Fig 2: Circulating ILC Populations Decrease during the Course of Immune Maturation(A) Gating strategy including lineage markers (CD3, CD4, CD11c, CD14, CD19, CD34, CD303, TCR?d, TCRaß) to identify two dominant NK populations defined by CD56high(green) and CD16high (purple) and three ILC subsets: ILC1 (orange), ILC2 (red), and ILCP (light blue).(B) Principal component analysis (PCA) and heatmap shown for each replicate for each participant (see Table S1).(C) DEGs among ILC2, ILCP, CD56high (NKCD56), and CD16high (NKCD16) NK cell populations from four HIV-negative and six HIV-positive pediatric subjects.(D) Frequencies of total helper ILC subsets as defined in (A), comparing HIV-negative newborn (NB) (n = 39), pediatric (2–5 years, n = 12), pediatric (>5 years, n = 25), and adult (n = 62) individuals expressed as percentage of total CD45+ lymphocytes.(E) As in (C) but showing frequencies of total NK and subset-specific differences between pediatric and adult subjects.p values by Dunn’s multiple comparisons test.

Fig 3: Characterization of Lin- lymphoid cells among human PBMCs A CD127+ and CD127- cells from Lin-CD56- population after gating on lymphoid, singlet, live, CD45+ cells of PBMCs. Lineage (Lin) markers include antibodies against: CD3, CD4, TCRaß, TCR?d, CD19, CD20, CD22, CD34, FceRIa, CD11c, CD303, CD123, CD1a, and CD14.B Heatmap of differentially expressed genes by RNA-Seq, sorted Lin-CD56-CD16-CD127+ versus Lin-CD56-CD16-CD127- cells, from PBMCs of five donors (log2 fold change > 1, Padj < 0.01 determined by DESeq2, the postsort profiles are shown in Appendix Fig S1).C Normalized counts of Lin-CD56-CD127+ (red) and Lin-CD56-CD127- (blue) cell-related genes from (B) by DESeq2 (n = 5).D, E Reactome analysis based on enriched transcripts of Lin-CD56-CD127+ cells (D) or Lin-CD56-CD127- cells (E).F Gating of Lin-CD56hi NK, Lin-CD56dim NK, Lin-CD56- NK cells, and Lin-CD56-CD16-CD127+ILCs.G The indicated populations in (F) were detected with isotype controls or antibodies against TBX21, CRTH2, and ROR?T. Data information: All data were generated using blood from healthy HIV-1-negative donors.