Figure 1. CCR5 segregates Th17 cells into IFNγ⁻IL-17⁺ and IFNγ⁺IL-17⁺.

(a) Resting CD4⁺, CD45RO⁺, CCR6⁺ or CCR6⁻ T cells were purified as described (methods) and stained with CCR5-PE or CXCR4-PerCP-cy5.5 antibodies (blue) or isotype-matched controls (red). (b) Percent of memory CCR5⁺ cells in CD4⁺ cells stained for CCR5 and CCR6. Each circle is representative of one adult healthy donor. (c) Resting memory CD4⁺ T cells were stained with CCR6-biotin and CCR5-PE (d) Stained cells in (c) were sorted into four memory (CD45RO⁺) subsets: CCR6⁺CCR5⁻, CCR6⁺CCR5⁺, CCR6⁻CCR5⁺, and CCR6⁻CCR5⁻. To detect IL-17 and IFNγ producing cells; we activated sorted subsets using PMA, Ionomycin and Golgi stop. Cells were fixed, permeabilized and stained with IFNγ-PE-cy7 and IL-17-FITC antibodies. An isotype-matched control did not show any background staining (data not shown). (e) Percent of IL-17⁺, IFNγ⁺ and IFNγ⁺IL-17⁺ in memory CCR6⁺CCR5⁻, CCR6⁺CCR5⁺, CCR6⁻CCR5⁺, and CCR6⁻CCR5⁻ T cell subsets. Each symbol represents one adult healthy donor. P values were calculated using the Two-Sample Student’s t-test.