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ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage.

Egawa T, Littman DR - Nat. Immunol. (2008)

Bottom Line: In ThPOK-deficient mice, such expression was derepressed in MHC class II-selected thymocytes, which contributed to their redirection to the CD8(+) T cell lineage.In the absence of both ThPOK and Runx, redirection was prevented and cells potentially belonging to the CD4(+) lineage, presumably specified independently of ThPOK, were generated.Our results suggest that MHC class II-selected thymocytes are directed toward the CD4(+) lineage independently of ThPOK but require ThPOK to prevent Runx-dependent differentiation toward the CD8(+) lineage.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Program, The Helen and Martin Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
The transcription factor ThPOK is required and sufficient for the generation of CD4(+)CD8(-) thymocytes, yet the mechanism by which ThPOK orchestrates differentiation into the CD4(+) helper T cell lineage remains unclear. Here we used reporter mice to track the expression of transcription factors in developing thymocytes. Distal promoter-driven expression of the gene encoding the transcription factor Runx3 was restricted to major histocompatibility complex (MHC) class I-selected thymocytes. In ThPOK-deficient mice, such expression was derepressed in MHC class II-selected thymocytes, which contributed to their redirection to the CD8(+) T cell lineage. In the absence of both ThPOK and Runx, redirection was prevented and cells potentially belonging to the CD4(+) lineage, presumably specified independently of ThPOK, were generated. Our results suggest that MHC class II-selected thymocytes are directed toward the CD4(+) lineage independently of ThPOK but require ThPOK to prevent Runx-dependent differentiation toward the CD8(+) lineage.

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CD8SP lineage-specific Runx3 expression from its distal promoter is required for Cd4 silencing and CD103 expression in CD8+ T cells(a) Runx3 and Runx1 protein expression in purified CD8+ cells from Runx3dYFP/YFP and Runx3dYFP/+ mice is shown with anti-HMG1 blot as loading control. Lysates from Cd4-cre+Runx1F/F CD4+ T cells and Cd4-cre+Runx3F/F CD8+ T cells were used as negative controls for Runx1 and Runx3 expression, respectively. (b,c) CD4 and CD8 (b) or CD8 and CD103 (c) expression in TCRβ+ lymph node (LN) cells from Runx3dYFP/YFP and Runx3dYFP/+ mice. In the right panel in (b), CD4 expression in CD8+ T cells from Runx3dYFP/YFP (open histogram) and Runx3dYFP/+ (shaded histogram) mice is shown. Mean fluorescent intensity: Runx3dYFP/+ 308, Runx3dYFP/YFP 1767. Data shown are representative of more than 3 independent experiments.
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Figure 1: CD8SP lineage-specific Runx3 expression from its distal promoter is required for Cd4 silencing and CD103 expression in CD8+ T cells(a) Runx3 and Runx1 protein expression in purified CD8+ cells from Runx3dYFP/YFP and Runx3dYFP/+ mice is shown with anti-HMG1 blot as loading control. Lysates from Cd4-cre+Runx1F/F CD4+ T cells and Cd4-cre+Runx3F/F CD8+ T cells were used as negative controls for Runx1 and Runx3 expression, respectively. (b,c) CD4 and CD8 (b) or CD8 and CD103 (c) expression in TCRβ+ lymph node (LN) cells from Runx3dYFP/YFP and Runx3dYFP/+ mice. In the right panel in (b), CD4 expression in CD8+ T cells from Runx3dYFP/YFP (open histogram) and Runx3dYFP/+ (shaded histogram) mice is shown. Mean fluorescent intensity: Runx3dYFP/+ 308, Runx3dYFP/YFP 1767. Data shown are representative of more than 3 independent experiments.

Mentions: To visualize thymocytes expressing Runx3d mRNA, we generated a reporter allele (Runx3dYFP) by replacing the first coding exon utilized by the Runx3 distal promoter with the yellow fluorescent protein (YFP) coding sequence (Supplementary Fig. 1a, online). In accordance with previous analyses of Runx protein and mRNA expression10, YFP expression was detected in CD8+ T cells, but not in CD4+ T cells or B220+ cells in peripheral blood lymphocytes from Runx3dYFP/+ mice (Supplementary Fig. 1b, online). To determine whether Runx3 distal promoter activity is required for Runx3 protein expression in CD8+ T cells, we generated homozygous Runx3dYFP/YFP mice, in which distal promoter-derived Runx3 expression was eliminated. Whereas germline deletion of both proximal promoter- and distal promoter-derived Runx3 results in neonatal lethality in the 129 or C57BL6 genetic background16,17, Runx3dYFP/YFP mice were viable and fertile, and showed no gross abnormality (data not shown). Runx3 protein was almost undetectable in CD8+ T cells, and was markedly reduced in T helper type (TH1)-polarized activated CD4+ T cells from Runx3dYFP/YFP compared to Runx3dYFP/+ mice (Fig. 1a and Supplementary Fig. 1c,d, online). Runx1 was up-regulated in Runx3-deficient CD8+ T cells from both Runx3F/FCd4-cre+ and Runx3dYFP/YFP mice (Fig. 1a and Supplementary Fig. 1d, online). This result indicates that most of Runx3 protein is derived from the distal promoter-driven transcript both in CD8+ T cells and in TH1-polarized CD4+ T cells, and that only a small amount of Runx3 protein is expressed from the proximal promoter in T cells. In CD8+ T cells from Runx3dYFP/YFP mice, Cd4 silencing was incomplete and there was no up-regulation of CD103 (integrin αE) expression, which was previously shown to be dependent on Runx3 (ref. 18) (Fig. 1b,c). Thus, Runx3 expression from the distal promoter is required for Cd4 silencing and CD103 expression in CD8SP thymocytes.


ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage.

Egawa T, Littman DR - Nat. Immunol. (2008)

CD8SP lineage-specific Runx3 expression from its distal promoter is required for Cd4 silencing and CD103 expression in CD8+ T cells(a) Runx3 and Runx1 protein expression in purified CD8+ cells from Runx3dYFP/YFP and Runx3dYFP/+ mice is shown with anti-HMG1 blot as loading control. Lysates from Cd4-cre+Runx1F/F CD4+ T cells and Cd4-cre+Runx3F/F CD8+ T cells were used as negative controls for Runx1 and Runx3 expression, respectively. (b,c) CD4 and CD8 (b) or CD8 and CD103 (c) expression in TCRβ+ lymph node (LN) cells from Runx3dYFP/YFP and Runx3dYFP/+ mice. In the right panel in (b), CD4 expression in CD8+ T cells from Runx3dYFP/YFP (open histogram) and Runx3dYFP/+ (shaded histogram) mice is shown. Mean fluorescent intensity: Runx3dYFP/+ 308, Runx3dYFP/YFP 1767. Data shown are representative of more than 3 independent experiments.
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Figure 1: CD8SP lineage-specific Runx3 expression from its distal promoter is required for Cd4 silencing and CD103 expression in CD8+ T cells(a) Runx3 and Runx1 protein expression in purified CD8+ cells from Runx3dYFP/YFP and Runx3dYFP/+ mice is shown with anti-HMG1 blot as loading control. Lysates from Cd4-cre+Runx1F/F CD4+ T cells and Cd4-cre+Runx3F/F CD8+ T cells were used as negative controls for Runx1 and Runx3 expression, respectively. (b,c) CD4 and CD8 (b) or CD8 and CD103 (c) expression in TCRβ+ lymph node (LN) cells from Runx3dYFP/YFP and Runx3dYFP/+ mice. In the right panel in (b), CD4 expression in CD8+ T cells from Runx3dYFP/YFP (open histogram) and Runx3dYFP/+ (shaded histogram) mice is shown. Mean fluorescent intensity: Runx3dYFP/+ 308, Runx3dYFP/YFP 1767. Data shown are representative of more than 3 independent experiments.
Mentions: To visualize thymocytes expressing Runx3d mRNA, we generated a reporter allele (Runx3dYFP) by replacing the first coding exon utilized by the Runx3 distal promoter with the yellow fluorescent protein (YFP) coding sequence (Supplementary Fig. 1a, online). In accordance with previous analyses of Runx protein and mRNA expression10, YFP expression was detected in CD8+ T cells, but not in CD4+ T cells or B220+ cells in peripheral blood lymphocytes from Runx3dYFP/+ mice (Supplementary Fig. 1b, online). To determine whether Runx3 distal promoter activity is required for Runx3 protein expression in CD8+ T cells, we generated homozygous Runx3dYFP/YFP mice, in which distal promoter-derived Runx3 expression was eliminated. Whereas germline deletion of both proximal promoter- and distal promoter-derived Runx3 results in neonatal lethality in the 129 or C57BL6 genetic background16,17, Runx3dYFP/YFP mice were viable and fertile, and showed no gross abnormality (data not shown). Runx3 protein was almost undetectable in CD8+ T cells, and was markedly reduced in T helper type (TH1)-polarized activated CD4+ T cells from Runx3dYFP/YFP compared to Runx3dYFP/+ mice (Fig. 1a and Supplementary Fig. 1c,d, online). Runx1 was up-regulated in Runx3-deficient CD8+ T cells from both Runx3F/FCd4-cre+ and Runx3dYFP/YFP mice (Fig. 1a and Supplementary Fig. 1d, online). This result indicates that most of Runx3 protein is derived from the distal promoter-driven transcript both in CD8+ T cells and in TH1-polarized CD4+ T cells, and that only a small amount of Runx3 protein is expressed from the proximal promoter in T cells. In CD8+ T cells from Runx3dYFP/YFP mice, Cd4 silencing was incomplete and there was no up-regulation of CD103 (integrin αE) expression, which was previously shown to be dependent on Runx3 (ref. 18) (Fig. 1b,c). Thus, Runx3 expression from the distal promoter is required for Cd4 silencing and CD103 expression in CD8SP thymocytes.

Bottom Line: In ThPOK-deficient mice, such expression was derepressed in MHC class II-selected thymocytes, which contributed to their redirection to the CD8(+) T cell lineage.In the absence of both ThPOK and Runx, redirection was prevented and cells potentially belonging to the CD4(+) lineage, presumably specified independently of ThPOK, were generated.Our results suggest that MHC class II-selected thymocytes are directed toward the CD4(+) lineage independently of ThPOK but require ThPOK to prevent Runx-dependent differentiation toward the CD8(+) lineage.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Program, The Helen and Martin Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
The transcription factor ThPOK is required and sufficient for the generation of CD4(+)CD8(-) thymocytes, yet the mechanism by which ThPOK orchestrates differentiation into the CD4(+) helper T cell lineage remains unclear. Here we used reporter mice to track the expression of transcription factors in developing thymocytes. Distal promoter-driven expression of the gene encoding the transcription factor Runx3 was restricted to major histocompatibility complex (MHC) class I-selected thymocytes. In ThPOK-deficient mice, such expression was derepressed in MHC class II-selected thymocytes, which contributed to their redirection to the CD8(+) T cell lineage. In the absence of both ThPOK and Runx, redirection was prevented and cells potentially belonging to the CD4(+) lineage, presumably specified independently of ThPOK, were generated. Our results suggest that MHC class II-selected thymocytes are directed toward the CD4(+) lineage independently of ThPOK but require ThPOK to prevent Runx-dependent differentiation toward the CD8(+) lineage.

Show MeSH
Related in: MedlinePlus