Molecular mechanisms for the regulation of the Il-10 gene in CD4 T cells : comparison of IL-10-T_regand CD25+T_reg.

Shoemaker, John (2006). Molecular mechanisms for the regulation of the Il-10 gene in CD4 T cells : comparison of IL-10-T_regand CD25+T_reg. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f67c

Abstract

This thesis outlines and provides direct evidence for transcription factor-mediated instructive regulation of the 11-10 gene in CD4 T cells. Firstly, I describe transcription factors that are differentially expressed in populations of IL-10-producing CD4 T cells. Genome-wide mRNA profiles (GeneChip) are used from six populations of CD4 T populations which represent both temporal and lineage-specific time-points within naive, effector and regulatory CD4 T cell populations. Secondly, validation experiments (qPCR) verified whether transcription factors isolated from the genome-wide profiling experiments (GeneChip) were quantitatively expressed in IL-10-producing populations. Thirdly, comparative genomics was used to analyze in silico the 11-10 locus for conserved putative transcription factor binding sites at potential regulatory regions. Selected transcription factors were retro-virally transduced into primary naive CD4 T cells and analyzed for their ability to induce IL-10 expression in a variety of differentiation conditions. Finally, the role of functionally validated transcription factors inducing IL-10 production was investigated at the molecular level, via their presence at specific locations at the 11-10 locus in vivo and in chromatin modifications. One example, GATA-3, shown here to be differentially expressed in IL-10-producing CD4 T cell populations, instructing modifications at the 11-10 locus and inducing IL-10 production in CD4 T cells.

The second aim of this thesis was to investigate — at the transcription factor-level -- the comparison of two phenotypically different CD4 regulatory T cell populations (CD25+T_reg and IL-10-T_reg). On one hand, both CD4 T regulatory cell populations are similar in that they do not produce effector upon secondary stimuli, both are anergic, and both suppress CD4 T cell proliferation in vitro and in vivo (as shown by their ability to abrogate multiple autoimmune and allergic disease animal models). On the other hand, CD25+T_reg are phenotypically described by their expression of the lineage-specific transcription factor, FoxP3, and IL-10- T_reg do not express FoxP3. Therefore, the molecular mechanisms as to how IL-10- T_reg do not express effector cytokines, are anergic and suppress CD4 T cell proliferation in vitro and in vivo without the expression of FoxP3 is investigated in this thesis. This thesis concludes by providing strong evidence that IL-10-T_reg down-regulate key NF-kB and AP-1 family transcripts and inhibit NFAT transcriptional activity, thereby compensating for FoxP3 expression, which is necessary in natural occurring CD25+T_reg.

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