Fig. 1: Validation of A_2AR eGFP reporter mice.

A Immunofluorescence staining of sectioned spleens obtained from C57BL/6 A_2AR eGFP reporter or C57BL/6 WT mice (n = 1 experiment of 2 individual mice). Image shows A_2AR expression (GFP), CD45 (Red) or CD8 (Red) and DAPI (Blue) at 20x magnification (Left) or 10x magnification (Right). Section on right depicts GFP (A_2AR) expression on cells located within the arterioles. B A_2AR (GFP) expression on indicated splenic immune subsets from A_2AR eGFP or C57BL/6 WT mice as determined by flow cytometry. Representative experiment of n = 3 C Percentage ( ± SEM) of indicated CD8⁺ subsets expressing A_2AR in spleens of n = 5 naïve mice. D A_2AR mRNA expression in FACS sorted GFP⁺ and GFP^- cells isolated from A_2AR eGFP⁺ reporter mouse spleens. Data represent the mean ± SD of 3 samples obtained from independent mice. A_2AR expression is plotted relative to L32. Statistical significance determined using unpaired two-sided t test. ****p < 0.0001 E, F. Expression of GFP (A_2AR), PD-1 and CD44 in CD8⁺ and CD4⁺ lymphocytes activated for 24 h with anti-CD3 (0.5 μg/ml) and anti-CD28 (0.5 μg/ml). Data represent the mean ± SD of triplicate samples from a representative experiment of n = 3. G Splenocytes from WT or A_2AR eGFP reporter mice were stimulated for 72 h with anti-CD3 (0.5 μg/ml) and anti-CD28 (0.5 μg/ml) in the presence or absence of NECA (1 µM) or SCH58261 (1 µM). Data represent the mean ± SD of triplicate samples. H, I. Anti-Her2 CAR T cells were generated from A_2AR eGFP mouse splenocytes and cocultured with indicated tumor cells. GFP Mean Fluorescence Intensity (MFI) of CD8⁺ T cells and CD4⁺ T cells (bottom) was measured by flow cytometry following 16 h co-culture with indicated Her2 expressing tumor lines, AT3 Her2, E0771 Her2 and MC38 Her2. I Data represent the mean ± SD of triplicate samples from a representative experiment of n = 3. Source data are provided as a Source Data file.