Fig 2: Systemic buffering negated the effect of anti-PD1 antibody in a mouse hepatocellular carcinoma (HCC) model. A, Liver weight of mice with doxycycline (DOX)-repressible MYC–driven HCC fed with regular or sodium bicarbonate (NaHCO3)-containing drinking water and treated with anti-PD1 antibody (iPD1) or isotype control (IgG). Each dot represents an individual mouse liver weight in the different groups, all determined upon euthanasia at 12 weeks after birth (N = 10, 43, 43, 30, and 31 animals for H2O + DOX, H2O + IgG, NaHCO3 + IgG, H2O + iPD1, and NaHCO3 + iPD1 groups, respectively; unpaired t test). B, Representative microscopic images of the mTORC1 target pS6-stained tumor slices. Scale: 100 µm (inset: 40 µm). C, Quantification of pS6-positive objects in images in B (N = 5 whole-tumor scans from different animals, one-way ANOVA with multiple comparisons). *, P < 0.05; ***, P < 0.001.

Fig 3: Increased proportion of responders in response to anti-PD1 treatmentTg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 µg of [4Y] MBP. 24 h later mice were randomized to receive 0.5 mg isotype or 0.5 mg anti-PD1 30 min prior to re-challenge with 8 µg [4Y] MBP peptide. Cells are gated on live CD4+Vß8.1/8.2+ T cells and analyzed for expression of Nr4a3-Blue vs. Nr4a3-Red 4 h after peptide rechallenge. Gates are set on the responder T cell population.

Fig 4: Strong TCR signaling signatures in tumors of anti-PD-L1-treated mice(A) 0.25 M MC38 cells were injected s.c. into Nr4a3-Tocky Ifng-YFP mice. CD4+ and CD8+ TILs were analyzed for Nr4a3-Blue versus Red (top), PD1 versus Lag3 (middle), or Nr4a3-Blue versus Ifng-YFP (bottom) expression.(B–D) Summary data of percentage of TIL for (B) Nr4a3-Blue+ (blue) or Nr4a3-Red+Blue− (red), (C) PD1+ (black) or Lag3+ (white), and (D) Ifng-YFP+ (black) or percentage of Ifng+Nr4a3-Blue+ (white), n = 3. Circles represent mean ± SEM. Statistical analysis by two-way Anova with Sidak’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.(E) Heatmap of log2 transformed and normalized counts for genes significantly upregulated (>1.5-fold and adjusted p value < 0.05) in C57BL/6 mice injected with 0.5 M MC38 cells then treated with isotype or anti-PD-L1 every 3 to 4 days before whole tumors were excised and 3′ mRNA-seq was performed (GEO: GSE93018) (Efremova et al., 2018).(F) Z score heatmap analysis of log2 transformed and normalized counts for genes pre-selected from Figure S5A and also expressed in GEO: GSE93018 (Efremova et al., 2018).See also Figure S6.

Fig 5: Strong TCR signaling drives high amounts of immune checkpoint expression(A) Heatmap comparing key inhibitory receptors and their relationships to Nr4a expression from Figure 2G.(B) PD1 expression on live CD4+Nr4a3-Timer+ T cells 12 h following immunization, n = 3. Bars represent mean ± SEM. Statistical analysis by one-way Anova with Tukey’s multiple comparisons test. **p < 0.01, ***p < 0.001; ns, not significant.(C and D) Lag3 (C, n = 6) or Tigit (D, n = 6) expression on live CD4+Nr4a3-Timer+ T cells 24 h after immunization. Statistical analysis by one-way Anova with Tukey’s multiple comparisons test. Bars represent mean ± SEM. **p < 0.01, ****p < 0.0001; ns, not significant.(E) CD4 versus intracellular CTLA-4 expression in live CD4+ T cells 24 h after immunization with the stated doses.(F) Lag3 and Tigit expression on live CD4+Il10-GFPhi (green) or Il10-GFPlo (gray) Nr4a3-Timer+ T cells 24 h after immunization with 80 µg [4Y] MBP.(G) Summary data of (F), n = 3. Statistical analysis by unpaired t test. Bars represent mean ± SEM. **p < 0.01, ***p < 0.001