Cd28 cd28.2

Polychromatic flow cytometry and cell sorting were performed on stained mononuclear cells as previously described^{15 (link)}. Antibodies against the following antigens were used for staining at predetermined concentrations: CD4 (clone OKT4), CD8 (SK1), IFNγ (4S.B3), IL-17 (eBio64DEC17), IL-21 (3A3-N2), IL-22 (IL22JOP), TNFα (MAb11), and PD-1 (J105) from eBioscience; CCR5 (3A9), CD3 (SP34-2), CD8 (SK1), CD16 (HI149), CD20 (2H7), CD23 (M-L233), CD45 (D058-1283), CD123 (7G3), and HLA-DR (L243) from BD; c-Kit (104D2), CD1a (HI149), CD11c (3.9), CD14 (M5E2), CD34 (561), CD95 (DX2), and IL-2 (MQ1-17H12) from Biolegend; CD127 (eBioRDR5) and CD218a (H44) from Thermo; NKp44 (2.9) from Miltenyi; and CD28 (CD28.2) from Beckman Coulter. CD4+ and CD8+ TM were defined as CD95+ singlet, clean, live, CD3+ lymphocytes. NKp44+ ILC3s were defined as CD45+ singlet, clean, live, lineage-negative (CD1a, CD3, CD8, CD11c, CD14, CD16, CD20, CD23, CD34, CD123), NKp44+CD127+CD218+ lymphocytes. Positive/negative gating based on clearly grouped populations, historical-determined expression, and the use of internal controls (Supplementary Fig. 8). A threshold of 100 collected events in the parent population was utilized for all subset expression analysis.

Cryopreserved samples of PBMC, lymph node, and colorectal biopsies were thawed at 37°C in RPMI 1640 containing 10% fetal bovine serum (FBS) and benzonase (Millipore) at 50 U/ml. Cells were resuspended in 1× phosphate-buffered saline (PBS) containing LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies) for 20 min at room temperature in the dark. Cells were washed and resuspended in wash buffer containing the following fluorescently conjugated antibodies: CD45 (D058-1283), CD28 (CD28.2, Beckman Coulter), CD4 (L200), CCR7 (3D12), CD95 (DX2), CD3 (SP34-2), and CD8 (SK1). After 15 min in the dark, cells were washed and resuspended in ice-cold 1× PBS. Cells were sorted using a BD FACSAria II. Sorted CD4⁺ T cells were FSC singlets, live, CD45⁺CD3⁺CD4⁺CD8⁻ lymphocytes, and subsets were defined as follows: naïve (CD95⁻CD28⁺ CCR7⁺), central memory (CD95⁺CD28⁺CCR7⁺), transitional memory (CD95⁺CD28⁺CCR7⁻), and effector memory (CD95⁺CD28⁻). All antibodies are from BD Biosciences unless otherwise indicated. In study 1, sorted memory populations were pooled into a single memory population and subject to proviral DNA analyses due to limiting cell numbers.