Fig 2: ILC1-like cells have a unique gene signature distinct from NK cells.(a) CB mononuclear cells (MNCs) were enriched prior to sorting via biotin-labelled antibodies (anti-CD3/CD19/CD14/CD66b) and sorted for ILC1-like cells, CD56dim, and CD56bright NK cells (see Figure 1—figure supplement 1 for sorting strategy). RNA sequencing was done on the Illumina platform. The heat map indicates the top 100 differentially expressed genes between ILC1-like cells and CD56bright NK cells including CD56dim NK cells. (b) A two-dimensional principle component analyses based on the top 2000 differentially transcribed genes of CD56bright NK cells, CD56dim NK cells, and ILC1-like cells is shown. (c) A four-way plot with a cut off at a log2 fold change ±1 (dotted lines) and adjusted p-values of 0.05 showing differently expressed genes of CB CD56bright NK cells compared to CD56dim NK cells and ILC1-like cells. Blue dots represent genes with an adjusted p-value<0.05 with a fold change >1. Green dots represent genes with an adjusted p-value <0.05 with a fold change between >1 (x-axis) and <1 (y-axis). Grey dots represent genes with an adjusted p-value >0.05. Red dots represent genes with an adjusted p-value<0.05 with fold rates < 1 (x-axis) and >1 (y-axis). Selected genes differentially expressed between NK cells subsets and ILC1-like cells are highlighted. (d) CB MNCs (n = 3) gated on ILC1-like cells, CD56bright NK cells, and CD56dim NK cells, respectively were analyzed by flow cytometry for selected NK, T, and ILC markers. Representative histograms for NK cell-specific factors containing CD94, NKG2A, CD56, and EOMES (upper panel), ILC1-specific factors containing CD117, CD127, CD7, CD161, and TBET (middle panel), and T cell-specific factors CD6, CD5, CD28, as well as a Mix of CD3/TCRaß/TCR?d (bottom panel). (e) Representative dot plot of the expression of CD5 and CD161 within CB ILC1-like cells with representative quantification of CD5+ (green bar and box) and CD161+ (yellow bar and box), (n = 5). The height of the bar represents the mean ± SEM. Levels of significance were calculated with a non-parametric t test (Mann-Whitney), ** p-value <0.01. Data represent at least three different donors and experiments. Figure 1—source data 1.R code for RNAseq data analyses used in Figure 1.This file contains the R code to generate the heatmap, PCA, and Four- Way plot displayed in Figure 1.

Fig 3: Isolation of naive lymphocytes using an ALFA-tagged nanobody recognizing CD62L. Total human PBMCs were left untreated (Before sorting) or isolated using an ALFA SelectorPE resin loaded with an ALFA-tagged anti-human CD62L nanobody (After sorting). a A sketch of the affinity purification strategy. b Cells were stained with an anti-CD62L antibody and analyzed by flow cytometry. c The same cells as in b were stained with antibodies directed against CD3, CD19 and CD62L, and analyzed by flow cytometry. A forward scatter/side scatter gate was set on lymphocytes in all analyses (Supplementary Fig. 9). Color code used in a: NbALFA (orange), ALFA-tag (blue), CD62L (light gray), NbCD62L (dark gray), CD62L+ T-lymphocyte (yellow)

Fig 4: ILC1-like cells possess high NKP potential without previous epigenetic priming for NK cell receptor expression.(a) Scheme of the experimental set-up. CB MNCS were freshly isolated, enriched using biotinylated antibodies (anti-CD3, CD14, CD19, CD66b), and sorted for CD5+ILC1-like cells, CD5-ILC1-like cells, CD56bright NK cells, and ILC2. One day prior to sorting, OP9-DL1 cells were plated in 96-well flat-bottom plates. Cells were either supplemented with IL-7 and FLt3L for the T cell condition or IL-2, IL-7, and IL-15 for the NK cell condition and cultured for 8 days with medium change at day 5. (b) Exemplary dot plots and quantification of CD94 expression together with either NKG2A or KIR2DL2/L3/S2 expression after 8 days of co-culture on OP9-DL1 from left to right for CD5+ILC1-like cells (n = 7), CD5-ILC1-like cells (n = 7), CD56bright NK cells (n = 5), and ILC2 (n = 6). (c) Comparative analysis of regions with open chromatin by ATAC sequencing for KLRD1 (CD94), KLRC1 (NKG2A), and KIR2DL3. For ATAC sequencing, 5000 CB-derived ILC1-like (top row) and NK cells (bottom row) were flow cytometrically sorted to >99% purity (n = 3). Arrows underneath the ATAC data indicate orientation and start of gene transcription. Heights of the bars represent mean ± SEM. Levels of significance were calculated with a One-Way ANOVA with a multiple comparison post-test (Kruskal-Wallis test), * p-value<0.05, ** p-value<0.01, *** p-value<0.001. Data represent at least three different donors and experiments (a–b). Data represent two experiments with three donors (c).

Fig 5: NK cell subpopulations in fresh PBMC.Freshly isolated PBMC from HIV-LTBI-, HIV-LTBI+ individuals, HIV+LTBI- and HIV+LTBI+ patients (30 donors in each group) were stained with Abs to CD3, CD56, CD16, CD27 and KLRG1. Flow cytometry was used to measure the percentages of A. CD3-CD56+ B. CD16+CD56+ C. CD3-CD27+CD56+ and D. CD3- CD27+KLRG1+CD56+cells. P values across the groups were derived using Kruskal Wallis One-way ANOVA and unpaired T test. Data is shown as median and interquartile range. Boxes show the median and interquartile range, and whiskers show the 5th and 95th percentile values. ns denotes not significant, * denotes p < 0.05, ** denote p < 0.01, *** denote p < 0.001, **** denote p < 0.0001.