Figure 1: CD39 marks a subset of exhausted CAR T cells with features of regulatory T cells.

A. Flow cytometric analysis of exhaustion marker expression in gated CD19.28z-CAR⁺ (CD19) (blue) and HA.28z-CAR⁺ (HA) T cells (red) at D0 (grey) and at each indicated time-point post-activation. Representative histogram shown of n = 3 donors.

B. (Left) Representative flow cytometry contour plot of CD39 expression in HA-CAR⁻ vs. CAR⁺ T cells on day 10 post-activation. (Right) Percent of CD39⁺ cells in HA-CAR− vs. CAR⁺ T cells from n=12 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

C. (Left) Representative flow cytometry analysis of CD39 expression in CD8⁺ vs. CD4⁺ HA-CAR T cells 10 days post-activation. (Right) Percent of CD39⁺ cells in CD4⁺ vs. CD8⁺ HA-CAR T cells from n = 11 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

D. Luminex analysis of cytokines secreted by CD39⁺ vs. CD39⁻CD8⁺ HA-CAR T cells sorted on day 14 and stimulated with Nalm6-GD2 for 24hrs at 1:1 E:T ratio. Data are mean ± s.e.m. of n= 3 donors. P values determined by ratio paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

E. Gene Set Enrichment Analysis (GSEA) of bulk RNA-seq collected 14 days post-activation of CD39⁺compared to CD39⁻HA-CAR T cells using the publicly available GSE25087 gene collection.

F. CyTOF analysis of CD4⁺ (bottom) and CD8⁺ (top) HA-CAR T cells gated based on CD39 expression 10 days post-activation. Heat map represents expression of the indicated markers as median Arcsinh relative to the total CD4⁺ or CD8⁺ sample. Representative donor shown of n=3 donors.

G. Schematic of immune suppression mediated by purinergic pathway. ATP- adenosine triphosphate; ADP- adenosine diphosphate; AMP- adenosine monophosphate; ADO- adenosine (red); A2aR- adenosine 2a receptor; CD39− ecto-ATP diphosphohydrolase-1; CD73– 5ʹ-ectonucleotidase; NF-κB- Nuclear Factor kappa B.

H. (Left) Representative flow cytometry contour plots showing expression of CD39 and CD73 in gated CD4⁺ or CD8⁺ HA-, CD19-CAR or mock T cells 14 days post-activation. (Right) Percent of CD39⁺CD73⁺ T cells in CD4⁺ vs. CD8⁺ by day 14 HA-, CD19-CAR or mock T cells in 6 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

I. Concentration of adenosine (ADO) produced by mock or CAR-expressing T cells with or without CD39 or CD73 spiked with 20uM of ATP at day 17 post-activation. Representative data from n=3–5 donors. Average purity of the knock-out cells was > 90%. P values determined by unpaired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

J. (Left) schematic of experimental design of immunosuppression co-culture assays. (Right) Control or A2aR KO CD19.bbz-CAR T cells were activated with Nalm6 at 1:1 ratio in the presence of HA- or CD39KO HA-CAR T cells. IL-2 was measured 24hrs post-stimulation. Data are mean ± s.e.m. from triplicate wells. P values determined by unpaired two-tailed t-tests. Representative result of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001