CD4+CD8+ thymocytes are susceptible to DNA fragmentation induced by phorbol ester, calcium ionophore and anti‐CD3 antibody

Abstract

Stimulation of murine thymocytes with phorbol ester or calcium ionophore for 18–24 h resulted in 70%–80% fragmentation of DNA into 180–200-bp multiples, followed by cell death. Experiments with fractionated subpopulations by panning or flow cytometry revealed that DNA fragmentation was selectively observed in CD4⁺CD8⁺ cells and in a portion of CD4⁻CD8⁺ cells. To investigate whether DNA cleavage is also inducible via antigen-specific receptors, thymocytes were incubated in wells precoated with anti-CD3 antibody. An approximately 20% increase of DNA fragmentation was constantly observed when unseparated thymocytes were stimulated with anti-CD3 antibody. In this anti-CD3-induced DNA degradation, CD4⁺CD8⁺ cells are probably the target cells, since (a) fetal thymocytes at day 18 of gestation were found vulnerable to anti-CD3-induced DNA cleavage and (b) flow cytometry analysis of viable cells recovered after cultivation in the anti-CD3-coated wells revealed that CD4⁺CD8⁺ cells were preferentially decreased. Further experiments with purified CD4⁺CD8⁺ cells, however, could not define a clear-cut increase of DNA fragmentation when isolated CD4⁺CD8⁺ cells were stimulated with anti-CD3 antibody. Addition of interleukin (IL) 1, IL 2, IL 3, IL 4 or interferon-γ to the CD4⁺CD8⁺ cell cultures failed to yield a DNA cleavage similar to that of unseparated thymocytes.