Figure 1

Adenosine-based recall antigen assay. (A) HLA-A*02:01 donor #1 PBMCs treated either with peptide alone (CMV pp65 NLVPMVATV) or peptide plus adenosine (ADO) on indicated days (x axis) and assayed on day 7. ADO treatments on day 1 were 1 mM final concentration while day 3 and day 5 ADO treatments are at a final ADO concentration of 0.5–0.75 mM. Data are representative of 3 independent experiments, except for the D3 (n of 2) and displayed as percent tetramer fold-change (FC) relative to peptide-only treated cells within each respective experiment. Dotted line used for comparison. (B) Assay setup whereby donor PBMCs are treated with ADO days 1, 3 and 5 and assayed for CD8⁺ tetramer positive T cells. ADO results in significantly reduced tetramer positive T cell expansion. Drug arm uses the same dosing regimen as ADO. (C) Donor #1 PBMCs were treated with NLVPMVATV or (D) HLA-A*11:01 donor #3 PBMCs treated with EBV EBNA-3B peptide IVTDFSVIK, EBV EBNA4 peptide AVFDRKSDAK, or CMV pp65 peptide ATVQGQNLK. The percent positive range of tetramer positive cells (among live T cells) for peptide-only stimulated wells is indicated with dotted arrow. GS-5734 and GS-441524 (parent nucleoside of GS-5734) were used at increasing doses of 500 nM, 5 µM and 50 µM. 50 µM GS-5734 treatment is indicated with striped bar to denote its high toxicity. DMSO was administered at the same concentrations as drugs; 0.001%, 0.01% or 0.1% final. ADO resulted in almost complete suppression of peptide-stimulated antigen-specific T cell expansion (third lane of each figure). Data are representative of 4 (C) or 3 (D) independent experiments, each experiment derived from a separate cryovial (same donor and lot number) and each condition was performed in duplicate or triplicate. (A,C,D) Ratios of all combinations of treated sample relative to peptide-only treated sample were calculated and median ratio values for each experiment are plotted. Median values were log transformed for one-way ANOVA and subsequent Dunnett’s multiple comparison test using peptide-only treated samples as the control group. *, adj p value ≤ 0.05; **, adj p value ≤ 0.01; ***, adj p value ≤ 0.001; ****, adj p value ≤ 0.0001.