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-gamma to the CD4+CD8+ cell cultures failed to yield a DNA cleavage similar to that of unseparated thymocytes.

Murine Macrophages Produce Secretory Leukocyte Protease Inhibitor During Clearance of Apoptotic Cells: Implications for Resolution of the Inflammatory Response

“…Preferential apoptosis of the CD4 ϩ CD8 ϩ thymocyte subset by anti-CD3 Ab has been well-documented in vivo as well as in vitro (31)(32)(33)(34). Previous studies indicate that thymic macrophages are the principal stromal cells responsible for phagocytosis of dying thymocytes and that the number of TUNEL-positive thymocytes declines to the original basal level after apoptotic cells are phagocytosed by macrophages in the thymus following administration of anti-CD3 Ab or hydrocortisone (28,35).…”

Macrophages are involved in DNA degradation of apoptotic cells in murine thymus after administration of hydrocortisone

“…29 In brief, thymocytes were lysed in the buffer containing 10 mM Tris-HCl (pH 8.0), 0.5% N-lauroylsarcosine and 0.1 M EDTA, and treated with 100 mg/ml proteinase K and 50 mg/ml RNase A. The DNA was extracted by phenol/chloroform and precipitated in isopropyl alcohol containing 0.5 M NaCl.…”

Susceptibility to cell death is a dominant phenotype: triggering of activation-driven T-cell death independent of the T-cell antigen receptor complex.

“…Stimuli that are normally mitogenic for T cells can induce cell death in transformed T-cell hybridomas (1,20,21,23,26,33) and in untransformed T cells (19,30,34). This process, activation-driven cell death, can be triggered by the presentation of appropriate antigen as well as by treatment with lectins and antibodies specific for the T-cell receptor complex (TCR) or other activation structures on the T-cell surface, such as Thy-1 and Ly-6 (20,21,33).…”

“…Negative selection in vivo has been shown to act preferentially on immature CD4+ CD8+ T cells (7,13). In thymic organ culture (6,27) and in vitro (19,30), these double-positive cells also appear to be more sensitive to TCR-mediated activation-driven cell death than are more mature T cells. Conversely, while mature T cells are susceptible to activation-driven cell death triggered via phosphatidylinositol-linked membrane proteins Thy-i and Ly-6 (34), it has been reported that stimulation via Thy-i does not induce T-cell death in thymic organ cultures (27).…”