Impaired negative selection of CCR7-deficient thymocytes in vivo. Nine-week-old C57BL/6 (+/+) and CCR7^−/− (−/−) mice were intravenously injected with PBS or 200 μg of anti-CD3 mAb. Forty-two hours after injection, thymocytes were quantified, stained with antibodies to CD3, CD4, CD24, and CD8, and analyzed by flow cytometry. CD24 expression was analyzed to identify the semimature CD4⁺CD8⁻CD24^high population undergoing negative selection after TCR ligation. (A) Proportions of CD4 SP and DP cells in the thymus of mice injected with PBS and mice injected with anti-CD3 mAb, demonstrating the depletion of the DP population in wild-type but not in mutant animals. (B) Proportions of CD4⁺CD8⁻CD24^high cells in the thymi of mice injected with anti-CD3 mAb. Note that in contrast to wild-type mice, only a minority of CD4⁺CD8⁻CD24^high cells have been depleted in CCR7-deficient animals after antibody administration. (C) Bone marrow of CD45.2 CCR7^−/− mice was mixed with bone marrow of CD45.1 wild-type mice at ratio of 1:1 and transferred into irradiated CD45.1 wild-type recipients. Lack of depletion of CCR7-deficient DP and CD4⁺CD8⁻CD24^high cells in a wild-type thymus environment indicates that an intrinsic defect in these thymocytes is responsible for the resistance of these cells to undergo depletion after TCR stimulation.