EDTA-anticoagulated whole blood samples were stained within 6 h of collection. Briefly, 100 μl of whole blood was stained with an antibody cocktail, diluted in Brilliant Stain Buffer (BD Biosciences). Red blood cells were lysed with FACS lysing solution (BD Biosciences), samples washed and resuspended in FACSflow. Two different antibody panels were used. One panel contained CD8 PerCP (SK1), CD4 FITC (SK3), CD3 APC-H7 (SK7), PD-1 BV786 (EH12.1) and ICOS BV650 (DX29) from BD Biosciences and the other panel contained CD8 Alexa Fluor 700 (RPA-T8), CD4 BV786 (L200), CD3 APC-H7 (SK7), TIGIT APC (MBSA43), CCR5 PE (2D7) from BD Biosciences and HLA-DR PE-Cy5.5 (TU36) from Invitrogen (Waltham, USA). CCR5 density was calculated using BD QuantibriteTM beads (BD Biosciences) [34 (link)]. Samples were acquired on a four laser BD LSRFortessa X-20 (BD Biosciences) within 4 h. CS&T beads and mid-range Rainbow Fluorescent Particles (BD Biosciences) were run before sample acquisition. Compensation was performed for each experiment using BD CompBeads (BD Biosciences). Samples were analyzed using FlowJo software version 9.9.6 (BD Biosciences).
The gating strategy is shown in Supplemental Figure 1A. A representative example of expression of these markers in a mother–infant pair is shown in Supplemental Figure 1B.
The gating strategy is shown in Supplemental Figure 1A. A representative example of expression of these markers in a mother–infant pair is shown in Supplemental Figure 1B.