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. 2020 Jul 27;130(9):4587–4600. doi: 10.1172/JCI137786

Figure 1. NaCl promotes the Th17 cell program in human effector memory Th cells independently of polarizing cytokines.

Figure 1

(A–C) Human effector memory Th cells were FACS sorted from fresh human PBMCs as CD4+CD14–CD45RA–CCR7– T cells and stimulated for a total culture period of 5 days in low- or high-NaCl conditions with CD3 and CD28 mAbs (48 hours plate-bound). (A) Intracellular staining and FACS on day 5 after PMA and ionomycin restimulation for 5 hours. FACS staining of an individual experiment (left) and cumulative data are shown. Each circle indicates an individual donor. gMFI, geometric MFI; max, maximum. (B) ELISA analysis of cell culture supernatants analyzed on day 5 after stimulation with PdBU and CD3 mAb for 8 hours (n = 3). (C) Transcriptome analysis and GSEA (GSE52260) of genes related to the Th17 signature in Th17 cells stimulated as in A (67). (D) Skin CD3+ T cells were isolated from human abdominal skin by overnight collagenase digestion followed by FACS sorting. The cells were stimulated for 48 hours with CD3 and CD28 mAbs in low- or high-NaCl conditions followed by intracellular cytokine staining after PMA and ionomycin restimulation. A representative experiment (left) and cumulative data are shown (middle). ELISA analysis (right) of cell culture supernatants from skin CD3+ T cells restimulated with PdBU and CD3 mAbs for 8 hours after 48 hours of CD3 and CD28 mAb stimulation in low- and high-NaCl conditions. Data were normalized to 20,000 T cells. Each circle indicates an individual donor. (E) FACS analysis performed as in A in the absence or presence of Th17-polarizing cytokines. The data are representative of 3 donors. (A, B, and D) A 2-tailed, paired Student’s t test was performed for comparisons between 2 groups.