Cell cycle regulation of T cell receptor activation-induced cell death

Strong stimulation of the T Cell Receptor (TCR) on cycling, peripheral T cells leads to negative selection by apoptosis, termed TCR-Activation-Induced Cell Death (TCR-AICD), that deletes potentially harmful self-reactive T cells. Studies were undertaken to examine a potential cell cycle specificity for this process. By analyzing cell cycle profiles, the status of cell cycle regulated proteins, BrdU labeled cells, and elutriated G₁ and G₂/M phase Jurkat T cells, it appeared that apoptosis preferentially occurred from the late G₁ phase. Cyclin E protein levels failed to be induced and cyclin-dependent kinase (Cdk) 2 failed to be activated, indicating a failure of the cells to transit the late G₁ Restriction Point. Also, the retinoblastoma tumor suppressor protein, pRB, was in its active, hypo-phosphorylated conformation that is only present in the G₁ phase. To test the requirement for pRB and the late G₁ position during TCR-AICD, the method of TAT-mediated direct protein transduction was used to introduce the HPV E7 oncoprotein into cells, to sequester and inactivate pRB, and the p16 tumor suppressor protein, to cause an early G₁ arrest and block progression to the late G₁ position. Both of these treatments protected TCR-stimulated cells from undergoing apoptosis and demonstrated the requirements for both active pRB and the late G₁ position in executing TCR-AICD.; The E2F-1 transcription factor is a direct target to pRB. Mice carrying a nullizygous deletion of E2F-1 suffer from splenomegaly and an excess of peripheral T cells. To examine a role for E2F-1 in TCR-AICD, a dominant negative E2F-1 protein was introduced by TAT transduction into cells stimulated to undergo apoptosis. In both Jurkat T cells and primary murine T cells, transduction of this protein protected cells from TCR-AICD. In addition, splenocytes from E2F-1 nullizygous mice were defective in their ability to undergo apoptosis, confirming the cell culture results.; TCR-AICD has previously been shown to occur in a p53-independent manner. The ability of a p53 family member, p73, was tested for its ability to affect apoptosis. p73 protein levels were induced during T cell apoptosis. Use of a dominant negative TAT-p73 protein protected cells and indicated that TCR-AICD occurs in a p73-dependent manner in both Jurkat T cells and in primary murine T cells. T cells from p73 nullizygous mice were impaired in their ability to undergo TCR-mediated death. In conclusion, TCR-AICD occurs in a cell cycle regulated fashion that requires pRB, E2F-1, and p73 for its execution.