Fig. 4: PD-L1 blockade results in the emergence of a PD-1⁺A_2AR⁻ subset of CD8⁺ T cells that correlates with therapeutic outcomes.

A–G. A_2AR eGFP reporter mice were injected with 5 × 10⁵ AT-3 ova tumors subcutaneously and where indicated treated at days 14 and 18 post tumor inoculation with anti-PD-L1 (200 µg/ mouse) and anti-CTLA-4 (150 µg/ mouse). A Tumors were excised 21 days post-tumor inoculation and fixed with 4% paraformaldehyde followed by 30% sucrose overnight. Immunofluorescence staining was performed on tumor slides. Staining for CD45 (red), DAPI (blue) and A_2AR (green) is shown. Slides imaged using ×4 and ×10 objective lens. Representative image from one mouse is shown. B Percentage of live, CD45.2⁺ cells that were A_2AR⁺ CD8⁺ T cells (left) and A_2AR⁺ CD4⁺ T cells (right). Data represent the mean ± SEM of individual mice pooled from 3–7 experiments (control and anti-PD-L1 n = 39, 7 experiments, anti-CTLA-4 n = 23, 4 experiments, anti-PD-L1 and anti-CTLA-4 n = 18, 3 experiments). C Percentage of CD8⁺ T cells (left) or CD4⁺ T cells (right) that are A_2AR⁺ following treatment. Data represent the mean ± SEM of individual mice pooled from 4 to 7 experiments (CD8⁺: control n = 39, 7 experiments, anti-PD-L1 n = 40, 7 experiments, anti-CTLA-4 n = 17, 3 experiments anti-PD-L1 and anti-CTLA4 n = 24, 4 experiments, CD4⁺: control n = 34, and anti-PD-L1 n = 34, anti-CTLA-4 n = 17, anti-PD-L1 and anti-CTLA4 n = 18) D Percentage of CD8⁺ T cells exhibiting a PD-1⁺A_2AR^- or PD-1⁺A_2AR⁺ phenotype at day 7 post treatment. Data represent the mean ± SEM of individual mice pooled from 2 to 6 experiments (control n = 33, 6 experiments, anti-PD-L1 n = 28, 5 experiments anti-CTLA-4 n = 11, 2 experiments, anti-PD-L1 and anti-CTLA4 n = 12, 2 experiments). E Correlation of the frequency of CD8⁺ T cells exhibiting PD-1⁺A_2AR^- or PD-1⁺A_2AR⁺ phenotypes with therapeutic efficacy. Therapeutic efficacy calculated as 100-(tumor weight in test sample/ average tumor weight in control group*100). n = 27 mice. F Representative flow cytometry staining of CD8⁺ T cells derived from non-treated mice (left) and mice treated with anti-PD-L1 that either elicited a therapeutic response (right) or no response (center). Data points represented as non-responders and responders are depicted as red in (E). G Proportion of CD8⁺ T cells isolated from tumor-draining lymph nodes exhibiting an A_2AR⁺tetramer⁺ phenotype (left) or absolute numbers of these cells (right). Data represent the mean ± SEM of 29 (non-treated) or 30 (anti-PD-L1) mice pooled from 5 individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-sided t test (B, D, G) or Linear regression analysis (E). Source data are provided as a Source Data file.