Fig 2: 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 3: Initial Immunological analysis of Salt-Losing Tubulopathy patients.a IL-17-related infection score in SLT patients (n = 47), healthy controls (n = 24), and disease (n = 22) controls: HC 1.0 (0.0–2.8); SLT 5.0 (1.0–8.0); DC 0.0 (0.0–2.3), p = 0.0006. Groups are compared using a Kruskal–Wallis test with multiple comparison testing. Error bars represent the interquartile range around the median. b Total immunoglobulin E (IgE) levels in SLT patients (n = 30). Red line depicts upper limit of normal at 120 KUL; 10/30 (33%) SLT patients had IgE > 120 KUL. c NK cells expressed as a proportion of lymphocytes in SLT patients (n = 32). Red line depicts lower limit of normal at 7%; 13 (40.6%) SLT patients had NK cells < 7%. d. T-cell activation analysis in SLT patients (n = 3) and healthy controls (n = 7). Proportion of CD4+ cells expressing CD25 after 72 h anti-CD3/anti-CD28 stimulation: HC 89.5% (84.9–95.4), SLT 89.1% (87.3–93.5), p = 0.99. Groups are compared with a two-sided Mann–Whitney test. Error bars within the graph represent the interquartile range around the median. Representative FACS contour plots of CD4 and CD25 staining have cell proportions as percentages within each quadrant. e T-cell proliferation analysis in SLT patients (n = 6) and healthy controls (n = 6). Proportion of CD4+ cells expressing Ki67 after 72 h anti-CD3/anti-CD28 stimulation: HC 83.6% (68.7–88.2) and SLT 76.2% (70.4–85.6), p = 0.48. Groups are compared with a two-sided Mann–Whitney test. Error bars within the graph represent the interquartile range around the median. Representative FACS contour plots of CD4 and Ki67 staining have cell proportions as percentages within each quadrant. f. Varicella zoster and pneumococcal-specific immunoglobulin G levels in SLT patients (n = 11). Findings in SLT patients are consistent with the healthy adult population as per assay interpretation data. ns not significant (p > 0.05), *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001. HC healthy control, SLT salt-losing tubulopathy. Source data are provided as a Source data file.

Fig 4: Pro-inflammatory and senescent S100A8+ immune cells accumulate in brown adipose tissue with age.a Bioinformatics analysis of scRNA-seq of BAT from young (5-month-old) and aged (27-month-old) rats. b GO analysis of differentially expressed genes in T cells (left) and neutrophils (right) from aged rats. c Violin plots for gene expression of S100a8 in cell populations of BAT from young and aged rats. d Relative cell proportion of S100A8+ and S100A8- cell populations in the BAT of young and aged rats. e Violin plots for gene expression of S100a8 in T cell subclusters of young and aged rats. f UMAP plot shows clustering of macrophages based on gene expression. g Violin plots for gene expression of S100a8 in macrophage subclusters of young and old. h, i Representative flow cytometry plots and quantification of the frequencies of S100A8+ cells in CD45+ CD3+ T cells (h), and CD45+CD11b+ myeloid cells (i), in the stromal vascular fractions (SVFs) of BAT from 2-, 8- and 15-month-old mice (n = 3–5/group). j Representative images of CD3 (green) and S100A8 (red) and staining in the BAT of young (2 months) and aged (15 months) mice. Scale bar, 50 µm. k Representative images of CD11b (green) and S100A8 (red) staining in the BAT of young (2 months) and aged (15 months) mice. Scale bar, 50 µm. Data shown are representative of three independent experiments with similar results. n indicates the number of biologically independent samples examined. Quantitative data are shown as mean ± SEM. Statistical differences were supposed to be significant when P < 0.05. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison test (h, i). Source data are provided as a Source Data File.

Fig 5: Initial investigation of impaired IL-17 responses in salt-losing tubulopathy patients.a Phosphorylation of STAT3 in CD4+ cells after stimulation with IL-6 and IL-21 in SLT patients (n = 13). Phosphorylation is expressed as ratio of upregulation of pSTAT3 in SLT compared to the HC assessed in the same experiment (n = 4 HCs assessed across 4 experiments). Red line drawn at ratio of 1 representing no difference between SLT and HC. b Phosphorylation of STAT1 in CD4+ cells after stimulation with IFNa in SLT patients (n = 13). Phosphorylation is expressed as ratio of upregulation of pSTAT1 in SLT compared to the HC assessed in the same experiment (n = 4 HCs assessed across 4 experiments). Red line drawn at a ratio of 1 representing no difference between SLT and HC. c Th17 and Tc17 polarisation, and supernatant IL-17 concentration in cell culture experiments (11 experiments in 11 healthy controls) with the addition of hydrochlorothiazide (HCT) 20 µM to culture conditions. Each variable is expressed as a ratio to the readout in standard culture conditions; red line drawn at ratio of 1 representing no difference in readouts with the addition of HCT. d Area under the calcium flux curve in healthy controls (n = 13) and Gitelman syndrome patients (n = 5) (as determined using the ratiometric calcium dye Indo-1 gated on CD4+ cells) after T-cell activation with anti-CD3: HC 275.9 AU (265.9–279.9); and Gitelman Syndrome (GS) 277.8 AU (257.5–280.5), p = 0.94. Compared with a two-sided Mann–Whitney test. Error bars represent interquartile range around the median. e Representative calcium flux curves in CD4+ cells after T-cell activation in a healthy control and two patients with GS. ns not significant (p > 0.05), *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, SLT salt-losing tubulopathy, HC healthy control, DC disease control, GS Gitelman syndrome. Source data are provided as a Source data file.