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A ThPOK-LRF transcriptional node maintains the integrity and effector potential of post-thymic CD4+ T cells

Nature Immunology volume 15, pages 947–956 (2014)Cite this article

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Abstract

The transcription factor ThPOK promotes CD4+ T cell differentiation in the thymus. Here, using a mouse strain that allows post-thymic gene deletion, we show that ThPOK maintains CD4+ T lineage integrity and couples effector differentiation to environmental cues after antigenic stimulation. ThPOK preserved the integrity and amplitude of effector responses and was required for proper differentiation of types 1 and 2 helper T cells in vivo by restraining the expression and function of Runx3, a nuclear factor crucial for cytotoxic T cell differentiation. The transcription factor LRF acts redundantly with ThPOK to prevent the transdifferentiation of mature CD4+ T cells into CD8+ T cells. As such, the ThPOK-LRF transcriptional module was essential for CD4+ T cell integrity and responses.

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Figure 1: Post-thymic ThPOK maintains CD4+ lineage integrity.
Figure 2: ThPOK is not needed for TH17 differentiation.
Figure 3: ThPOK 'protects' TH2 responses.
Figure 4: ThPOK 'protects' TH2 differentiation by restraining Runx functions.
Figure 5: ThPOK antagonism of Runx activity is required for proper TH1 differentiation.
Figure 6: ThPOK is important for CD4+ T cell responses in vivo.
Figure 7: ThPOK and LRF redundantly prevent CD4+ to CD8+ transdifferentiation.

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Acknowledgements

We thank T. Ciucci for experimental assistance and discussions; T.-A. Lewis for animal care and genotyping; M. McGinty and Q. Xiao for technical assistance; X. Wu for microarray analyses; D. Littman, P.P. Pandolfi and S. Reiner for mice; M. Grigg for the T. gondii strain; J. Grainger, T. Hand and S. Spencer for discussions; and J. Ashwell and J. Brenchley for reading the manuscript. Supported by the US National Institutes of Health Intramural Research Programs of the National Cancer Institute, Center for Cancer Research, the National Institute of Allergy and Infectious Diseases and the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

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Author notes

  1. Lie Wang, Elizabeth Wohlfert & Ki-Duk Song

    Present address: Present addresses: Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, China (L.W.), Department of Microbiology and Immunology, University at Buffalo School of Medicine and Biomedical Sciences, Buffalo, New York, USA (E.W.), Center for Agricultural Biomaterials, Seoul National University, Seoul, South Korea (K.-D. S.).,

Authors and Affiliations

  1. Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Melanie S Vacchio, Lie Wang, Andrea C Carpenter, Yumei Xiong, Linus C Williams & Rémy Bosselut

  2. Mucosal Immunology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Nicolas Bouladoux, Elizabeth Wohlfert & Yasmine Belkaid

  3. Section on Cellular and Developmental Biology, Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

    Ki-Duk Song & Paul E Love

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Contributions

M.S.V., L.W., Y.B. and R.B. designed the research; M.S.V., L.W., N.B, A.C.C., Y.X., L.C.W. and E.W. performed and analyzed experiments; K.-D.S. and P.E.L. constructed and provided CD2-Cre mice; R.B. supervised the research; M.S.V. and R.B. wrote the manuscript.

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Correspondence to Rémy Bosselut.

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Integrated supplementary information

Supplementary Figure 1 Analyses of resting cell populations in Thpokpd mice.

(a) Percentage of Rosa26YFP-expressing cells among CD4 or CD8SP thymocytes (Th) or spleen T cells (Spl) from CD2-Cre transgenic mice. (b,c) Plots indicate numbers (× 106) of total thymocytes (b, top), gated thymocytes (b, bottom three rows) and gated splenocytes (c). Each symbol represents one 6–8 week-old mouse. (a–c) Data are representative of four independent experiments. Thpok +/+ and Thpokpd mice are littermates to control for background effects. Significance was determined by Student’s t-test (*, P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, not significant in other cases). (d) Percent of Rosa26YFP-expressing cells among Foxp3-expressing CD4SP thymocytes or CD4+ spleen T cells from Thpokpd mice. Each symbol represents one mouse; data are from three experiments. (e) Immunoblot analysis of Runx protein expression in sorted spleen naïve CD4+ or CD8+ T from Thpokpd or Thpok+/+ littermate mice. The same membrane was subsequently stripped and reprobed for β-actin expression to evaluate lane loading. Representative of three independent experiments. (f) Contours plots show CD4 versus CD8 expression on total (left) or TCRβhiCD24lo (right) thymocytes from 6–9 week-old littermate mice of the indicated genotype. Representative of three experiments. (g) Absolute numbers of mature (TCRhi CD24lo) CD4SP thymocytes in mice of the indicated genotype. Each symbol represents one mouse. No significant difference among genotypes.

Supplementary Figure 2 TH17 differentiation of Thpokpd cells.

(a,b) Graphs show cumulative results for colon length (a) and mouse weight (b) from two independent C. rodentium infection experiments. Mean ± s.e.m. is shown. Each symbol represents a mouse. No significance by t-test. (c) ELISA quantification of IL-17 secretion by Thpokpd and Thpok+/+ cells activated under non-polarizing (THN), TH1 or TH17 conditions. Each symbol represents a separate culture. Data are from three mice of each genotype analyzed separately. Bars indicate mean ± s.e.m. No significance by t-test between Thpok+/+ and Thpokpd. (d) Histograms show GzmB expression in T effector cells obtained after 5-day cultures in TH17 conditions of sorted CD4+CD44lo spleen T cells from Thpok+/+ (solid grey) or Thpokpd (plain line) mice. The dashed line histogram shows GzmB expression on wild-type CD8+ effector cells cultured under THN conditions. Representative of three independent experiments.

Supplementary Figure 3 TH2 differentiation of Thpokpd cells.

(a) ELISA quantification of IL-4 secretion by Thpokpd and Thpok+/+ cells activated under nonpolarizing conditions as in Fig. 3e. Each symbol represents a separate culture. Data are from three distinct sorting experiments, each with a distinct mouse from each genotype. The average IL-4 concentrations were 0.17 ng/ml for Thpok+/+ cells and <0.01 ng/ml for Thpokpd cells (*P < 0.005 by paired t-test). (b) Scatter plots show the percentage of IL-4 or IFNγ producers among Thpokpd or Thpok+/+ cells cultured under non polarizing conditions as in Fig. 3e. Each symbol represents a culture derived from a single mouse. Statistical significance was determined by t-test (*P < 0.005; **P < 10–4).

Supplementary Figure 4 Functional relationships between ThPOK and Eomes and Cbfβ.

(a) Scatter plots show the percentage of IL 4- or IFNγ-producing cells among Thpok+/+, Thpokpd, Thpokpd Cbfbpd or Thpokpd Eomespd cells cultured under nonpolarizing conditions as in Fig. 3e. Each symbol represents a culture derived from a single mouse. Statistical significance was determined by t-test (*P <0.05; **P < 0.025; ***P < 0.01) on pooled data from three independent experiments. Contours plots (b) show CD4 versus CD8 expression on total (left) or TCRβhiCD24lo (center) thymocytes, or on TCRβ+ spleen cells (right) from 5 week old Thpok+/+Eomes+/+, Thpokpd Eomes+/+ or ThpokpdEomespd mice. Representative of three or four mice for each genotype from two independent experiments. (c) Graphs show the absolute numbers of CD4+CD8– and CD8+ T cells in the spleens from mice shown in b. There was no significant difference in the size of either subset among the three genotypes.

Supplementary Figure 5 Analyses of gene expression in Thpokpd effector cells.

(a) Contour plots show CD8 versus CD4 expression in Thpok+/+ and Thpokpd effector T cells derived from naïve CD4+CD8– cells as in Fig. 5b. (b) Heat map indicates relative expression (log2 values) on genes with 3-fold or greater change in expression in Thpokpd CD4+CD8+ vs. wild-type CD4+CD8– effector T cells.

Supplementary Figure 6 Post-thymic ThPOK restrains cytotoxic gene expression during in vivo TH1 responses.

(a) Contour plots assess uninfected (left) or day 10-infected (right) Thpok+/+ or Thpokpd spleen CD4+ T cells for expression of intra-cellular IFN-γ versus IL-17A. (b) Parasite load was quantified as percentage of RFP+ splenocytes in mice infected with RFP-expressing T. gondii. Each symbol represents one mouse; data are from two independent experiments. No significant difference by t-test. (c) Contour plots of Eomes versus T-bet expression in spleen CD4+ T cells from uninfected (left) or day 10-infected (right) Thpok+/+ or Thpokpd mice. Data (a,c) are representative of three experiments for each panel.

Supplementary Figure 7 Overlapping functions of LRF and ThPOK in CD4 expression.

(a) Histogram overlays show CD4 expression on sorted CD4lo (1), CD4+ CD8– (2) CD44lo spleen T cells from Thpokpd mice or their Thpok+/+ CD4+ CD8- counterparts (3) ex vivo (left panel), or after 5 d culture under TH1 conditions (right). Representative of three experiments. (b,c) Contours plots assess 6–8-week-old littermates of the indicated genotype for expression of CD4 and CD8 on total (left) and TCRβhiCD24lo (right) thymocytes (b), or (c) on total (left) or gated TCR+ YFP+ CD44lo (right) spleen T cells. Figures are representative of three experiments, summarized in d. (d) Plot summarizes numbers (× 106) of mature (TCRhi CD24lo) CD4 and CD8SP thymocytes (top) and of CD4+ (including CD4lo and pDP) and CD8+ T cells from 6–8 week-old mice of the indicated genotype. Significance was determined by Student’s t-test (*P < 0.05, ** P < 0.01; not significant otherwise). (e) Immunoblot analysis of Runx protein expression in effector T cells obtained after 5-d culture under TH1 conditions from naïve CD8+ T cells from Thpok+/+ Zbtb7a+/+ mice or CD4+ T cells from Thpokpd Zbtb7a+/+ or Thpokpd Zbtb7apd mice. The same membrane was subsequently stripped and reprobed for β-actin expression to evaluate lane loading. Representative of two immunoblots.

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Vacchio, M., Wang, L., Bouladoux, N. et al. A ThPOK-LRF transcriptional node maintains the integrity and effector potential of post-thymic CD4+ T cells. Nat Immunol 15, 947–956 (2014). https://doi.org/10.1038/ni.2960

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