The thymus generates major histocompatibility complex (MHC)-restricted alphabetaT cells that only recognize antigenic ligands in association with MHC or MHC-like molecules. We hypothesized that MHC specificity might be imposed on a broader alphabetaTCR repertoire during thymic selection by CD4 and CD8 coreceptors that bind and effectively sequester the tyrosine kinase Lck, thereby preventing T cell receptor (TCR) signaling by non-MHC ligands that do not engage either coreceptor. This hypothesis predicts that, in coreceptor-deficient mice, alphabeta thymocytes would be signaled by non-MHC ligands to differentiate into alphabetaT cells lacking MHC specificity. We now report that MHC-independent alphabetaT cells were indeed generated in mice deficient in both coreceptors as well as MHC ("quad-deficient" mice) and that such mice contained a diverse alphabetaT cell repertoire whose MHC independence was confirmed at the clonal level. We conclude that CD4 and CD8 coreceptors impose MHC specificity on a broader alphabetaTCR repertoire during thymic selection by preventing thymocytes from being signaled by non-MHC ligands.

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells1. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks2–4, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma5,6, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL–AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4−/− MLL–AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL–AF9 blasts, which requires cyclin-dependent kinase inhibitor p21Cip1 (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

SummaryCD4+CD8 + thymocytes expressing self-reactive T cell antigen receptors (TCR) are deleted in the thymus as a consequence of TCIL/sdf-antigen/major histocompatibility complex interactions. However, the signals that are necessary to initiate clonal deletion have not yet been clarified. Here we demonstrate that TCR engagement does not efftdently induce apoptosis of CD4+CD8 + thymocytes, although it generates signals that increase expression of CD5, a thymocyte differentiation marker. In fact, TCR signals fail to induce thymocyte apoptosis even when augmented by simultaneous engagement with CD4 or lymphocyte function 1-associated molecules. In marked contrast, signals generated by engagement of both TCR and the costimulatory molecule CD28 potently induce apoptosis of CD4 +CD8 + thymocytes. Thus, the present results define a requirement for both TCR and costimulatory signals for thymocyte apoptosis and identify CD28 as one molecule that is capable of providing the necessary costimulus. These results provide a molecular basis for differences among cell types in their ability to mediate negative selection of developing thymocytes.T olerance to self-proteins is maintained among T ceils through the elimination or inactivation of clones which express antigen receptors reactive to self-antigen/MHC complexes (1, 2). Elimination of stir-reactive immature T cells takes place in the thymus by donal deletion which occurs via apoptosis (3-5). CD4+CD8 + thymocytes, the major targets of donal deletion, can be induced to undergo apoptoffs both in vivo and in vitro through engagement of their antigen receptors by either intrathymic self-ligands or by antireceptor antibodies (6-13). However, it is not known whether induction of apoptosis requires signals in addition to those transduced by the TCR. In fact, donal deletion of thymocytes is usually assayed in the presence of dedicated APCs that are capable of providing ligands for costimulatory molecules present on thymocytes. Whether APCs bearing costimulatory ligands are uniquely capable of mediating TCRdriven apoptosis of CD4+CD8 + thymocytes, or whether any cell type capable of presenting self-antigen/MHC complexes can mediate negative selection (14) is not dear.In this report, we show that isolated TCR signals do not efficiently drive apoptosis of CD4+CD8 + thymocytes even when enhanced by coengagement with CD4 or LFA-1. However, TCR signals deliver a potent apoptotic stimulus when combined with signals provided by the costimulatory molecule, CD28. These results demonstrate that both TCR and costimulatory signals are necessary to induce thymocyte apoptosis and indicate that only ceUs expressing costimulatory ligands can mediate negative selection. Materials and MethodsIsolation of CD4 + CD8 + Thymoqtes. CD4+CD8 + thymocytes were purified from young adult C57BL/6 thymuses by panning on anti-CD8 coated phtes (15). More than 95% of the harvested cells were CD4+CD8 +.Culture Conditions and Antibodies. 24-wen tissue cuhure plates were coated with antibody incubating them overnight...