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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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De-repression of CD8+ lineage-specific genes in MHCII-restricted cells in the absence of ThPOK or in the presence of insufficient amount of ThPOK(a) MHCII-restricted HSAhiCD69+CD4+CD8lo/−ThPOK-GFPhi thymocytes from Zbtb7bGFP/+ (black columns) or Zbtb7bGFP/− (white columns) were purified using gates as shown in Fig. 3, and expression of CD4+ lineage-specific genes (Cd4, St8si6, Zbtb7b and Gata3) and CD8+ lineage-specific genes (Cd8a, Itgae, Prf1, Gpr114, Nkg7 and Cd160) was examined by qRT-PCR. mRNA expression of individual genes was normalized against Hprt1 expression and average expression in Zbtb7bGFP/+ cells was set as 1. (b) Intracellular staining for IFN-γ and IL-4 in Zbtb7bFN/− CD4+CD8− T cells following 3 days of stimulation with anti-CD3 and anti-CD28 in the absence of IL-12. (c) Expression of the CD8+ lineage-specific IFN-γ regulator Eomes in CD4+ T cells from wild-type and Zbtb7bFN/− mice and in wild-type CD8+ T cells, as quantified by q-RT-PCR. Hprt1-normalized Eomes mRNA expression is shown as average and standard deviations from three independent samples. Statistical difference was tested by two-tailed T test with assumption of unequal variance. Genes that showed a P value smaller than 0.05 are marked with asterisks in (a). Actual P values for individual genes were as follows: Cd4: 0.28, St8sia6: 0.13, Zbtb7b: 0.01, Gata3: 0.5, Cd8a: 0.04, Itgae: 0.04, Prf1: 0.01, Gpr114: 0.09, Nkg7: 0.04, Cd160: 0.02.
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Figure 6: De-repression of CD8+ lineage-specific genes in MHCII-restricted cells in the absence of ThPOK or in the presence of insufficient amount of ThPOK(a) MHCII-restricted HSAhiCD69+CD4+CD8lo/−ThPOK-GFPhi thymocytes from Zbtb7bGFP/+ (black columns) or Zbtb7bGFP/− (white columns) were purified using gates as shown in Fig. 3, and expression of CD4+ lineage-specific genes (Cd4, St8si6, Zbtb7b and Gata3) and CD8+ lineage-specific genes (Cd8a, Itgae, Prf1, Gpr114, Nkg7 and Cd160) was examined by qRT-PCR. mRNA expression of individual genes was normalized against Hprt1 expression and average expression in Zbtb7bGFP/+ cells was set as 1. (b) Intracellular staining for IFN-γ and IL-4 in Zbtb7bFN/− CD4+CD8− T cells following 3 days of stimulation with anti-CD3 and anti-CD28 in the absence of IL-12. (c) Expression of the CD8+ lineage-specific IFN-γ regulator Eomes in CD4+ T cells from wild-type and Zbtb7bFN/− mice and in wild-type CD8+ T cells, as quantified by q-RT-PCR. Hprt1-normalized Eomes mRNA expression is shown as average and standard deviations from three independent samples. Statistical difference was tested by two-tailed T test with assumption of unequal variance. Genes that showed a P value smaller than 0.05 are marked with asterisks in (a). Actual P values for individual genes were as follows: Cd4: 0.28, St8sia6: 0.13, Zbtb7b: 0.01, Gata3: 0.5, Cd8a: 0.04, Itgae: 0.04, Prf1: 0.01, Gpr114: 0.09, Nkg7: 0.04, Cd160: 0.02.

Mentions: The finding of Runx3d de-repression in ThPOK-deficient MHCII-restricted thymocytes raises the possibility that other CD8+ lineage-specific genes may also be de-repressed in these cells. To assess this possibility, we compared gene expression between CD4+CD8−HSAhiGFP+ thymocytes from Zbtb7bGFP/− and Zbtb7bGFP/+ mice. qRT-PCR analysis showed that Zbtb7bGFP/− cells expressed significantly higher amounts of transcripts that are normally expressed in the CD8SP lineage of wild-type mice, including Cd8a, Itgae, Nkg7, Cd160 and Prf1 (Fig. 6a). In addition, unlike wild-type CD4+ T cells, a majority of Zbtb7bFN/− CD4+ T cells produced IFN-γ when stimulated with anti-CD3 and anti-CD28 in the absence IL-12 (Fig. 6b). Expression of the CD8 lineage-specific IFN-γ–regulating transcription factor Eomes was significantly elevated in Zbtb7bFN/− compared to wild-type CD4+ T cells (Fig. 6c). These findings suggest that a high amount of ThPOK expression is required during development of CD4+ T cells to block the CD8SP lineage-specific gene expression program.


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)

De-repression of CD8+ lineage-specific genes in MHCII-restricted cells in the absence of ThPOK or in the presence of insufficient amount of ThPOK(a) MHCII-restricted HSAhiCD69+CD4+CD8lo/−ThPOK-GFPhi thymocytes from Zbtb7bGFP/+ (black columns) or Zbtb7bGFP/− (white columns) were purified using gates as shown in Fig. 3, and expression of CD4+ lineage-specific genes (Cd4, St8si6, Zbtb7b and Gata3) and CD8+ lineage-specific genes (Cd8a, Itgae, Prf1, Gpr114, Nkg7 and Cd160) was examined by qRT-PCR. mRNA expression of individual genes was normalized against Hprt1 expression and average expression in Zbtb7bGFP/+ cells was set as 1. (b) Intracellular staining for IFN-γ and IL-4 in Zbtb7bFN/− CD4+CD8− T cells following 3 days of stimulation with anti-CD3 and anti-CD28 in the absence of IL-12. (c) Expression of the CD8+ lineage-specific IFN-γ regulator Eomes in CD4+ T cells from wild-type and Zbtb7bFN/− mice and in wild-type CD8+ T cells, as quantified by q-RT-PCR. Hprt1-normalized Eomes mRNA expression is shown as average and standard deviations from three independent samples. Statistical difference was tested by two-tailed T test with assumption of unequal variance. Genes that showed a P value smaller than 0.05 are marked with asterisks in (a). Actual P values for individual genes were as follows: Cd4: 0.28, St8sia6: 0.13, Zbtb7b: 0.01, Gata3: 0.5, Cd8a: 0.04, Itgae: 0.04, Prf1: 0.01, Gpr114: 0.09, Nkg7: 0.04, Cd160: 0.02.
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Figure 6: De-repression of CD8+ lineage-specific genes in MHCII-restricted cells in the absence of ThPOK or in the presence of insufficient amount of ThPOK(a) MHCII-restricted HSAhiCD69+CD4+CD8lo/−ThPOK-GFPhi thymocytes from Zbtb7bGFP/+ (black columns) or Zbtb7bGFP/− (white columns) were purified using gates as shown in Fig. 3, and expression of CD4+ lineage-specific genes (Cd4, St8si6, Zbtb7b and Gata3) and CD8+ lineage-specific genes (Cd8a, Itgae, Prf1, Gpr114, Nkg7 and Cd160) was examined by qRT-PCR. mRNA expression of individual genes was normalized against Hprt1 expression and average expression in Zbtb7bGFP/+ cells was set as 1. (b) Intracellular staining for IFN-γ and IL-4 in Zbtb7bFN/− CD4+CD8− T cells following 3 days of stimulation with anti-CD3 and anti-CD28 in the absence of IL-12. (c) Expression of the CD8+ lineage-specific IFN-γ regulator Eomes in CD4+ T cells from wild-type and Zbtb7bFN/− mice and in wild-type CD8+ T cells, as quantified by q-RT-PCR. Hprt1-normalized Eomes mRNA expression is shown as average and standard deviations from three independent samples. Statistical difference was tested by two-tailed T test with assumption of unequal variance. Genes that showed a P value smaller than 0.05 are marked with asterisks in (a). Actual P values for individual genes were as follows: Cd4: 0.28, St8sia6: 0.13, Zbtb7b: 0.01, Gata3: 0.5, Cd8a: 0.04, Itgae: 0.04, Prf1: 0.01, Gpr114: 0.09, Nkg7: 0.04, Cd160: 0.02.
Mentions: The finding of Runx3d de-repression in ThPOK-deficient MHCII-restricted thymocytes raises the possibility that other CD8+ lineage-specific genes may also be de-repressed in these cells. To assess this possibility, we compared gene expression between CD4+CD8−HSAhiGFP+ thymocytes from Zbtb7bGFP/− and Zbtb7bGFP/+ mice. qRT-PCR analysis showed that Zbtb7bGFP/− cells expressed significantly higher amounts of transcripts that are normally expressed in the CD8SP lineage of wild-type mice, including Cd8a, Itgae, Nkg7, Cd160 and Prf1 (Fig. 6a). In addition, unlike wild-type CD4+ T cells, a majority of Zbtb7bFN/− CD4+ T cells produced IFN-γ when stimulated with anti-CD3 and anti-CD28 in the absence IL-12 (Fig. 6b). Expression of the CD8 lineage-specific IFN-γ–regulating transcription factor Eomes was significantly elevated in Zbtb7bFN/− compared to wild-type CD4+ T cells (Fig. 6c). These findings suggest that a high amount of ThPOK expression is required during development of CD4+ T cells to block the CD8SP lineage-specific gene expression program.

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