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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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Runx3d and ThPOK reporter expression in developing thymocytes(a) Distal promoter-derived Runx3 expression determined by the Runx3d-YFP reporter in thymocyte subpopulations as defined in Supplementary Fig. 2, online. YFP+ cells are gated with orange rectangles. (b) ThPOK-GFP reporter expression in developing thymocytes. GFPhi and GFPlo populations are gated with dark and light blue rectangles, respectively. Percentages indicate frequencies of YFP+, GFPhi, and GFPlo cells among thymocyte subsets marked at top, except for those in the CD4+CD8+ subpopulation, for which percentages correspond to frequencies of gated cells among CD4+CD8+TCRβhi thymocytes (marked with asterisks). (c) ThPOK-GFP reporter expression in MHCI- and MHCII-restricted CD4+CD8lo thymocyte subpopulations. The percentages of total GFP+ cells (bottom gate) and GFPhi cells (top gate) are shown. (d) Mutually exclusive high ThPOK-GFP and Runx3d-YFP expression during thymocyte differentiation. ThPOK-GFP and Runx3d-YFP expression in developing thymocytes from indicated mice. Numbers indicate percentages of cells within gates. (e) Continued ThPOK-GFP expression in redirected MHCII-restricted ThPOK-deficient CD8+ T cells. ThPOK-GFP expression in CD8+ T cells from Zbtb7bGFP/+ or Zbtb7bGFP/− mice. Numbers indicate percentages of GFP+ cells. Data shown are representative of more than 3 independent experiments.
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Figure 2: Runx3d and ThPOK reporter expression in developing thymocytes(a) Distal promoter-derived Runx3 expression determined by the Runx3d-YFP reporter in thymocyte subpopulations as defined in Supplementary Fig. 2, online. YFP+ cells are gated with orange rectangles. (b) ThPOK-GFP reporter expression in developing thymocytes. GFPhi and GFPlo populations are gated with dark and light blue rectangles, respectively. Percentages indicate frequencies of YFP+, GFPhi, and GFPlo cells among thymocyte subsets marked at top, except for those in the CD4+CD8+ subpopulation, for which percentages correspond to frequencies of gated cells among CD4+CD8+TCRβhi thymocytes (marked with asterisks). (c) ThPOK-GFP reporter expression in MHCI- and MHCII-restricted CD4+CD8lo thymocyte subpopulations. The percentages of total GFP+ cells (bottom gate) and GFPhi cells (top gate) are shown. (d) Mutually exclusive high ThPOK-GFP and Runx3d-YFP expression during thymocyte differentiation. ThPOK-GFP and Runx3d-YFP expression in developing thymocytes from indicated mice. Numbers indicate percentages of cells within gates. (e) Continued ThPOK-GFP expression in redirected MHCII-restricted ThPOK-deficient CD8+ T cells. ThPOK-GFP expression in CD8+ T cells from Zbtb7bGFP/+ or Zbtb7bGFP/− mice. Numbers indicate percentages of GFP+ cells. Data shown are representative of more than 3 independent experiments.

Mentions: To investigate regulation of Runx3d expression in developing thymocytes, we next examined YFP expression in a series of intermediate thymocyte subsets defined by CD4, CD8, TCRβ, CD69 and CD24 (HSA) expression (Fig. 2a and Supplementary Fig. 2a,b, online). YFP+ cells were not detected in pre-selected CD69− DP thymocytes (data not shown). Following positive selection, MHCI- and MHCII-selected thymocytes both transit through an intermediate CD69+HSAhiTCRβintCD4+CD8lo (referred to hereafter as CD4+CD8lo) stage (Supplementary Fig. 2c, online) 19–25. At this stage, we observed 2–5 % of cells expressing YFP (Fig. 2a and Supplementary Fig. 2d, online). These YFP+ cells were absent in Runx3dYFP/+ mice on a B2m−/− background, indicating that Runx3d transcription is activated only in positively selected MHCI-restricted thymocytes (Supplementary Fig. 2d, online). Following the CD4+CD8lo stage, MHCI-selected thymocytes become CD4+CD8+CD69+TCRβhi, and such cells are absent in mice lacking β2-microglobulin, an essential component of MHCI molecules (B2m−/−) (Supplementary Fig. 2e, online). In contrast, MHCII-restricted thymocytes continue to down-regulate surface CD8 expression to become CD4+CD8− thymocytes before down-regulation of HSA. A majority of CD4+CD8+CD69+TCRβhi thymocytes expressed YFP, and cells with the highest surface TCRβ expression were brightest for YFP (Fig. 2a). Mature HSAlo/− CD8SP thymocytes expressed a uniformly high amount of YFP (Fig. 2a). YFP expression was not detected in the MHCII-restricted CD4+CD8−HSAhi population. These results indicate that Runx3d-YFP expression is highly restricted to MHCI-selected thymocytes, that it occurs largely following transit from the CD4+CD8lo stage, and that it specifically marks developing 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)

Runx3d and ThPOK reporter expression in developing thymocytes(a) Distal promoter-derived Runx3 expression determined by the Runx3d-YFP reporter in thymocyte subpopulations as defined in Supplementary Fig. 2, online. YFP+ cells are gated with orange rectangles. (b) ThPOK-GFP reporter expression in developing thymocytes. GFPhi and GFPlo populations are gated with dark and light blue rectangles, respectively. Percentages indicate frequencies of YFP+, GFPhi, and GFPlo cells among thymocyte subsets marked at top, except for those in the CD4+CD8+ subpopulation, for which percentages correspond to frequencies of gated cells among CD4+CD8+TCRβhi thymocytes (marked with asterisks). (c) ThPOK-GFP reporter expression in MHCI- and MHCII-restricted CD4+CD8lo thymocyte subpopulations. The percentages of total GFP+ cells (bottom gate) and GFPhi cells (top gate) are shown. (d) Mutually exclusive high ThPOK-GFP and Runx3d-YFP expression during thymocyte differentiation. ThPOK-GFP and Runx3d-YFP expression in developing thymocytes from indicated mice. Numbers indicate percentages of cells within gates. (e) Continued ThPOK-GFP expression in redirected MHCII-restricted ThPOK-deficient CD8+ T cells. ThPOK-GFP expression in CD8+ T cells from Zbtb7bGFP/+ or Zbtb7bGFP/− mice. Numbers indicate percentages of GFP+ cells. Data shown are representative of more than 3 independent experiments.
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Figure 2: Runx3d and ThPOK reporter expression in developing thymocytes(a) Distal promoter-derived Runx3 expression determined by the Runx3d-YFP reporter in thymocyte subpopulations as defined in Supplementary Fig. 2, online. YFP+ cells are gated with orange rectangles. (b) ThPOK-GFP reporter expression in developing thymocytes. GFPhi and GFPlo populations are gated with dark and light blue rectangles, respectively. Percentages indicate frequencies of YFP+, GFPhi, and GFPlo cells among thymocyte subsets marked at top, except for those in the CD4+CD8+ subpopulation, for which percentages correspond to frequencies of gated cells among CD4+CD8+TCRβhi thymocytes (marked with asterisks). (c) ThPOK-GFP reporter expression in MHCI- and MHCII-restricted CD4+CD8lo thymocyte subpopulations. The percentages of total GFP+ cells (bottom gate) and GFPhi cells (top gate) are shown. (d) Mutually exclusive high ThPOK-GFP and Runx3d-YFP expression during thymocyte differentiation. ThPOK-GFP and Runx3d-YFP expression in developing thymocytes from indicated mice. Numbers indicate percentages of cells within gates. (e) Continued ThPOK-GFP expression in redirected MHCII-restricted ThPOK-deficient CD8+ T cells. ThPOK-GFP expression in CD8+ T cells from Zbtb7bGFP/+ or Zbtb7bGFP/− mice. Numbers indicate percentages of GFP+ cells. Data shown are representative of more than 3 independent experiments.
Mentions: To investigate regulation of Runx3d expression in developing thymocytes, we next examined YFP expression in a series of intermediate thymocyte subsets defined by CD4, CD8, TCRβ, CD69 and CD24 (HSA) expression (Fig. 2a and Supplementary Fig. 2a,b, online). YFP+ cells were not detected in pre-selected CD69− DP thymocytes (data not shown). Following positive selection, MHCI- and MHCII-selected thymocytes both transit through an intermediate CD69+HSAhiTCRβintCD4+CD8lo (referred to hereafter as CD4+CD8lo) stage (Supplementary Fig. 2c, online) 19–25. At this stage, we observed 2–5 % of cells expressing YFP (Fig. 2a and Supplementary Fig. 2d, online). These YFP+ cells were absent in Runx3dYFP/+ mice on a B2m−/− background, indicating that Runx3d transcription is activated only in positively selected MHCI-restricted thymocytes (Supplementary Fig. 2d, online). Following the CD4+CD8lo stage, MHCI-selected thymocytes become CD4+CD8+CD69+TCRβhi, and such cells are absent in mice lacking β2-microglobulin, an essential component of MHCI molecules (B2m−/−) (Supplementary Fig. 2e, online). In contrast, MHCII-restricted thymocytes continue to down-regulate surface CD8 expression to become CD4+CD8− thymocytes before down-regulation of HSA. A majority of CD4+CD8+CD69+TCRβhi thymocytes expressed YFP, and cells with the highest surface TCRβ expression were brightest for YFP (Fig. 2a). Mature HSAlo/− CD8SP thymocytes expressed a uniformly high amount of YFP (Fig. 2a). YFP expression was not detected in the MHCII-restricted CD4+CD8−HSAhi population. These results indicate that Runx3d-YFP expression is highly restricted to MHCI-selected thymocytes, that it occurs largely following transit from the CD4+CD8lo stage, and that it specifically marks developing 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