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FoxA1 directs the lineage and immunosuppressive properties of FoxA1+ regulatory T cells in EAE and MS

ABSTRACT: Affymetrix gene expression analysis was carried out to investigate the differences in gene profile of MBP89-101-reactive encephalitogenic T cells before and after co-culture with cerebellar granular neurons (CGNs). Co-culture of MBP89-101-reactive encephalitogenic T cells with CGNs leads to generation of T cells with regulatory T cells phenotype (CD4+CD25+membrane bound TGF-b+ T cells) or a new regulatory phenotype (CD4highPD-L1high T cells). CGN-induced CD4+CD25+membrane bound TGF-b+ T regulatory cells, CD4highPD-L1high T cells were purified by FACSAria. IFN-beta induced T lymphocytes (CD4highPD-L1high T cells) were also FACSAria purified. All these populations were compared to MBP89-101-reactive encephalitogenic T cells. Samples were prepared from biological triplicates for each FACSAria sorted population.

ORGANISM(S): Mus musculus

PROVIDER: GSE54490 | GEO | 2014/02/16

SECONDARY ACCESSION(S): PRJNA236639

REPOSITORIES: GEO

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Project description:TGF-b is an important pleiotropic cytokine with potent immunoregulatory properties. Although many previous reports have been proposed for the immunoregulatory functions of TGF-b on T cells, such as the suppression of cell proliferation, cytokine production and cytokine signaling, as well as the induction of apoptosis, it is not well elucidated whether the each effect of TGF-b on T cells is dependent on Smad signaling or Smad-independent other signaling pathways. The aim of the study was to clarify the involvement of Smad signaling and to investigate the redundancy of Smad2 and Smad3 on various TGF-b-mediated regulation of gene expression in CD4+ T cells. We used microarrays to detail the global program of gene expression regulated by TGF-b in CD4+ T cells, and identified distinct classes of up/down-regulated genes which are dependent on or independent of TGF-b-Smad signaling. Most of genes regulated by TGF-b were redundantly dependent on Smad2 and Smad3, including Foxp3 and IL-2. In addition, some genes were sufficiently regulated via Smad2 or Smad3 signaling alone. In contrast, TGF-b-mediated RORgt induction was independent of Smad signaling. CD4+CD25-CD44loCD62Lhi T cells (naive) were isolated from the spleens in wild-type (WT), T cell-specific Smad2 conditional knockout (Smad2KO or Smad2del/del), Smad3 knockout (Smad3KO or Smad3-/-) or Smad2del/delSmad3+/- mice by using a BD FACS ariaTM cell sorter (BD Bioscience) (purity: >98%). Freshly purified cells were then stimulated with anti-TCR stimuli in the absence or presence of TGF-b for 24 hr, respectively. A complete and precise experimental procedure is given in the "treatment protocol". It was very difficult to obtain the enough number of CD4+CD25-CD44loCD62Lhi naive T cells from Smad2del/delSmad3-/- mice because alomost all of CD4+ T cells were activated in Smad2del/delSmad3-/- mice. We confirmed that the several known Smad-regulated genes were almost out of control in Smad2del/delSmad3+/- CD4+ T cells by using quantitative RT-PCR. Furthermore, previous studies have reported the similar results in other cell types deficit in two alleles of Smad2 and one allele of Smad3. For these reasons, we substituted Smad2del/delSmad3+/- naive T cells for Smad2/3-deficient naive T cells. Cells were quickly collected 24 hr after culture for RNA extraction and hybridization on Affymetrix microarrays.

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FoxA1 directs the lineage and immunosuppressive properties of FoxA1+ regulatory T cells in EAE and MS

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