As previously described (6 (link)), PBMC were washed and stained with 5 μL Live/Dead Violet (Invitrogen) followed by surface staining with the following titrated antibodies: 0.5 μL CD3-AF700 (clone UCHT1, eBioscience), 2 μL CD4-APC (clone RPA-T4, BioLegend), 2 μL CD8-eFluor605 (clone RPA-T8, eBioscience), 2 μL CD14-PE/Cy7 (clone HCD14, BioLegend), 2 μL CD16-AF488 (clone 3G8, BioLegend), 1 μL CD19-PE/Cy5 (clone HIB19, BioLegend), 2 μL CD25-APC/Cy7 (clone BC96, BioLegend), 2 μL CD127-NC650 (clone eBioRDR5, eBioscience), and 15 μL HLA-DR–PE (clone L243, BioLegend). Fluorescence minus one (FMO) controls were used to set gates for CD25, CD127, and HLA-DR. Samples were acquired on a BD LSR II flow cytometer. Results are presented as percentages of cells after excluding dead cells and doublets. CD4+ and CD8+ T cells were identified as CD3+ cells, while CD14+/− and CD16+/− cells were identified as CD3− and CD19− populations. CD25+CD27− populations were gated on the CD4+ cells. The network representation of cell populations positively correlated among all infants was done using the igraph package in R. To identify closely related clusters (communities) within the network, the cluster_optimal function was used, implementing an algorithm described in Brandes et. al.(26 (link)).
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