Spontaneous apoptosis in human thymocytes.

Apoptosis seems to be involved in different stages of immune cell development. In particular, experimental evidence suggests that it is a major form of cell death in the thymus. The present analysis of human thymocytes reveals that a fraction of these cells, cultured in vitro, undergoes spontaneous apoptosis. This observation is based both on molecular (DNA fragmentation) and morphological (electron microscopic) investigations of the cells. The apoptotic thymocytes are CD3- or CD3lo, CD4lo, and CD8lo and do not express Bcl-2 protein. Furthermore, thymocytes die by apoptosis when exposed to pharmacological stimuli, such as tumor necrosis factor-alpha, dexamethasone, ATP, or Ca++ ionophore. Thus the apoptotic machinery in thymocytes can be triggered by an imbalance in growth factors in the in vitro culture media and can be modulated by various biochemical signals. The process of spontaneous apoptosis is independent of mRNA or protein synthesis, as actinomycin D and cycloheximide fail to inhibit this phenomenon. Furthermore, apoptosis seems to require active oxidative phosphorylation, as it is prevented by incubation of the cells with inhibitors of the respiratory chain.

A novel 120-kDa antigen shared by immature human thymocytes and long-term-activated T cells.

In this study we report the characterization of monoclonal antibody (mAb) 8B4/20, raised against immature human thymocytes, that identifies a novel leukocyte antigen. The molecular characterization of the antigen by immunoprecipitation and immunoblotting yields, under nonreducing conditions, a specific band of 120 kDa which, under reducing conditions, displays a slightly lower molecular mass (110 kDa. mAb 8B4/20 detects a molecule found on the majority of thymocytes with an inverted gradient of expression when compared to CD3. It appears at high density on the CD3-/low thymocytes, at reduced density on the CD3med and double-positive thymocytes, and is absent on CD3hi and single-positive thymocytes and on peripheral blood T cells. Immunohistochemistry on frozen sections demonstrates cortical staining of the thymic lobules. Flow cytometric analysis of the different subsets of peripheral blood mononuclear cells shows that mAb 8B4/20 detects an antigen expressed only on CD56+/CD16+ natural killer cells and on a fraction of CD14+ monocytes. T cells, B cells, erythrocytes, granulocytes and platelets are consistently negative. The expression of the molecule on tumor cell lines does not show lineage restriction. Analysis of phytohemagglutinin plus recombinant interleukin-2-activated peripheral blood lymphocytes shows that mAb 8B4/20 identifies an antigen expressed on CD3+ cells by week 3 of culture. Thus, it recognizes a very late activation antigen (VLA) on mature T cells. The cell distribution and the electrophoretic pattern of the molecule identified by mAb 8B4/20 is distinct from that of known CD and of integrin/VLA molecules. Its function on thymocytes is so far unknown; however, the binding of mAb 8B4/20 to tumor lines induces changes in the morphology and adhesive properties of the 8B4/20+ cells growing in suspension. We suggest that mAb 8B4/20 recognizes a molecule that may be involved in interactions between thymocytes and other thymic structures that may be relevant for the selection process.