Fig 2: 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 3: Co-inhibitory receptors exert distinct quantitative and qualitative control over T cell re-activation(A) Experimental design for blockade of co-inhibitory receptors.(B) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later mice were randomized to receive either 0.5 mg isotype pool (1:1 ratio of rat IgG1 and rat IgG2a), anti-Lag3, or anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells were analyzed for Nr4a3-Blue versus Nr4a3-Red analysis 4 h after peptide re-challenge.(C and D) Summary data from (B) detailing the percentage of responders (percent of Nr4a3-Blue+Red+, C) or median Nr4a3-Blue within Nr4a3-Blue+Red+ CD4+ T cells (D) in isotype (n = 5), anti-Lag3 (n = 6), or anti-PD1 (n = 6) treated mice. Bars represent mean ± SEM, dots represent individual mice. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test.(E) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later, mice were randomized to receive 0.8 mg isotype pool, 0.8 mg anti-Lag3, or 0.8 mg anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells expression of Nr4a3-Blue versus Nr4a3-Red 4 h after peptide re-challenge in pre-sorted (top) and sorted (bottom) populations.(F) RNA was extracted from the sorted populations and 3′ mRNA-seq performed. PCA of the normalized expression data identified 3 clusters, n = 2.(G) KEGG pathway analysis of DEG between isotype and anti-PD1 treated groups.(H) Z score heatmap analysis of log2 transformed and normalized counts displaying the 69 DEG between isotype and anti-PD1 groups, in isotype, anti-PD1, or anti-Lag3 groups.(I) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later mice received 0.5 mg rat IgG2a or or anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells were analyzed for Nr4a3-Blue versus Nr4a3-Red analysis 4 h after peptide re-challenge. Gates were set on responding T cells.(J) Summary data from (I) for cells responding to dose 2 (n = 4). Bars represent mean ± SEM. ∗p < 0.05 by unpaired t test.(K) Histograms showing expression of Nr4a3, OX40, GITR and ICOS in responding T cells (indicated by gates in I) between isotype (gray)- or anti-PD1 (blue)-treated cells.(L) Summary data of the median of expression of the stated markers in responding T cells, n = 4. Bars represent mean ± SEM. Statistical analysis by unpaired t test. ∗∗p < 0.01, ∗∗∗p < 0.001.(M) Analysis of intracellular IRF8 in CD4+ Tg4 T cells from isotype (gray)- or anti-PD1 (blue)-treated mice, n = 4. Bars represent mean ± SEM. Statistical analysis by unpaired t test. ∗p < 0.05..(N) Analysis of intracellular STAT4 in isotype or anti-PD1 treated mice. Bars represent mean ± SEM, n = 4.Please also see Figures S4 and S5 and Table S3.