Apoptosis plays a crucial role in shaping the T cell repertoire during T cell development in the thymus. The observed disappearance in the thymus of CD4+ CD8+ thymocytes with a specific TCR, and the lack of CD4+ or CD8+ single positive mature cells expressing the same TCR specificity in the periphery have led to the conclusion that deletion occurs at the CD4+ CD8+ double positive stage; however, there is no direct evidence demonstrating apoptotic CD4+ CD8+ cells in situ. Apoptosis of thymocytes in situ at other stages of T cell development has also not been reported. Using three-color immunofluorescence and flow cytometric assays on frozen human thymic tissue and freshly isolated human thymocytes respectively, we directly identify CD4+ CD8+ and CD4- CD8- thymocytes in newborn human thymus that contain intracellular fragmented DNA and are therefore apoptotic. We determine that 75% of the apoptotic thymocytes are CD4+ CD8+ double positive apoptotic thymocytes, and interestingly, that 13% are CD4- CD8- double negative thymocytes. The majority of apoptotic thymocytes in situ are detected at the cortical-medullary junction; however, apoptotic thymocytes are also found scattered throughout the cortex. Furthermore, we determine that within the apoptotic thymocyte population, 54% express the apoptotic regulatory protein bcl-2 in vivo, whereas 32% are bcl-2 negative. Thus, our in vivo data directly demonstrate that both CD4+ CD8+ and CD4- CD8- human thymocytes die in situ via an apoptotic process, and that expression of the bcl-2 protein in situ does not prevent immature thymocytes from apoptosis.

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