In Vivo Differentiated Cytokine-Producing CD4⁺ T Cells Express Functional CCR7

Chemotactic response profile of cytokine producing murine CD4⁺ T cells toward CCL21. The response of pooled splenic CD62L^low CD4⁺ cells in an in vitro chemotaxis assay was measured by flow cytometry. For cytokine analysis input and migrated cells were stimulated for 4 h with PMA and ionomycin and stained intracellularly for IL-4, IFN-γ, and IL-10. Results are expressed as the percentage of cells of the respective subset that migrated to the lower chamber. A, Flow cytometric analysis of input and migrated cells (gated on CD4). B, Response profiles of the different cytokine subsets. Subsets marked “single” are positive for the indicated and negative for the other analyzed cytokines; nonproducers are negative for IL-10, IL-4, and IFN-γ. The means ± SD of four independent experiments are shown.

Chemotactic response profile of cytokine-producing CD4⁺ T cells from spleens of CCR7^−/− and CCR7^+/+ littermates. The responses of pooled splenic CD4⁺ cells of each group in an in vitro chemotaxis assay to CCL21 (A), medium alone (B), and CXCL12 (C) were measured by flow cytometry. Results are expressed as the percentage of cells of the respective subset that migrated to the lower chamber. A–C, For cytokine analysis input and migrated CCR7^−/− and CCR7^+/+ cells were stimulated for 4 h with PMA and ionomycin and intracellularly stained for IL-4, IFN-γ, and IL-10. D, Migration of CD62L^low- vs CD62L^high-expressing CCR7^−/− CD4⁺ T cells toward CCL19 and CCL21. The dotted line indicates the medium control. Data represent the mean ± SD of three independent experiments (A–C) or one representative experiment of three performed (D).

Chemotactic response profile of human cytokine-producing CD4⁺ T cells to CCL19. Human PBMCs from healthy volunteers were subjected to an in vitro chemotaxis assay and analyzed by flow cytometry. For cytokine analysis, input and migrated cells were stimulated for 4 h with PMA and ionomycin and intracellularly stained for IL-4 and IFN-γ. Results are expressed as the percentage of cells of the respective subset that migrated to the lower chamber. A, Flow cytometric analysis of input and migrated cells (gated on CD4⁺and CD3⁺). B, Response profiles of the different cytokine subsets. Each donor was individually analyzed, except the medium control, where equal numbers of cells of each donor were pooled prior to migration. Data represent the mean ± SD of nine volunteers from two independent experiments.

Flow cytometric analysis of CCR7-surface expression of naive and effector/memory CD4⁺ T cells from human peripheral blood and murine spleen. A–C, MACS-sorted CD4⁺ T cells from peripheral blood of healthy volunteers were stained for CD45RA and CCR7. A, Expression of CCR7 on unstimulated naive and memory T cells. B, Effect of PMA/ionomycin stimulation on CCR7 expression. CD4⁺ T cells were stimulated for 4 h with PMA and ionomycin or were mock-treated and stained for CCR7 and CD45RA, fixed, and permeabilized. C, CCR7 expression and intracelular cytokine staining after stimulation, separately given for CD45RA⁻ (upper panels) and CD45RA⁺ (lower panels) cells. D–F, Murine CD4⁺ T cells from BALB/c wild-type or CCR7-deficient animals were stained (D) or MACS-sorted (E and F) for CD4, and binding of CCL19-Fc was tested. D, CCL19-Fc binding and CD62L expression of CD4⁺T cells from spleens of BALB/c mice. E, CD4⁺ T cells from CCR7-wild-type and -deficient animals were stimulated for 4 h with PMA and ionomycin or mock-treated, stained with CCL19-Fc, fixed, and permeabilized and (F) additionally stained intracellularly for IFN-γ. Data are representative for eight individually analyzed donors in three independent experiments (A–C) and three independent experiments with pooled spleen cells of two or three animals per group (D–F).