Human memory T cells (TM cells) that produce IL-17 or IL-22

Human memory T cells (TM cells) that produce IL-17 or IL-22 are currently defined as Th17 or Th22 cells, respectively. higher in CCR6+IL-17+ cells compared with either CCR6? or CCR6+IL-17? cells, irrespective of the activating stimuli (i.e., aCD3/aCD28 or PMA and ionomycin). However, both CCR6+ T cell subsets displayed elevated expression levels that ranged between 80- and 400-fold higher than those observed in CCR6? TM cells (Fig. 1 B and Fig. S1 B; Singh et al., 2008). PLA2G4C Given that was highly expressed in CCR6+ TM cells independent of ex vivo IL-17 production, we next cultured these T cell subsets in IL-2Csupplemented medium for 6 d to ask whether CCR6+IL-17? cells could up-regulate IL-17 expression. As expected, a large majority of cells initially sorted as CCR6+IL-17+ maintained high-level IL-17 expression upon restimulation, whereas CCR6?IL-17? cells remained largely IL-17 negative (Fig. 1 C and Fig. S1 C). Remarkably, 20C40% of the CCR6+ cells initially sorted as IL-17? expressed IL-17 after culture with IL-2 (Fig. 1 C and Fig. S1 C). The appearance of IL-17Cproducing T cells within ex vivoCisolated CCR6+IL-17? cultures was not a result of selective Rucaparib outgrowth of residual IL-17+ cells, as FACS-sorted CCR6+IL-17? and CCR6+IL17+ cells proliferated equally well in response to IL-2 stimulation, as Rucaparib seen by fluorescent dye dilution and mixed co-culture experiments (Fig. S1, DCG). Figure 1. De novo expression of IL-17 in CCR6+IL-17? human Rucaparib TM cells is induced by c-cytokines. (A) Total CD4+ TM cells (CD45RO+CD25?) were stimulated for 18C24 h with aCD3/aCD28 beads and then were FACS sorted into CCR6? … A more comprehensive analysis of cytokine gene expression by human CCR6+IL-17? and CCR6+IL-17+ cells revealed that these two cell types were nearly indistinguishable after culture with IL-2. Specifically, several proinflammatory cytokines canonically associated with the Th17 lineage Rucaparib ((Fig. 1 D). Given that IL-2 is the prototype of the IL-2 family of cytokines, all of which signal through cytokine receptors comprised in part by the c subunit, we next asked whether other c-cytokines could also induce de novo IL-17 production by CCR6+IL-17? TM cells. Both IL-7 and IL-15 induced similar levels of IL-17 expression in CCR6+IL-17? T cells, whereas IL-23, which is known to enhance Th17 cell differentiation (Ivanov et al., 2007), did not influence IL-17 expression in the absence of IL-2 (Fig. 1 E). In contrast, and as observed for IL-2, Rucaparib neither IL-7 nor IL-15 induced IL-17 expression in CCR6? TM cells. In addition, CCR6+, but not CCR6?, IL-17? TM cells isolated from peripheral lymphoid organs of wild-type C57B/6 mice were capable of producing IL-17 after 6 d in culture with IL-2 (Fig. S2). These findings suggest that ex vivo analyses of IL-17 expression underestimate the frequency of TM cells that can express IL-17 in inflammatory settings. Because several studies have investigated changes in Th17 frequencies within autoimmune patient cohorts, we asked whether CCR6+IL-17? TM cells isolated from the peripheral blood of patients with RA could be similarly induced to express IL-17 by IL-2 stimulation. Indeed, we observed that CCR6+, but not CCR6?, IL-17? TM cells from RA patients up-regulated IL-17 after culture with IL-2 to similar levels as those observed in healthy adult donors (Fig. 1 F). Collectively, these data demonstrate that CCR6+ TM cells are uniquely poised to express IL-17 in response to IL-2 stimulation irrespective of their IL-17 phenotype ex vivo. These results also indicate that this inflammatory feature of CCR6+ TM cells is conserved between humans and mice. IL-17 induction in response to c-cytokine stimulation is conserved in heterogeneous CCR6+ TM cell subsets Human CCR6+ Th17 cells have been reported to be enriched within CXCR3? or CD161+ subsets as cells of these subphenotypes produce more.

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