RESUMO
During thymic T cell development, immature CD4+CD8+ double-positive (DP) thymocytes develop either into CD4+CD8- Th cells or CD4-CD8+ CTLs. Differentially expressed primary factors inducing the fate of these cell types are still poorly described. The transcription factor Runx3/AML-2 Runx, runt [corrected] dominant factor; AML, acute myeloid leukemia is expressed specifically during the development of CD8 single-positive (SP) thymocytes, where it silences CD4 expression. Deletion of murine Runx3 results in a reduction of CD8 SP T cells and concomitant accumulation of CD4+CD8+ T cells, which cannot down-regulate CD4 expression in the thymus and periphery. In this study we have investigated the role of Runx3 during thymocyte development and CD4 silencing and have identified integrin alpha(E)/CD103 on CD8 SP T cells as a new potential target gene of Runx3. We demonstrate that Runx3 is necessary not only to repress CD4, but also to induce CD103 expression during development of CD8 SP T cells. In addition, transgenic overexpression of Runx3 reduced CD4 expression during development of DP thymocytes, leading to a reduced number of CD4 SP thymocytes and an increased number of CD8 SP thymocytes. This reversal is not caused by redirection of specific MHC class II-restricted cells to the CD8 lineage. Overexpression of Runx3 also up-regulated CD103 expression on a subpopulation of CD4 SP T cells with characteristics of regulatory T cells. Thus, Runx3 is a main regulator of CD4 silencing and CD103 induction and thus contributes to the phenotype of CD8 SP T cells during thymocyte development.
Assuntos
Antígenos CD/biossíntese , Antígenos CD4/biossíntese , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/fisiologia , Cadeias alfa de Integrinas/biossíntese , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD4/genética , Relação CD4-CD8 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD8/biossíntese , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core , Cruzamentos Genéticos , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Inativação Gênica , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Antígenos de Histocompatibilidade Classe II/genética , Células Matadoras Naturais/citologia , Linfopenia/genética , Linfopenia/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Timoma/genética , Timoma/imunologia , Timo/citologia , Timo/imunologia , Timo/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
During thymic T cell development, immature CD4(+)/CD8(+) thymocytes develop into either CD4(+)/CD8(-) helper or CD4(-)/CD8(+) CTLs. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. Here we developed a novel approach to investigate gene function during thymocyte development. We transfected ex vivo isolated immature thymocytes with gene-specific morpholino antisense oligonucleotides and induced differentiation in cell or organ cultures. A morpholino oligonucleotide specific for CD8alpha strongly reduces CD8 expression. To our knowledge, this is the first demonstrated gene knockdown by morpholino oligonucleotides in primary lymphocytes. Using this approach, we show here that the transcription factor Runx3 is involved in silencing of CD4 expression during CD8 T cell differentiation. Runx3 protein expression appears late in thymocyte differentiation and is confined to mature CD8 single-positive thymocytes, whereas Runx3 mRNA is transcribed in mature CD4 and CD8 thymocytes. Therefore, Runx3 protein expression is regulated at a post-transcriptional level. The knockdown of Runx3 protein expression through morpholino oligonucleotides inhibited the development of CD4(-)/CD8(+) T cells. Instead, mature cells with a CD4(+)/CD8(+) phenotype accumulated. Potential Runx binding sites were identified in the CD4 gene silencer element, which are bound by Runx protein in EMSAs. Mutagenesis of potential Runx binding sites in the CD4 gene silencer abolished silencing activity in a reporter gene assay, indicating that Runx3 is involved in CD4 gene silencing. The experimental approach developed here should be valuable for the functional analysis of other candidate genes in T cell differentiation.
