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. 2014 Jan 13;211(1):89–104. doi: 10.1084/jem.20130301

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© 2014 Ramesh et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — Th17 cytokines and IL23R are independently regulated in human T cell subsets. (A) CD4⁺CD25⁻ memory (CD45RO⁺) T cell subsets from healthy adult donor peripheral blood were analyzed by flow cytometry. CCR6⁺ or CCR6⁻ cells were gated as CCR7^hi (central memory; T_CM), CCR7^int (CCR7-intermediate), or CCR7^lo (effector memory; T_EM) cells, and CCR4 and CXCR3 expression was analyzed. Data represent >20 stains performed on individual donors or donor pools. (B) FACS-sorted T_CM (CCR7^hi) or T_EM (CCR7^lo) subsets were stimulated with PMA and ionomycin (P + I) and production of IFN-γ and IL-17A was determined by intracellular cytokine staining. Th1, CCR6⁻CCR4^loCXCR3^hi; Th2, CCR6⁻CCR4^hiCXCR3^lo; Th17, CCR6⁺CCR4^hiCXCR3^lo; Th17.1, CCR6⁺CCR4^loCXCR3^hi. Representative flow cytometry plots from 3 experiments performed on different pools of healthy adult donor blood are shown, and each donor pool contained blood from 2–4 individual donors. (C) IFN-γ and IL-17A production by FACS-sorted T_CM or T_EM cells was determined by intracellular cytokine staining as in B on 3 different pools of healthy adult donor peripheral blood. Each donor pool contained blood from 2–4 different donors. Individual and mean percentages of IL-17A⁺ (left), IFN-γ⁺ (middle), or IL-17A⁺/IFN-γ⁺ (right) T cells are shown, and data from each donor pool is color-coded. *, P < 0.05 by paired Student’s t test. (D) T_EM Th1, Th2, Th17, or Th17.1 subsets were FACS-sorted as in B from two different pools of healthy donor peripheral blood. Both donor pools contained blood from 2–4 individual donors. Sorted cells were stimulated with anti-CD3/anti-CD28 for 72 h and expression of the indicated genes was measured by nanostring. Data are shown as fold change in gene expression within each donor pool; data from the two donor pools are color-coded. Horizontal bars in C and D represent the mean values. (E) Expression of pathogenic (red) or nonpathogenic (blue) murine Th17-signature genes (Lee et al., 2012) was determined in T_EM Th17 and Th17.1 cells by nanostring as in D. Data are shown as mean relative (Log₂ fold change) mRNA expression ± SD from 2 experiments on cells from different pools of healthy adult donor blood as in D. (F) T_EM Th1, Th2, Th17, or Th17.1 cells (FACS-sorted as in B) were stimulated with anti-CD3/anti-CD28 and cultured for 6 d with or without IL-23. Cells were then restimulated with PMA and ionomycin and IFN-γ and IL-17A expression was determined by intracellular cytokine staining and FACS analysis. Data represent 3 experiments performed on independent donor pools, with each pool containing blood from 2–4 individual donors.