Genetic analysis of T-cell differentiation

Basic Sciences

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Abstract

We are interested in intrathymic T-cell development, an attractive model to study cell differentiation and death/survival decisions in mammals. Our research focuses on the differentiation of immature T-cell precursors (thymocytes) that express both CD4 and CD8 (double positive, DP), into mature T-cells that express either CD4 or CD8 but not both (single positive, SP). The survival and differentiation of thymocytes during this step requires signaling through their T-cell receptor (TCR) upon triggering by Major Histocompatibility Molecules (MHC). We have been studying this process using two main approaches. [unreadable] We have generated models in which intrathymic TCR signaling is prematurely downregulated in vivo during the differentiation of DP into SP thymocytes. This was achieved by using gene regulatory elements that are extinguished during this step to express Zap70, a tyrosine kinase required for TCR signaling. Using this system, we had shown that such premature termination of signaling prevents differentiation into the CD4-lineage and impairs the survival of CD8-lineage cells (although not their CD8-lineage differentiation). We have further documented that CD4-lineage commitment requires TCR signaling to persist until CD4-differentiating thymocytes have terminated CD8 expression. [unreadable] In a second approach, we have used high-throughput gene expression analyses to identify genes potentially involved in differentiation and survival as DP thymocytes differentiate into mature T-cells. We have shown that one such gene, encoding the zinc finger transcription factor cKrox (also known as Zfp67 or Thpok) is upregulated during the differentiation of CD4 but not of CD8-lineage cells. We have found that enforced cKrox expression in thymocytes precludes CD8 but promotes CD4 T-cell differentiation. In fact, cKrox expression in thymocytes that recognize class I-MHC peptide complexes and normally become cytotoxic CD8 T-cells makes them differentiate into helper CD4 T-cells, demonstrating that cKrox imposes CD4-lineage differentiation in the thymus. We are currently exploring several aspects of cKrox function in thymocytes, including the regulation of its expression, how it affects other effector genes of CD4-CD8 lineage differentiation and whether cKrox expression can by-pass the requirement for TCR signaling during CD4 differentiation. We are also deriving a conditional cKrox allele to study cKrox function in more detail.