Abstract
Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD+8+ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.
Highlights
Intrathymic T-cell development is the consequence of an orderly series of molecular events initiated by contact between bone marrow derived precursors and thymic stromal cells
TCR ligation effectively reduces CD4 and CD8 levels while increasing CD5 and CD69 expression even when apoptosis is not a consequence of cross-linking the TCR complex (Page et al, 1993; Swat et al, 1993; Kearse et al, 1995). These findings suggest that a TCR-mediated signal transduction pathway is operative in DP thymocytes regardless of whether the signal culminates in DNA fragmentation and cell death
Thymocytes from female H-2b H-Y transgenic mice were cultured for various intervals in the presence or absence of plate-bound anti-TCR( chain antibody (T3.70) to determine whether signaling through the TCR alone could induce phenotypic alterations and/ or apoptosis
Summary
Intrathymic T-cell development is the consequence of an orderly series of molecular events initiated by contact between bone marrow derived precursors and thymic stromal cells. Some studies conclude that antibody-mediated TCR cross-linking alone is sufficient to stimulate DNA fragmentation and/or cell death (McConkey et al, 1989a; Carlow et al, 1992; Migita et al, 1994), other groups report that anti-CD3-stimulated DP thymocytes fail to undergo apoptosis in the absence of APCs, suggesting that, in addition to TCR engagement, a second signal is required fo induce apoptosis in the DP subset (Page et al, 1993; Punt et al, 1994; Kearse et al, 1995). TCR ligation effectively reduces CD4 and CD8 levels while increasing CD5 and CD69 expression even when apoptosis is not a consequence of cross-linking the TCR complex (Page et al, 1993; Swat et al, 1993; Kearse et al, 1995) These findings suggest that a TCR-mediated signal transduction pathway is operative in DP thymocytes regardless of whether the signal culminates in DNA fragmentation and cell death. Similar experiments carried out with lpr and gld mutant mice demonstrate that Fas/FasL interactions are not responsible for CsA-dependent depletion of TCRactivated DP thymocytes
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