Fig 2: Metabolite concentration impact on CD49a+CD103+ TRM-like cell differentiation and maintenance in vitro.CD49a+CD103+ cells per microliter (A) and percent CD49a+ (B) were assessed in metabolite alteration assays where both lipid (0×–4×) and glucose (4.5–24.5 mg/mL) were altered after activation in the presence of IL-2 and cultured with IL-7 and TGF-β. Small circles represent individual data points. Data are generated from two independent experiments of n = 5 mice per experiment.

Fig 3: Metabolite concentration impact on TRM-like cell differentiation and maintenance in vitro.CD49a+ cells per microliter (A) and percent CD49a+ (B) were assessed in metabolite alteration assays where both lipid (0×–4×) and glucose (4.5–24.5 mg/mL) were altered after activation in the presence of IL-2 and cultured with IL-7 and TGF-β. Small circles represent individual data points. Data generated from two independent experiments of n = 5 mice per experiment.

Fig 4: CD73 accelerates the exhaustion process in CD8+ T cells in vitro. (A) CD73- and CD73+ OT-I cells were sorted and activated with the SIINFEKL peptide and IL-7/IL-15 for 2 or 5 d. The bar graph shows the absolute numbers of total cells in the cultures following chronic activation of CD73− (gray) and CD73+ (blue) CD8+ T cells. The dotted line represents the number of cells at day 0 (input). (B) Bar graphs showing CD25 and CD69 mean fluorescence intensity on CD73− (gray bars) and CD73+ (blue bars) on OT-I cells chronically activated for 2 d. (C) Bar graphs showing mean fluorescence intensity of CD25 and CD69, and the frequency of granzyme B (GZMB) expressing cells on CD73− (gray bars) and CD73+ (blue bars) OT-I cells that were chronically activated for 5 d. A–C, each dot represents an independent experiment (n = 3). (D) OT-I and OT-I/CD73KO cells were chronically activated with the SIINFEKL peptide and IL-7/IL-15 for 5 d. Dot plots depict PD-1 and TIM3 expression in OT-I and OT-I/CD73KO cells following 5 d of chronic stimulation. The gates show the frequency of Tpex and Tex cells. The bar graph shows the frequency of Tex cells after 5 d of chronic activation of OT-I (gray bar) and OT-I/CD73KO (red bar) cells. Data are presented as mean ± SD. Each dot in the bar graphs represents an independent experiment (n = 9–15). P values were calculated using the Student's paired t-test (A–C) and unpaired t-test with Welch's correction (D). *p ≤ 0.05, **p ≤ 0.01.

Fig 5: CD73 deficiency promotes a stem-like transcriptional profile in exhausted CD8+ T cells. OT-I and OT-I/CD73KO cells were activated for 5 d under chronic stimulation with the SIINFEKL peptide in the presence of IL-7 and IL-15 and analyzed by RNAseq. (A) PCA analysis of WT and CD73KO RNAseq samples. Each dot represents an independent experiment (WT n = 3, CD73KO n = 3). (B) Heat map showing a set of genes upregulated and downregulated in WT cells compared to CD73KO cells. (C) Gene set enrichment analysis for functional pathways was performed using the expression matrix of the data and the hallmarks of the MsigDB as a reference. (D) Gene set enrichment analysis for function-associated signatures.