Molecular Mechanisms of Pre-T Cell Receptor-induced Survival

En route to maturing as T cell receptor (TCR) αβ-expressing cells, the development of thymocytes is contingent on expression of a pre-TCR complex comprising a TCRβ chain paired with a surrogate TCRα chain, pre-Tα (pTα). The pre-TCR has been proposed to promote cell survival, proliferation, differentiation, and lineage commitment. However, the precise molecular mechanisms governing this variety of effects remain elusive. Here, we present a cellular system designed to biochemically dissect signals elicited upon pre-TCR expression. Using the T cell line 4G4 stably transfected with one of the two known pTα isoforms or selective pTα deletion mutants and TCRβ, we were able to observe that expression of a functional pre-TCR complex is sufficient to control the levels of surface Fas protein, the stimulation of mitogen-activated and stress-regulated kinases, and the activation status of the p53 antioncogene. We demonstrate that this regulation has a major impact on the expression of important regulators of apoptosis, such as Bcl-2 family members, and the cell cycle, such as p21WAF. Furthermore, we show here that cells expressing a functional pre-TCR are more resistant to different types of DNA damage-induced apoptosis and that these effects are contingent on an intact cytoplasmic tail of pTα. We finally propose that the presence of a functional pre-TCR complex triggers many intracellular pathways capable of driving and ensuring thymocyte survival in the presence of DNA damage. En route to maturing as T cell receptor (TCR) αβ-expressing cells, the development of thymocytes is contingent on expression of a pre-TCR complex comprising a TCRβ chain paired with a surrogate TCRα chain, pre-Tα (pTα). The pre-TCR has been proposed to promote cell survival, proliferation, differentiation, and lineage commitment. However, the precise molecular mechanisms governing this variety of effects remain elusive. Here, we present a cellular system designed to biochemically dissect signals elicited upon pre-TCR expression. Using the T cell line 4G4 stably transfected with one of the two known pTα isoforms or selective pTα deletion mutants and TCRβ, we were able to observe that expression of a functional pre-TCR complex is sufficient to control the levels of surface Fas protein, the stimulation of mitogen-activated and stress-regulated kinases, and the activation status of the p53 antioncogene. We demonstrate that this regulation has a major impact on the expression of important regulators of apoptosis, such as Bcl-2 family members, and the cell cycle, such as p21WAF. Furthermore, we show here that cells expressing a functional pre-TCR are more resistant to different types of DNA damage-induced apoptosis and that these effects are contingent on an intact cytoplasmic tail of pTα. We finally propose that the presence of a functional pre-TCR complex triggers many intracellular pathways capable of driving and ensuring thymocyte survival in the presence of DNA damage. T cell receptor double negative double positive 2-(4-amidinophenyl)-6-indolecarbamidine terminal deoxinucleotidyltransferase-mediated dUTP-fluorescein isothiocyanate nick end labeling c-Jun N-terminal kinase hemagglutinin pre-TCRα chain fluorescence-activated cell sorting Development of immature thymocytes is contingent on passage through at least two major checkpoints: positive selection mediated by interactions of TCRαβ with proteins of the major histocompatibility complex, and the β-selection point mediated by major histocompatibility complex-independent signaling through the pre-TCR1 (1Mombaerts P. Clarke A.R. Rudnicki M.A. Iacomini J. Itohara S. Lafaille J.J. Wang L. Ichikawa Y. Jaenisch R. Hooper M.L. Tonegawa S. Nature. 1992; 360: 225-231Crossref PubMed Scopus (965) Google Scholar, 2Shinkai Y. 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Nature. 2000; 406: 521-527Crossref Scopus (139) Google Scholar), consistent with the idea that the main role for the pre-TCR is to facilitate pairing with the CD3 complex. However, unlike TCRαβ, the pre-TCR has an additionally extended cytoplasmic tail encoded by the pTα gene. Within the tail two proline-rich motifs can be identified by sequence similarity to motifs in the cytoplasmic tail of human CD2 that mediate binding to CD2BP2, an adaptor molecule involved in intracellular signaling (8Saint-Ruf C. Ungewiss K. Groettrup M. Bruno L. Fehling H.J. von Boehmer H. Science. 1994; 266: 1208-1212Crossref PubMed Scopus (358) Google Scholar, 16Sunder-Plassmann R. Reinherz E.L. J. Biol. Chem. 1998; 273: 24249-24257Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Heretofore, the importance of the intracytoplasmic region of the pre-TCR has been uncertain, but very recently the expression of pTα mutants in retrovirally transduced T cell precursors and cell lines showed that the pTα cytoplasmic tail, in particular the proline-rich domain, plays a crucial role in pre-TCR signal transduction (17Aifantis I. Borowski C. Gounari F. Lacorazza H.D. Nikolich-Zugich J. von Boehmer H. Nat. Immunol. 2002; 3: 483-488Crossref PubMed Scopus (70) Google Scholar). Based on the analysis of cells on either side of the DN III to DN IV transition, the pre-TCR has been hypothesized to regulate thymocyte survival, proliferation, differentiation, and lineage commitment (18Dudley E.C. Girardi M. Owen M.J. Hayday A.C. Curr. Biol. 1995; 5: 659-669Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 19Hoffman E.S. Passoni L. Crompton T. Leu T. Schatz D. Koff A. Owen M.J. Hayday A.C. 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Immunity. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), changes were noted in the DNA binding of a known of signaling from death receptor for a gene by a in DN IV cells in DN III and in DP cells Barber D.F. M. Hayday A.C. S. Immunity. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). apoptosis by DNA an intact p53 in thymocytes I. Genes Dev. 1996; 10: PubMed Scopus Google and via activation of the kinase R. H. N. T. L. A. A. 1999; PubMed Scopus Google Scholar). of p53 in a cell death in DN thymocytes and the in pre-TCR cell by this pre-TCR P. Immunity. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The analysis of expressing of either the pre-TCR or of molecules associated with the that thymocytes that in a functional TCRβ chain through the transition from DN III to DN However, the of these in is a and the pre-TCR expression and proposed is of some regulators of thymocyte survival and are in and any of which be for changes in cells as the DN III to DN IV transition T. T. D. D. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J.J. A. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the very levels of expression of the pre-TCR in thymocytes very to and even more to biochemically this of these the for to pTα this we one such by expression of the pre-TCR to the that this molecule on some important regulators of cell death and thymocyte Here, we the development and of a variety of stably transfected expressing different pre-TCR in a T cell line able to a functional pre-TCR complex upon of the pTα and TCRβ D.F. Passoni L. Wen L. Geng L. Hayday A.C. J. Immunol. 1998; 161: 11-16PubMed Google Scholar). The of the pre-TCR to regulate the expression of Fas, Bcl-2 family members, and the of molecules such as p53 and stress-regulated is We in this system the of pre-TCR expression in the regulation of cell death by DNA damage. the in this to that expression of a and functional pre-TCR complex is able to regulate signals that the of activation of mitogen-activated kinases, and of gene such as Fas, and 4G4 cells were with a TCRβ chain or with and and were in as D.F. Passoni L. Wen L. Geng L. Hayday A.C. J. Immunol. 1998; 161: 11-16PubMed Google Scholar). transfected with either one of the pTα chain of the TCRβ chain, the pTα as be by However, we were to any pTα in either by or by that the pre-Tα chain a complex when of TCRβ, consistent with has been M. Ungewiss Azogui O. R. Owen M.J. Hayday A.C. von H. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). of in which the cytoplasmic two proline-rich were with the a and a The into from which in into the expression that an N-terminal D. Douglas N.C. Barber D.F. Liu Q. Sullo R. Geng L. Fehling H.J. von Boehmer H. Hayday A.C. J. Exp. Med. 2001; 194: 695-703Crossref PubMed Scopus (26) Google Scholar). of transfected proteins by and by intracellular as D.F. Passoni L. Wen L. Geng L. Hayday A.C. J. Immunol. 1998; 161: 11-16PubMed Google Scholar). cells were by and in as C. L. R. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google the of of of cell were with The were by in in with and to a were with by and with a of TCRβ, the were by to and with an the of TCRβ from intracellular the cell were in for in and in a for were in The were and The cells were in and on a analysis to apoptosis in as The cells were on for to as C. L. R. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The cells were in a for and for in in in and for in in the an in cell death the that the labeling at of at the cells were on and a The of cells by and cells. Fas expression by the (22Ogasawara J. Watanabe-Fukunage R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. S. Nature. 1993; PubMed Scopus Google and in of the showed of Fas expression. cell were and as with or in of the activation of an kinase levels in the different were by analysis or a positive the were with a and a for activation or for stimulation to cell the to of the p53 in 4G4 were to and with for of in p53 found to at of a the cells were as and with of and to analysis with and levels in the different were by analysis of of cell and the of Bcl-2 in the of were two different and apoptosis the cells were with for to with by The of cells by of the The T cell line 4G4 D.F. Passoni L. Wen L. Geng L. Hayday A.C. J. Immunol. 1998; 161: 11-16PubMed Google to stably pre-TCR of were by selection and expression of TCRβ TCRβ either the or pTα and TCRβ a of in which two motifs in the cytoplasmic tail were by and the levels of expression different the the different gene were in and of the pTα tail expression or of pTα or TCRβ in these cells. the to whether the pre-TCR expression from cell we a system apoptosis in the upon with a of pre-TCR more resistant to apoptosis either TCRβ or cells expressing TCRβ the pTα tail for this death in of cells, in of cells apoptosis to this cell death has been to activation of Fas in T cells Y. D. D. T. J. Cell. Biol. 1998; PubMed Scopus Google Scholar, A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. Y. 1998; PubMed Scopus Google Scholar), and can be mediated by with this expressing a pre-TCR surface Fas expression as compared with cells or cells expressing the TCRβ chain changes in Fas levels were in expressing the cytoplasmic tail of pTα a of the pTα tail both to the regulation of surface Fas expression and to from The presence of DNA by has been the of the that to of of thymocytes I. Genes Dev. 1996; 10: PubMed Scopus Google Scholar). in the β-selection and can be by expression of a TCRβ chain paired with the pTα gene However, pre-TCR expression from DNA damage-induced apoptosis is still an unresolved an to this we a of as to to any were with a known to DNA through of II L. M. N. M. A. 2000; Google Scholar). expressing a of pre-TCR high levels of to either TCRβ or cells TCRβ the pTα tail for the death and of cells, of the were that expression of pre-TCR is sufficient cells from DNA damage-induced has been that the pre-TCR through the DNA of the DN to DP transition by p53 P. Immunity. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the to biochemically whether pre-TCR regulate p53 levels or we to whether the levels of p53 and two T. R. D. Cell. 1993; Full Text PDF PubMed Scopus Google and T. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar), were in expressing TCRβ or TCRβ any of the pTα isoforms and TCRβ in the of or in expressing either of the pTα. the of a gene in particular the a The levels of p53 were very in that changes in the of p53 to be the of the in and expression. expressing the of the TCRβ any changes in or when compared with control cells. at the that the p53 and p53 in cells. this we to the of the p53 that we of the of that to the activation status of the p53 of p53 has been to the to upon DNA Y. K. Genes Dev. 1997; PubMed Scopus Google and a for p53 activation upon DNA damage. cells with an of we observe a of p53 in cells expressing TCRβ that were as a control when the levels of p53 were into the However, we in the activation status of p53 in to in the different The of p53 in cells expressing TCRβ as compared with cells expressing TCRβ, this even more in cells expressing TCRβ, very levels of p53 be We that expressing the showed levels of p53 the importance of the cytoplasmic of the pTα for effects of the pre-TCR and that expression of a functional pre-TCR to of p53 upon DNA damage. 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Immunol. 1999; PubMed Scopus Google Scholar). but be the of Fas (22Ogasawara J. Watanabe-Fukunage R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. S. Nature. 1993; PubMed Scopus Google Scholar), the activation of Barber D.F. M. Hayday A.C. S. Immunity. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), the regulation of H. C. N. C. M. J. Exp. Med. 2000; PubMed Scopus Google Scholar), and the of p53 P. Immunity. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of these changes been to occur the pre-TCR and has been that the pre-TCR molecule with these intracellular to whether these changes are the of the expression of the pre-TCR molecule or in we and T cell stably expressing different of the pre-TCR complex. system to in the signaling that this molecule is able to and to defined of these been to show that activation in III thymocytes is on expression of pre-TCR Barber D.F. M. Hayday A.C. S. 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Immunol. 2002; 3: 483-488Crossref PubMed Scopus (70) Google by expression of different pTα mutants in retrovirally transduced T cell precursors and cell lines showed that the pTα cytoplasmic tail, in particular the proline-rich domain, plays a crucial role in pre-TCR signaling and T cell we progress one and the molecular for this by an role of the pTα tail in survival and We here that the pTα tail is for at least some of the elicited by the the cellular system we were able to the that pre-TCR expression has in the expression of Fas of some of Fas in DN thymocyte thymocytes from which show DN to DP when the is a Fas K. K.-I. H. Y. K. J. Immunol. 1997; Google Scholar). this is that the analysis of thymocyte subsets showed that the in Fas expression in DN cells to subsets I and Fas levels were high in DN II and DN III but showed a on DN IV cells (22Ogasawara J. Watanabe-Fukunage R. Adachi M. Matsuzawa A. Kasugai T. Kitamura Y. Itoh N. Suda T. S. 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Immunity. 1999; Full Text Full Text PDF PubMed Scopus Google p53 to be a very of the pre-TCR of signals elicited by expression of a single receptor complex in to the to which thymocytes are the β-selection ensuring the and of cellular and a of this at the of and of with the of is We are to J. at the of for and