FIG 2.

CD161⁺ CD4⁺ T cells are primarily of the memory phenotype with typical Th17 and pTFH characteristics. (A) Percentage of CD161-positive and -negative cells in different CD4⁺ T cell subsets and composition of CD161-positive and -negative CD4⁺ T cells among healthy individuals (n = 15). T_N, naive T cells; T_TD, terminally differentiated T cells; T_CM, central memory T cells; T_EM, effector memory T cells; T_TM, transitional memory T cells. (B and C) Representative FACS plots depicting the chemokine receptor profile, including CXCR5, CCR6, CCR4, and CXCR3 expression, on CD161⁻ CD4⁺ (left) and CD161⁺ CD4⁺ (right) T cells (B). Cumulative results from 6 independent experiments are shown in panel C. SSC, side scatter. (D) Cytokine production profile. PBMCs from healthy donors (n = 14) or HIV-1-infected subjects receiving ART for more than 2 years (n = 8) were stimulated with phorbol-12-myristate-13-acetate and ionomycin in the presence of brefeldin A for 8 h. Dot plots comparing cytokine production among CD161-positive or -negative CD4⁺ T cells. IFN-γ, gamma interferon, TNF-a, tumor necrosis factor alpha. (E and F) Frequency and CD161 expression levels of Th1, Th2, Th17, and Th1Th17 cells. CD45RO⁺ CD4⁺ T cells from healthy donors (n = 8) were analyzed for their differential expression of CCR6, CCR4, and CXCR3. The CCR6⁺ subsets included the CCR4⁺ CXCR3⁻ (Th17) and CCR4⁻ CXCR3⁺ (Th1Th17) subsets. The CCR6⁻ subsets included the CCR4⁺ CXCR3⁻ (Th2) and CCR4⁻ CXCR3⁺ (Th1) subsets. (G) CXCR5 and IL-21 expression in CD161-positive and -negative cells. CXCR5 expression was measured by FACS. For IL-21 measurement, FACS-purified CD161-positive or -negative CD4⁺ T cells from healthy donors were stimulated by anti-CD3/CD28 for 3 days, and the IL-21 mRNA level (n = 4) was measured by RT-qPCR as the level of expression relative to that for GADPH. IL-21 (n = 6) secretion was analyzed by intracellular staining as described in the legend to D. P values were determined by the Mann-Whitney test or Student's t test. The mean ± SEM is shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.