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Remy Bosselut, M.D., Ph.D.
Genetic analysis of intrathymic T-cell development
We investigate the transcriptional control of T cell development and function. We are specifically interested in the gene expression programs that control the 'choice' by T cell precursors of the CD4 or CD8 lineage, and that perpetuate lineage differentiation in mature T cells.
Most T cells recognize peptide antigens presented by class I (MHC-I) or class II (MHC-II) classical Major Histocompatibility Complex molecules. T cell recognition of MHC-peptide complexes is aided by two surface glycoproteins called coreceptors: CD4 which binds MHC-II and CD8 which binds MHC-I. Coreceptor expression on mature T cells is mutually exclusive and strongly correlates with both MHC specificity and functional differentiation. The general rule is that MHC I-specific T cells are CD4-CD8+ and cytotoxic (CD8 cells), whereas MHC II-specific T cells are CD4+CD8- and helper or regulatory (CD4 cells). This double correspondence is essential for the proper function of the immune system and is established in the thymus, where CD4 and CD8 T cells emerge as separate lineages from precursors expressing both CD4 and CD8 (double positive). CD4 T cells constitute an essential part of the adaptive immune system, as dramatically illustrated by the severity of the CD4 immunodeficiency induced by the Human Immunodeficiency Virus HIV.
Our research is directed towards the analysis of the transcriptional circuitry that decides CD4-CD8 lineage differentiation. Over the last few years, we have addressed three aspects of this question.
First, the function of two zinc finger transcription factors, Gata3 and Thpok (also known as Zbtb7b or cKrox) during CD4 cell differentiation in the thymus. While both Gata3 and Thpok had previously been shown to be necessary for the development of CD4 T cells, their role in that process remained unclear. To address this question, we generated mice carrying null alleles of Thpok, or a GFP-based bacterial artificial chromosome transgene to monitor Thpok expression. We also obtained mice deleting Gata3 in DP thymocytes, generated in the laboratory of Bill Paul (NIAID). Using these animals, we made the following findings (Wang et al, 2008, Nat. Immunol.). First, disruption of either Gata3 or Thpok blocks the development of CD4 T cells at an early stage of their differentiation, before they commit to the CD4 lineage (i.e. they lose the ability to adopt a CD8 fate). Second, the requirement for Gata3 precedes that for Thpok; notably, Gata3 is needed for Thpok expression, whereas the reverse is not true. We found that Gata3 binds within a region of the Thpok locus critical for Thpok expression, suggesting that Gata3 directly activates Thpok transcription. Third, Gata3 is needed for Thpok to promote CD4 differentiation, but not for Thpok to inhibit CD8 differentiation. Based on these findings, we proposed that Gata3 and Thpok acted as specification and commitment factors, respectively, during CD4-lineage differentiation in the thymus, in agreement with other reports published simultaneously. We are currently evaluating how these factors promote the expression of other genes characteristic of the CD4 lineage.
The hypothesis that Thpok serves primarily to repress the expression of CD8-lineage genes fits with results from our earlier experiments assessing the function of Thpok in mature T cells (Jenkinson et al., 2007). Using Thpok retroviral transduction into CD8 T cells (in which this factor is normally not expressed) we had found that Thpok represses the expression of CD8 and of characteristic cytotoxic genes, including those encoding perforin and granzyme B. Reciprocally, we have recently investigated the function of Thpok in mature CD4 T cells (in which it is normally expressed) using loss-of-function approaches, by generating an hypomorphic and a conditional (floxed) Thpok allele (Wang et al., 2008, Immunity). We found that Thpok maintains the 'CD4 identity' as its deletion in mature (post-thymic) CD4 T cells that had developed as Thpok-sufficient results in the re-expression of CD8 and of cytotoxic genes. We further showed that such aberrant expression depends on the activity of Runx transcriptional regulators; accordingly, Thpok-deficient CD4 cells abnormally express Runx3, a member of this family normally present in CD8 but not CD4 cells and essential to the proper differentiation of CD8-lineage cells in the thymus. Thus, Thpok functions in CD4 T cells to permanently repress expression of CD8 lineage genes.
In a third series of experiments, we analyzed the function of Ets1, the founding member of a large family of transcription factors that bind specific DNA sequences typically centered over a GGAA tetranucleotide motif, in CD8 differentiation. In agreement with the results of previous studies, we showed that Ets1 is required for the proper termination of CD4 expression during the differentiation of MHC I-restricted thymocytes; that is, Ets1-deficient thymocytes fail to normally silence Cd4. In contrast, Ets1 is not required for other events associated with MHC I-induced positive selection, including the initiation of cytotoxic gene expression, cortico-medullary migration or thymus exit. Previous studies had shown that Runx3 is required in developing CD8-lineage thymocytes for the proper silencing of Cd4. Indeed, we found that Ets1-deficient thymocytes failed to normally up-regulate Runx3. Furthermore, enforced Runx3 expression in Ets1-deficient MHC I-restricted thymocytes largely rescued their impaired Cd4 silencing, indicating that Ets1 is not required for Runx3 function. Finally, we documented that Ets1 binds at least two evolutionary conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription. These findings identify Ets1 as a key player during CD8-lineage differentiation and indicate that it acts at least in part by promoting Runx3 expression.
Altogether, these findings contribute to the assembly of the transcriptional regulatory network that govern CD4-CD8 choice in the thymus. Additionally, they demonstrate that similar (although not identical) circuitries control both the emergence of CD4 and CD8 lineages in the thymus, and their persistent separation in mature post-thymic T cells. We are currently examining whether and how Thpok contributes to the expression of CD4-lineage genes and to effector functions of CD4 peripheral T cells. We are also expanding these studies to identify factors that control the expression of Thpok and Runx3.
Collins, A., Littman, D. R., and Taniuchi, I. (2009). RUNX proteins in transcription factor networks that regulate T-cell lineage choice. Nat Rev Immunol 9, 106-115.
Ho, I. C., Tai, T. S., and Pai, S. Y. (2009). GATA3 and the T-cell lineage: essential functions before and after T-helper-2-cell differentiation. Nat Rev Immunol 9, 125-135.
Singer, A., Adoro, S., and Park, J. H. (2008). Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat Rev Immunol 8, 788-801.
Wang, L., and Bosselut, R. (2009). CD4-CD8 lineage differentiation: Thpok-ing into the nucleus. J Immunol 183, 2903-2910.
Original papers from the lab
Wang, L., Wildt, K. F., Castro, E., Xiong, Y., Feigenbaum, L., Tessarollo, L., and Bosselut, R. (2008b). The zinc finger transcription factor Zbtb7b represses CD8-lineage gene expression in peripheral CD4+ T cells. Immunity 29, 876-887.
Wang, L., Wildt, K. F., Zhu, J., Zhang, X., Feigenbaum, L., Tessarollo, L., Paul, W. E., Fowlkes, B. J., and Bosselut, R. (2008a). Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4(+) T cells. Nat Immunol 9, 1122-1130.
Zamisch, M., Tian, L., Grenningloh, R., Xiong, Y., Wildt, K. F., Ehlers, M., Ho, I. C., and Bosselut, R. (2009). The transcription factor Ets1 is important for CD4 repression and Runx3 up-regulation during CD8 T cell differentiation in the thymus. J Exp Med 206, 2685-2699.
This page was last updated on 2/19/2013.