Fig 2: Effect of altering extracellular ion concentration within a physiological range on CD4 subset balance.a Effect of NaCl (0–40 mM), KCl (0–2 mM), and MgCl2 (0–0.8 mM) during T-cell activation with anti-CD3/anti-CD28 on the ratio of expression of IFN?:IL-17 in CD4+ cells, representative of Th1:Th17 cells. Data shown represent dose–response in a healthy control. Mean of technical duplicates is plotted. b Effect of NaCl (0–40 mM), KCl (0–2 mM), and MgCl2 (0–0.8 mM) during T-cell activation with anti-CD3/anti-CD28 on the ratio of expression of IL-4:IL-17 in CD4+ cells, representative of Th2:Th17 cells. Data shown represent dose–response in a healthy control. Mean of technical duplicates is plotted. c Representative FACS dot plots of the effect of +40 mM NaCl on the expression of IFN?, IL-4, and IL-17 in CD4+ cells from a healthy control. Cell proportions as percentages are documented within each quadrant. d CD4+ subset balance after T-cell activation with anti-CD3/anti-CD28 in the presence of NaCl (40 mM), KCl (2 mM), and MgCl2 (1 mM) used alone and in combination. Data show subset balance reported as a ratio to standard conditions in healthy controls (n = 2). The median ratio is plotted. Lines drawn at a ratio of 1 represents no difference in subset balance in the presence of ions compared to standard conditions. Source data are provided as a Source data file.

Fig 3: 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.

Fig 4: CD20dim CD4+ T cells involve memory phenotypes with more activation than CD20- cells. PBMC from uninfected donors, ART-suppressed patients and viremic patients were stained with CD20, activation markers (HLA-DR, CD69, CD25), and T-cell differentiation markers (CCR7, CD45RO, CD27, CD95). a Gating strategy used to identify CD20dim CD4+ T cells. Previous sequential gates are represented in Supplementary Fig. 9. Viable CD4+ T cells (identified by CD3 and CD4 expression) were first selected for the analysis of CD20 expression, defined by its Fluorescence Minus One (FMO) control. T-cell memory subsets were selected as follows: CD4+ TNA (CCR7+, CD45RO-, CD27+, CD95-), CD4+ TSCM (CCR7+, CD45RO-, CD27+, CD95+); CD4+ TCM (CCR7+, CD45RO+), CD4+ TTM (CCR7-, CD45RO+, CD27+); CD4+ TEM (CCR7-, CD45RO+, CD27-), CD4+ TTD (CCR7-, CD45RO-). b, c Representative bright-field and pseudo-color fluorescence images of CD20dim CD4+ T cells (b) and B cells (c) from two ART-suppressed patients (#9 and #22) using the Amnis imaging flow cytometer technology. Scale bar 10 µm. d Percentage of CD20dim expression within CD4+ T cells in uninfected controls and the two patient cohorts. Mann–Whitney comparison was used to compare n = 6 uninfected controls, n = 21 ART-suppressed patients, n = 20 viremic patients. e Expression of the activation markers CD25, CD69 or HLA-DR in CD20dim and CD20- CD4+ T cells in different cohorts of HIV+ patients. Comparisons were performed using the two-tailed Wilcoxon test (n = 21 ART-suppressed patients and n = 20 viremic patients). f Distribution of CD20dim in CD4+ T-cell subsets in ART-suppressed (orange) and viremic (red) HIV-infected patients. NA, naive; SCM, memory stem cell; CM, central memory; TM, transitional memory; TD, terminal differentiated. ANOVA and Dunn’s multiple comparison test were performed, n = 10 ART-suppressed and n = 9 viremic patients. Median values and min and max ranks are represented in panels d–f. g Correlation of CD20dim CD4+ T cells with time on suppressive ART (left), CD4+ T-cell counts (middle) and plasma viral load (right) are shown. ART-suppressed patients in orange and viremic patients in red. Spearman’s nonparametric correlation coefficients and associated P values are shown. Panels (d) and (e) included patients #2–10, 15–26, 60–65, 67–74, and 76–81. Panel (f) patients #1–10, 60–65, and 67–69. Panel (g) patients #2–10, 15–26, 60–65, 67–74, and 76–81. Data underlying this Figure are provided as Source Data file

Fig 5: CD20 expression in T cells of intact human lymph nodes. Paraffin-embedded lymph node sections from HIV-infected patients were stained with anti-CD20 combined with anti-CD3 (a) or anti-p24 antibodies (b), alternatively sections were proceed for in situ RNA-hybridization of CD20-mRNA or mRNA and HIV RNA (c). a Representative micrograph of a lymph node section from one viremic HIV+ patient stained with anti-CD20 (green) and anti-CD3 (violet) antibodies. The discontinuous line indicates a B-cell follicle and the white arrows double positive cells. Right panels correspond to zoomed views from different patients. b Upper panel shows the number of cells per mm2 expressing CD20+ and CD3+ in lymph nodes (circles, each dot corresponds to one follicle) and in the extrafollicular region (triangles, each dot corresponds to one optical field). Two HIV-viremic (#84 and 85, in red) and two ART-suppressed patients (#31 and 32, in orange) are shown. Comparison between values inside B-cell follicles from viremic and ART-suppressed patients was performed using a Mann–Whitney test, and comparison within viremic 1 was performed with the Wilcoxon test. Lower panel shows the percentage of CD3+ cells within the B-cell follicle expressing CD20. Each dot corresponds to one follicle. Medians and min–max rank are represented. c Representative micrograph from a lymph node section from one HIV-viremic patient stained with anti-CD20 (green) and anti-p24 (violet) antibodies. High magnification images correspond to B-cell regions from different ART-suppressed and viremic patients, white arrows indicate double positive cells and the discontinuous line indicates B-cell follicle. Patients #31 and 82–84 are shown. d Representative image of a B-cell follicle section from one viremic patient (#82) subjected to the in situ hybridization technique. CD20-mRNA is represented in green and HIV RNA in violet. At the middle, zoomed images of double positive cells (white arrows) or HIV RNA single positive cells (orange arrows). Data underlying this Figure are provided as Source Data file