Direct visualization of thymocyte apoptosis in neglect, acute and steady-state negative selection

During thymocyte differentiation, the majority of the developing cells die in situ by apoptosis and are subsequently removed by macrophages, DNA fragmentation is one of the hallmarks of apotosis and can be detected in situ by TdT-mediated dUTP-biotin nick end labeling (TUNEL), We used TUNEL combined with immunohistology to determine the sites of thymocyte apoptosis in mice transgenic for a TCR (F5) which recognizes a peptide (NP68) of the influenza virus nucleoprotein (NP) presented on the MHC class I H-2D(b) molecule, Apoptosis due to neglect was studied in F5 mice expressing a neutral MHC haplotype (F5/H-2(q)) and in beta(2)-microglobulin-deficient F5 mice (F5/beta(2)m(-/-)). In both cases, the frequency of apoptotic cells was similar to that seen in F5/H-2(b) mice and non-transgenic C57BI/10 mice. Antigen-induced apoptosis was studied in F5 mice after i.p. injection of the cognate NP68 peptide and in F5/NP double-transgenic mice, Three hours after peptide injection, apoptosis was high throughout the thymus cortex and clusters of apoptotic cells formed due to tissue macrophage uptake, whereas the thymic medulla remained unaffected. Massive recruitment of inflammatory cells into the thymus was seen as early as 1 h after peptide injection, Nine hours after peptide injection changes were apparent in the cortical epithelium and, by 4 days, the cortical network had collapsed to give scattered, compacted epithelial cells, In contrast, in F5/NP double-transgenic mice, thymocyte apoptosis induced by cognate self-peptide was localized at the cortico-medullary junction with little change seen in the epithelium of the cortex.