Flow Cytometry
For phenotypic characterization, immunostaining of 200 μl of fresh
peripheral whole blood with following fluorescently labelled monoclonal
antibodies, anti-CD3 (Clone:SK7), anti-CD4 (Clone: RPA-T4), anti-CD8
(Clone: SK1), anti-CD25 (Clone: M-A251), anti-CD127 (Clone: HIL-7R-M21),
anti-CCR7 (Clone: 150503), anti-CD45RA (Clone: HI100), anti-CD14 (Clone:
M5E2 ) and anti-16 (Clone: 3G8) was performed using stain/lyse/wash
protocol as described earlier (11). Data acquisition was performed on BD
FACS Aria Fusion (SORP) flow cytometer (BD Biosciences) where at least
100,000 events in lymphocyte scatter gate were acquired. Data analysis
was carried out using FlowJo software. IL-6 determination was carried
out using a cytometric bead array (CBA, BD Biosciences) to quantify
cytokine levels in plasma of study participants as per manufacturers’
protocol.