Functional responsiveness of CCR4⁺ T cells to the murine CCR4 ligand MDC. (a) Chemotactic response of CCR4⁺CD4⁺ T cells to murine chemokines. The migration of FACS-sorted CCR4-negative and -positive BDC2.5 CD4⁺ T cells to murine MDC (15 ng/ml) was studied in a Transwell assay. The assay was also carried out in the presence of anti-human CCR4 and isotype control mAb’s (left). In addition, the chemotactic function of the various murine chemokines MDC, TARC, SLC, and MCP-1 was evaluated on CCR4-expressing BDC2.5 CD4⁺ T cells (right). Data representing the percentage of migrating sorted cells (left) or CCR4⁺CD4⁺ T cells are expressed as the mean of triplicates ± SEM. Left graph: black bar, CCR4^–; white bar, CCR4⁺; light gray bar, CCR4⁺/IgG1; dark gray bar, CCR4⁺/anti-CCR4 mAb. Right graph: black bar, murine MCP-1; white bar, murine MDC; light gray bar, murine TARC; dark gray bar, murine SLC. These data indicate that the human CCR4 mAb cross-reacts with murine CCR4. (b) Expression of CCR4 and CCR5 and activation molecules CD25 and CD69 in leukocyte subsets 8-week-old NOD/shi mice and their modulation through the activation or polarization of BDC2.5 CD4⁺ T cells. (c) Cytokine profile of polarized Th subsets. To assess the polarization of Th1 and Th2 cells in vitro, we have examined the double intracellular cytokine staining of the polarized Th effectors generated by short-term culture of highly purified naive BDC2.5 T cell populations. We demonstrated that Th1 and Th2 cells were well polarized and differentiated (n = 5).