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. 2022 Apr 11;21(6):948–959. doi: 10.1158/1535-7163.MCT-21-0802

Figure 3.

Figure 3. Pharmacologic inhibition of CD73 enhances activation and cytolytic capabilities of tumor-specific T cells. A, Human CD4+ and CD8+ T cells from a healthy donor were transduced with a Neo-12 TCR and examined for CD73 expression and antigen specificity (dextramer staining) by flow cytometry. Greater than 95% of transduced cells expressed CD73, and 48.8% of CD4+ and 51.3% of CD8+ T cells expressed the transduced TCR as measured by dextramer staining. B, T cells from A were cocultured with K562 cells expressing HLA-A2 alone or HLA-A2 with the Neo12 epitope in the presence of AMP + EHNA ± AB680 for 72 hours. Frequency of CD25+ CD69+ T cells and secretion of IL2 (CD4+ T cells) and IFNγ (CD8+ T cells) were quantified. ***, P < 0.001; ****, P < 0.0001, Dunnett multiple comparisons test versus AMP alone. C, Representative flow plots displaying activated phenotype (CD44+, CD25+, CD62Lhi) of mouse OT-I CD8+ T cells and frequency of CD73+ cells on both activated OT-I CD8+ T cells and EG7.OVA tumor cells. Stained samples (red) overlaid on the isotype controls (blue). D, CTL killing assay showing time course (top) and 48 hours quantification (bottom) of tumor cell growth measured by the IncuCyte of red fluorescently labeled EG7.OVA cell confluence when cocultured with preactivated OT-I CD8+ T cells in the presence of 50 μmol/L AMP + 2.5 μmol/L EHNA ± 50 nmol/L AB680. Data presented as mean ± SEM and representative of two independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001, Dunnett multiple comparisons test versus AMP alone.

Pharmacologic inhibition of CD73 enhances activation and cytolytic capabilities of tumor-specific T cells. A, Human CD4+ and CD8+ T cells from a healthy donor were transduced with a Neo-12 TCR and examined for CD73 expression and antigen specificity (dextramer staining) by flow cytometry. Greater than 95% of transduced cells expressed CD73, and 48.8% of CD4+ and 51.3% of CD8+ T cells expressed the transduced TCR as measured by dextramer staining. B, T cells from A were cocultured with K562 cells expressing HLA-A2 alone or HLA-A2 with the Neo12 epitope in the presence of AMP + EHNA ± AB680 for 72 hours. Frequency of CD25+ CD69+ T cells and secretion of IL2 (CD4+ T cells) and IFNγ (CD8+ T cells) were quantified. ***, P < 0.001; ****, P < 0.0001, Dunnett multiple comparisons test versus AMP alone. C, Representative flow plots displaying activated phenotype (CD44+, CD25+, CD62Lhi) of mouse OT-I CD8+ T cells and frequency of CD73+ cells on both activated OT-I CD8+ T cells and EG7.OVA tumor cells. Stained samples (red) overlaid on the isotype controls (blue). D, CTL killing assay showing time course (top) and 48 hours quantification (bottom) of tumor cell growth measured by the IncuCyte of red fluorescently labeled EG7.OVA cell confluence when cocultured with preactivated OT-I CD8+ T cells in the presence of 50 μmol/L AMP + 2.5 μmol/L EHNA ± 50 nmol/L AB680. Data presented as mean ± SEM and representative of two independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001, Dunnett multiple comparisons test versus AMP alone.