MDC acts directly on CCR4 expressing thymocytes. (A) The migration of thymocytes is MDC-dependent. Freshly isolated thymocytes from 3- to 6-week-old BALB/c mice were assayed for chemotaxis in response to murine MDC as follows. Thymocytes were cultured for 4 hours in the presence of 50 nmol/L MDC, and cells were removed by centrifugation. The resulting MDC-conditioned media was then used to stimulate the chemotaxis of fresh thymocytes in the presence or absence of a neutralizing antibody to MDC. The number of cells migrating was determined by FACS analysis. All assays were performed on samples in triplicate; data shown are the mean ± SEM and are representative of 3 independent experiments. (B) Expression of CCR4 by cells that had migrated in response to MDC was determined by RT-PCR.