD8+ T cells to analyze HBV-specific TCR.(B) Show immunohistochemistry. Stain liver slides with both fluorochrome-conjugated CD8 antibody and s183 TCR pentamer, and examine under a fluorescent microscope. Green color indicates CD8 and red color indicates HBV-specific TCR. Data are representative of five mice per group of three independent experiments.

Fig 3: In vivo antitumor effect of anti-PD-1 antibody. (A) Tumor volume and tumor weight in the MC38 tumor model. Anti-PD-1 mAb administration was 5 mg/kg, n = 4 mice/group. (B) Tumor volume and tumor weight of CT26 tumor model. Anti-PD-1 mAb administration was 5 mg/kg, n = 3 mice/group. (C, D) Flow cytometry analysis of tumor-infiltrating T cells for mice treated with PBS or anti-PD-1 antibody in MC38 tumor model (C) and in CT26 tumor model (D). (E, F) IHC staining of CD8a and Foxp3 in CT26 tumors. "*" means p-value < 0.05 and "**" means p-value < 0.01.

Fig 4: (A) IHC staining of Foxp3 in MC38 tumors treated with anti-PD-1 or anti-PCSK9 antibody and quantitative analysis of positive particles and (B) for CT26 tumors. (C) Flow cytometry analysis of CD4+ and Foxp3+ T-cell infiltration in MC38 tumors. (D, E) Tumor volume in the mice under the treatment of anti-PD-1 and anti-PCSK9 antibody with/without anti-CD8, anti-CD4, anti-NK1.1, and anti-CSF1R antibody. "*" means p-value < 0.05 and "**" means p-value < 0.01 while "ns" means not statistically significant.

Fig 5: EGR2 regulates the exhausted CD8+ T cell response to checkpoint blockade.WT or cKO mice were depleted of CD4+ T cells then infected with LCMV-Cl13, and from days 28-42 p.i. mice were treated with anti-PD-L1 or isotype control antibody every 3 days prior to analysis at day 42 p.i. a The sum of GP33–41 and GP276–286 specific CD8+IFN?+ T cell numbers (error bars show error of the summed numbers). For indicated significant differences, exact p values (left to right) = 0.0001, <0.0001, 0.0004. b %TIM3-TCF1+ cells within H2-DbGP33–41 tetramer-stained CD8+ T cells. For indicated significant differences, exact p values (left to right)= <0.0001, <0.0001, 0.0049, 0.0136. c, d Representative (c) and pooled (d) %TNFa+ and %TNFa+IL-2+ cells within the GP276–286 peptide-specific CD8+IFN?+ T cells. n = 7–9 mice per group from three independent experiments. For indicated significant differences, exact p values (left to right) for left graph = 0.0003, 0.0102, 0.0048, for right graph = 0.0347, 0.0385. All p-values in this figure were calculated using a one-way ANOVA with a Tukey’s post-test. Error bars depict SEM, *p < 0.05, **p < 0.01, ***p < 0.001.