Fig 2: Trp2/Kb-specific CD8+ T cells in pre-immune wild-type (WT) and Dct-/- mice share a naïve phenotype while showing modest differences in frequency and tetramer staining.(A) Tetramer enrichment was performed to enumerate Trp2/Kb-specific CD8+ T cells per mouse. Median tetramer fluorescence intensity (MFI) was used to estimate the avidity of enriched Trp2/Kb-specific (B) or B8R/Kb-specific cells (C). (D) CD44/CD122 expression of Trp2/Kb-specific cells. (E) CD69 expression of Trp2/Kb-specific cells. (F) PD-1, CTLA-4, LAG3, and TIM3 expression of Trp2/Kb-specific cells. (G) CD5 expression of Trp2/Kb-specific cells. Data are compiled from three independent experiments in A and D. Individual experiments are shown in B and C; results are representative of other experiments. The graphs in (E, F, and G) represent individual experiments with four mice per group. Squares indicate male animals. **p<0.01 by unpaired t test.Figure 1—source data 1.Data file related to Figure 1.Figure 1—source data 2.Primers used to screen prospective clones for homologous recombination of the Dct knockout construct.Figure 1—source data 3.Primers used to screen mice to confirm deletion of the Dct gene.

Fig 3: Enforcing expression of GLUT3 enables higher glucose uptake, survival, and resistance to exhaustion of TCM cells. (A) Schematic for the generation of GLUT3-TCM cells. (B) Evaluation of transduction efficiency for GLUT3 expression by anti-Flag antibody staining and flow cytometric analysis. (C) Viability of transduced T cells as measured by staining with a viability dye and flow cytometric analysis (days 9-12). (D) Percentage of CD44+ CD62L+ and CD44+ CD62L- T cells in culture. Left: summary of all donors tested. Right: Representative flow cytometry dot plot. (E) Seahorse analysis of the basal extracellular acidification rate (ECAR) of MOCK- and GLUT3-T cells with a metabolic perturbation assay. (F) Top Left: Percentage necrosis (AnnexinV-/7-AAD+) of MOCK- versus GLUT3-T cells over time in media with or without glucose (Glc).Bottom Left: evaluation of live (Annexin V-/7-AAD-), early apoptotic (Annexin V+/7-AAD-), late apoptotic (Annexin V+/7-AAD+), and necrotic (Annexin V-/7-AAD+) MOCK- and GLUT3-T cells after 72h in culture in the presence or absence of Glc. Right: representative dot plot of AnnexinV/7-AAD staining at 72h (AnnV=Annexin V). (G) Expression levels of PD1, LAG3, TIM3, TCF-1 and TOX upon repeated stimulation with SIINFEKL peptide for 5 days. Top: mean fluorescence intensity (MFI) levels are shown. MFI levels of GLUT3-T cells from each culture are normalized to corresponding MOCK-T cells. Bottom: representative histograms of PD1, LAG3, TIM3 TCF1 and TOX expression. Shown is average ± SD of different cultures. Statistical analysis by paired, two-tailed t test (E, G), two-way analysis of variance (ANOVA) with correction for multiple comparisons by post hoc Tukey’s test (F Top Left: Glc+ 48h p=0.0005, Glc- 48h p<0.0001, Glc+ 72h p<0.0001, Glc- 72h p<0.0001), two-way ANOVA with correction for multiple comparisons by post hoc Sidak’s test (F Bottom Left: Glc +, Necrosis MOCK vs GLUT3 p=0.0016; Glc +, Early Apoptosis MOCK vs GLUT3 p=0.0009; Glc -, Necrosis MOCK vs GLUT3 p=0.0456). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, non-significant.

Fig 4: 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.

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