Fig 2: Creatine uptake regulates CD8 T cell response to antigen stimulation. (A–N) CD8 T cells were purified from CrT-WT or CrT-KO mice and stimulated in vitro with plate-bound anti-CD3 (5 µg/ml; n = 3–4). The analyses of CrT mRNA expression (A), CrT protein expression (B), cell proliferation (C), cell survival (D), effector cytokine production (E–G and J–L), activation marker expression (H and I), and cytotoxic molecule production (M and N) are shown, either over a 4- to 5-d time course (A, C, D, E, and J) or 48 h after anti-CD3 stimulation (F–I and K–N). (O–S) CrT-KO CD8 T cells were stimulated in vitro with anti-CD3 and transduced with a MIG-CrT retrovector (O; n = 3). The analyses of retrovector transduction rate (P), CrT mRNA expression (Q), and IL-2 effector cytokine production (R and S) 96 h after stimulation are shown. Representative of two (O–S) and three (A–N) experiments, respectively. Data are presented as the mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test. See also Fig. S3.

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: 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+IFNγ+ 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) %TNFα+ and %TNFα+IL-2+ cells within the GP276–286 peptide-specific CD8+IFNγ+ 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.

Fig 5: How to generate HBV viral Ag-specific iPSC-CTLsTransduce mouse iPSCs with the following retroviral constructs: HBs183-91 TCR (MiDR-HBV s183 TCR) or OVA257–264 TCR (MiDR-OVA TCR), and co-culture the transduced iPSCs with OP9-DL1/DL4 stromal cells for T lineage differentiation.(A) Present the schematic representation of the retrovirus constructs expressing HBV s183 TCR. Ψ, packaging signal; 2A, picornavirus self-cleaving 2A sequence; LTR, long terminal repeats.(B) Visualize the HBV TCR-transduced iPSCs by a fluorescence microscope.(C) Transduce GFP+ iPSCs (left and middle, no transduction) with the retroviral construct MiDR or MiDR with HBV s183 TCR, and analyze and sort the GFP+ DsRed+ iPSCs by flow cytometry (right) and by a high-speed cell sorter.(D) Analyze HBV s183 TCR for Vβ28 gene expression by PCR.(E) Show morphology of T cell differentiation on various days.(F) Show flow cytometric analysis of the iPSC-derived cells on day 28, gating on CD3+CD8+ cells, and analyze for the expression of CD8 and TCRVβ28. Data shown are representative of three individual experiments.