*2.8. Flow Cytometry:*

For intracellular cytokine staining, two million cells were stimulated in 96-well plates with overlapping peptide pools of either HIV Env, Influenza HA1, or Zika prME protein, media alone (negative control), or phorbol 12-myristate 13-acetate (PMA) and ionomycin (BD Biosciences, San Jose, CA, USA) (positive control) for 6 h at 37 ◦C + 5% CO2 in the presence of GolgiPlug and GolgiStopTM (BD Biosciences, Franklin Lakes, NJ, USA). These are the same peptides pools described in the ELISpot sections. After 6 h, cells were collected and stained in FACS buffer with a panel of surface antibodies containing live dead eFluor V450, FITC anti-CD4, Alexa Fluor 700 anti-CD44, and APC-Cy7 anti-CD8 for 30 min at 4 ◦C. Cells were washed and then fixed with Foxp3/Transcription Factor Fixation/Permeabilization (ThermoFischer Scientific, Waltham, MA, USA) for 20 min at 4 ◦C. Cells were washed with Perm/Wash buffer before intracellular staining with PE-Cy7 anti-IL-2, PerCP-Cy5.5 anti-CD3ε, PE anti-TNFα, and APC anti-IFNγ for 1 h at 4 ◦C. Cells were then washed with Perm/Wash buffer before suspension in Perm/Wash buffer and acquisition on a BD LSRII. All results were analyzed using FlowJoTM v.10.0.
