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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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ThPOK is dispensable for differentiation of CD4SP thymocytes(a) HSA and TCRβ expression in total thymocytes (top), and CD4 and CD8 expression in HSAlo/− TCRβhi mature thymocytes (middle) and TCRβ+B220− peripheral T cells (bottom) in the absence of ThPOK, CBFβ, or both. The CD4−CD8+ cells shown with asterisks in Lck-cre+CbfbF/F and Lck-cre+CbfbF/FZbtb7bGFP/− panels are those that escaped Cre-mediated inactivation of Cbfb and hence have normal Cd4 silencing. Data shown here are representative from two independent experiments with similar results. (b) Absolute numbers of HSA+CD69+ positively selected thymocytes and HSAlo/− mature CD4SP thymocytes in the absence of ThPOK, CBFβ, or both. Each column shows cell numbers in a mouse with each of the genotypes.
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Figure 5: ThPOK is dispensable for differentiation of CD4SP thymocytes(a) HSA and TCRβ expression in total thymocytes (top), and CD4 and CD8 expression in HSAlo/− TCRβhi mature thymocytes (middle) and TCRβ+B220− peripheral T cells (bottom) in the absence of ThPOK, CBFβ, or both. The CD4−CD8+ cells shown with asterisks in Lck-cre+CbfbF/F and Lck-cre+CbfbF/FZbtb7bGFP/− panels are those that escaped Cre-mediated inactivation of Cbfb and hence have normal Cd4 silencing. Data shown here are representative from two independent experiments with similar results. (b) Absolute numbers of HSA+CD69+ positively selected thymocytes and HSAlo/− mature CD4SP thymocytes in the absence of ThPOK, CBFβ, or both. Each column shows cell numbers in a mouse with each of the genotypes.

Mentions: Our data indicated a correlation between Runx3 de-repression and redirection of MHCII-restricted thymocytes to the cytotoxic lineage. To determine whether Runx complexes are required for lineage redirection occurring in the absence of ThPOK, we generated mice doubly deficient for ThPOK and CBFβ. We used the Lck-cre transgene to conditionally inactivate Cbfb because its later inactivation in DP thymocytes with Cd4-cre allows generation of CD8+ mature thymocytes (which de-repress CD4)12, most likely due to long half-life of the CBFβ protein. Conditional inactivation of Cbfb with Lck-cre allows thymocytes to proceed through beta selection and phenocopies loss of both Runx1 and Runx3 at the DP stage. HSAhiCD69+ and HSAhiTCRβhi positively selected thymocytes were reduced by approximately 5-fold in Lck-cre+CbfbF/F mice and Lck-cre+CbfbF/FZbtb7bGFP/− mice as compared to littermate control or ThPOK-deficient mice (Fig. 5a,b). Similar to our previous observation in Runx1F/FRunx3F/FCd4-cre+ mice10, CD4−CD8+ mature thymocytes with intact CD4 silencing, that had escaped Cre-mediated inactivation of Cbfb, were present in Lck-cre+CbfbF/F mice. Although almost all of the mature thymocytes were redirected to the CD8SP subset in Lck-cre+Cbfb+/+Zbtb7bGFP/− mice, a majority of the mature HSAlo thymocytes in Lck-cre+CbfbF/FZbtb7bGFP/− mice were CD4+CD8− and were not redirected to the CD8 lineage (Fig. 5a). We also observed a CD4+CD8− T cell population not redirected to the CD8 lineage in lymph nodes of Lck-cre+CbfbF/FZbtb7bGFP/− mice (Fig. 5a). The number of CD4+CD8− thymocytes and peripheral T cells were reduced compared to wild-type mice but were similar in Lck-cre+CbfbF/FZbtb7bGFP/+ and Lck-cre+CbfbF/FZbtb7bGFP/− mice (Fig. 5b). We do not believe that these cells represent a rare population of unconventional CD4SP thymocytes, as iNKT cells do not develop in the absence of Runx complexes27 and Foxp3+ regulatory T cells are severely reduced in both CBFβ-deficient mice and ThPOK-deficient mice (unpublished results). In addition, CD40L (CD154) was up-regulated upon stimulation of CD4+ T cells lacking both transcription factors, consistent with their being helper lineage cells (Supplementary Fig. 6, online). Thus these findings indicate that Runx complexes are required for redirection of MHCII-restricted thymocytes towards the CD8SP lineage in the absence of ThPOK, and ThPOK is not required for CD4 expression in mature MHCII-restricted thymocytes and CD4+ peripheral T cells.


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)

ThPOK is dispensable for differentiation of CD4SP thymocytes(a) HSA and TCRβ expression in total thymocytes (top), and CD4 and CD8 expression in HSAlo/− TCRβhi mature thymocytes (middle) and TCRβ+B220− peripheral T cells (bottom) in the absence of ThPOK, CBFβ, or both. The CD4−CD8+ cells shown with asterisks in Lck-cre+CbfbF/F and Lck-cre+CbfbF/FZbtb7bGFP/− panels are those that escaped Cre-mediated inactivation of Cbfb and hence have normal Cd4 silencing. Data shown here are representative from two independent experiments with similar results. (b) Absolute numbers of HSA+CD69+ positively selected thymocytes and HSAlo/− mature CD4SP thymocytes in the absence of ThPOK, CBFβ, or both. Each column shows cell numbers in a mouse with each of the genotypes.
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Related In: Results  -  Collection

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Figure 5: ThPOK is dispensable for differentiation of CD4SP thymocytes(a) HSA and TCRβ expression in total thymocytes (top), and CD4 and CD8 expression in HSAlo/− TCRβhi mature thymocytes (middle) and TCRβ+B220− peripheral T cells (bottom) in the absence of ThPOK, CBFβ, or both. The CD4−CD8+ cells shown with asterisks in Lck-cre+CbfbF/F and Lck-cre+CbfbF/FZbtb7bGFP/− panels are those that escaped Cre-mediated inactivation of Cbfb and hence have normal Cd4 silencing. Data shown here are representative from two independent experiments with similar results. (b) Absolute numbers of HSA+CD69+ positively selected thymocytes and HSAlo/− mature CD4SP thymocytes in the absence of ThPOK, CBFβ, or both. Each column shows cell numbers in a mouse with each of the genotypes.
Mentions: Our data indicated a correlation between Runx3 de-repression and redirection of MHCII-restricted thymocytes to the cytotoxic lineage. To determine whether Runx complexes are required for lineage redirection occurring in the absence of ThPOK, we generated mice doubly deficient for ThPOK and CBFβ. We used the Lck-cre transgene to conditionally inactivate Cbfb because its later inactivation in DP thymocytes with Cd4-cre allows generation of CD8+ mature thymocytes (which de-repress CD4)12, most likely due to long half-life of the CBFβ protein. Conditional inactivation of Cbfb with Lck-cre allows thymocytes to proceed through beta selection and phenocopies loss of both Runx1 and Runx3 at the DP stage. HSAhiCD69+ and HSAhiTCRβhi positively selected thymocytes were reduced by approximately 5-fold in Lck-cre+CbfbF/F mice and Lck-cre+CbfbF/FZbtb7bGFP/− mice as compared to littermate control or ThPOK-deficient mice (Fig. 5a,b). Similar to our previous observation in Runx1F/FRunx3F/FCd4-cre+ mice10, CD4−CD8+ mature thymocytes with intact CD4 silencing, that had escaped Cre-mediated inactivation of Cbfb, were present in Lck-cre+CbfbF/F mice. Although almost all of the mature thymocytes were redirected to the CD8SP subset in Lck-cre+Cbfb+/+Zbtb7bGFP/− mice, a majority of the mature HSAlo thymocytes in Lck-cre+CbfbF/FZbtb7bGFP/− mice were CD4+CD8− and were not redirected to the CD8 lineage (Fig. 5a). We also observed a CD4+CD8− T cell population not redirected to the CD8 lineage in lymph nodes of Lck-cre+CbfbF/FZbtb7bGFP/− mice (Fig. 5a). The number of CD4+CD8− thymocytes and peripheral T cells were reduced compared to wild-type mice but were similar in Lck-cre+CbfbF/FZbtb7bGFP/+ and Lck-cre+CbfbF/FZbtb7bGFP/− mice (Fig. 5b). We do not believe that these cells represent a rare population of unconventional CD4SP thymocytes, as iNKT cells do not develop in the absence of Runx complexes27 and Foxp3+ regulatory T cells are severely reduced in both CBFβ-deficient mice and ThPOK-deficient mice (unpublished results). In addition, CD40L (CD154) was up-regulated upon stimulation of CD4+ T cells lacking both transcription factors, consistent with their being helper lineage cells (Supplementary Fig. 6, online). Thus these findings indicate that Runx complexes are required for redirection of MHCII-restricted thymocytes towards the CD8SP lineage in the absence of ThPOK, and ThPOK is not required for CD4 expression in mature MHCII-restricted thymocytes and CD4+ peripheral T cells.

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