IN-SITU DETECTION AND CHARACTERIZATION OF APOPTOTIC THYMOCYTES IN HUMAN THYMUS - EXPRESSION OF BCL-2 IN-VIVO DOES NOT PREVENT APOPTOSIS

Apoptosis plays a crucial role in shaping the T cell repertoire during T cell development in the thymus. The observed disappearance in the t hymus 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 developmen t has also not been reported. Using three-color immunofluorescence and flow cytometric assays on frozen human thymic tissue and freshly isol ated human thymocytes respectively, we directly identify CD4(+) CD8(+) and CD4(-) CD8(-) thymocytes in newborn human thymus that contain int racellular fragmented DNA and are therefore apoptotic. We determine th at 75% of the apoptotic thymocytes are CD4(+) CD8(+) double positive a poptotic thymocytes, and interestingly, that 13% are CD4(-) CD8(-) dou ble negative thymocytes. The majority of apoptotic thymocytes in situ are detected at the cortical-medullary junction; however, apoptotic th ymocytes are also found scattered throughout the cortex. Furthermore, we determine that within the apoptotic thymocyte population, 54% expre ss 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 apopt otic process, and that expression of the bcl-2 protein in situ does no t prevent immature thymocytes from apoptosis.