Surface markers were evaluated using combinations of fluorochrome-conjugated monoclonal antibodies that were each titrated individually for their optimal stain index. PBMCs were stained at 10 million cells/mL in 200uL PBS. All PBMCs were incubated for 10 minutes with an amine-reactive viability dye (LIVE/DEAD Aqua, Invitrogen), washed twice, and then stained for 15 minutes at room temperature with combinations of monoclonal antibodies. For ex vivo phenotyping, cells were stained with CD3-AF700 (UCHT1, BD), CD4-PECy5 (RPA-T4, BD), CD8-APC-AF750 (3B5, Invitrogen), CD45RO-PETR (UCHL1, Beckman Coulter), CCR7-BV421 (150503, BD), CXCR5-AF488 (RF8B2, BD), PD-1-PE (EH12.2H7, BioLegend), CD14-V500 (M5E2, BD), and CD19-V500 (HIB19, BD). In vitro phenotyping was performed with combinations of CD3-AF700, CD4-PECy5, CD8-APC-AF750, CD45RO-PETR, CXCR5-AF488, CD14-V500, CD19-V500, ICOS-PE (DX29, BD), CD40L-PE (TRAP1, BD) and PD-1-BV421 (EH12.2H7, BioLegend). All PBMCs were washed twice after staining, fixed with 2% paraformaldehyde, and analyzed on a BD LSR Fortessa (BD Biosciences) at the VMC Flow Cytometry Shared Resource.
Flow cytometry data was analyzed using BD Biosciences FACSDiva Software. In all experiments, forward and side scatter were used to identify lymphocytes and from that population non-viable, CD14+, CD19+, CD8+ cells were excluded from further analysis (Fig. S1 ).
Flow cytometry data was analyzed using BD Biosciences FACSDiva Software. In all experiments, forward and side scatter were used to identify lymphocytes and from that population non-viable, CD14+, CD19+, CD8+ cells were excluded from further analysis (