Impaired responsiveness of CCR7-deficient thymocytes to TCR stimulation in vitro. (A) Thymocytes of DO11.10/CCR7^+/+ (+/+) and DO11.10/CCR7^−/− (−/−) mice were coincubated with either unloaded or OVA-loaded DCs. Cells were harvested after 18 hours of incubation and analyzed by flow cytometry. CCR7-deficient thymocytes were less responsive to TCR stimulation as indicated by the lower number of DP cells with decreased expression of CD4 and CD8 after stimulation. (B) Thymocytes of wild-type (+/+) and CCR7-deficient (−/−) mice were incubated for either 12 or 24 hours in the absence (unstimulated) or in the presence (stimulated) of antibodies to CD3 and CD28. Similar expression levels of CD69 were observed in unstimulated thymocytes of both strains. Although TCR stimulation induces up-regulation of CD69 on thymocytes of both strains, mutant thymocytes respond more slowly and less efficiently (→). For panels A and B, pseudocolor density plots are gated on living thymocytes according to forward and side scatter (FSC/SSC) profile, numbers indicate the percentage of cells in the specified regions. (C) Impaired responsiveness of CCR7-deficient thymocytes to CD3/CD28 sti-mulation is also reflected by delayed kinetic of CD5 up-regulation and TCR down-regulation. … indicates unstimulated; ——, stimulated. Data shown are representative of 3 independent experiments.