title: In situ detection and characterization of apoptotic thymocytes in human thymus. Expression of bcl-2 in vivo does not prevent apoptosis.
abstract: 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.
authors.affiliation: Department of Cell Biology, Loyola University Medical Center, Maywood, IL 60153, USA.
authors.forename: P T
authors.initials: PT
authors.lastname: Le
authors.forename: H T
authors.initials: HT
authors.lastname: Maecker
authors.forename: J E
authors.initials: JE
authors.lastname: Cook
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D007231:Infant, Newborn; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000502:physiology*; D013950:Thymus Gland / Q000166:cytology*
chemical_list: D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2