Recall antigen assay

The recall antigen assay spanned a duration of 7 days. On day 0, donor PBMCs (purchased from Astarte Biologics, now part of Cellero, Lowell, MA) were washed twice after thaw and resuspended at 5 × 10⁶ viable cells per mL in AIM V medium (ThermoFisher Scientific, Waltham, MA, Cat # 12055091) with 5% human AB serum (Millipore Sigma, Burlington, MA, Cat # H3667). Control unstimulated cells were first plated in a round-bottom 96-well plate, 100 µL per well (500,000 cells). For the remainder of cells, 10 µg/mL peptide (final concentration unless otherwise stated) was added to cells to create a cell/peptide mixture. 100 µL (500,000 cells) of cell/peptide mixture was added to appropriate wells. NLVPMVATV peptide was purchased from Astarte Biologics. IVTDFSVIK, AVFDRKSDAK, and ATVQGQNLK peptides were purchased from MBLI (Chicago, IL). On day 1 of the recall antigen assay, each well was supplemented with 100 µL complete medium, 50U/mL IL-2 final concentration (Millipore Sigma, Cat # 11147528001) with or without treatments (i.e. adenosine, GS-5734 and GS-441524) for a final well volume of 200 µL. On days 3 and 5, 100 µL volume from each well was removed without disturbing cell pellet. This was replaced with fresh complete IL-2 supplemented media (50 U/mL final concentration), with or without treatments so that total well volume was maintained at 200 µL. Readout for recall antigen assay was flow cytometry on day 7. Tetramers were used in flow cytometric analysis that corresponded to the peptide used for stimulation. Tetramer positive cells correspond to memory T cells that expanded in response to peptide stimulation on day 0. For adenosine-treated wells, 1 mM adenosine (Millipore Sigma, Cat # A9251) was given on day 1 and 0.5–0.75 mM adenosine was given on days 3 and 5. Adenosine was dissolved in complete media unless otherwise noted. GS-5734 and GS-445124 (Cayman Chemical, Ann Arbor, MI) were dissolved in Hybri-Max DMSO (Millipore Sigma, Cat # D2650). Adenosine was also prepared in Hybri-Max DMSO and used as a control in initial experiments although at much lower concentration than adenosine dissolved in complete medium. Adenosine deaminase (ADA) (Cat #10102105001) and Inosine (Cat #I4125) were both purchased from Millipore Sigma. Inosine was prepared in complete media. Tofacitinib (Selleckchem, Cat # CP-690550) was prepared in DMSO.