For phenotypic analysis, PBMCs were washed before being stained for viability and surface phenotype. For intracellular cytokine analysis, PBMCs were washed, stained for viability and surface phenotype, and, following fixation and permeabilization, stained for intracellular cytokine production.

Details of the antibodies that were used are presented in Supplementary Materials and Methods. Cells were washed, resuspended, and stored in the absence of light at 4°C until data were acquired using a FACSAria III flow cytometer (BD Biosciences). Compensation beads (BD Biosciences) were used to create compensation matrices, and sequential cell isolation was used to identify populations of interest (Figs. 1 and 3). For phenotypic analysis, values were expressed as the median fluorescence intensity. For intracellular cytokine analysis, the frequency of each population positive for each cytokine above background was multiplied by the geometric MFI for each cytokine-positive population—this well-established metric is known as the iMFI (44). Full details are presented in Supplementary Materials and Methods.

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