Transcriptional reprogramming of mature CD4+ helper T cells generates distinct MHC class II–restricted cytotoxic T lymphocytes

Journal name:
Nature Immunology
Volume:
14,
Pages:
281–289
Year published:
DOI:
doi:10.1038/ni.2523
Received
Accepted
Published online

Abstract

TCRαβ thymocytes differentiate into either CD8αβ+ cytotoxic T lymphocytes or CD4+ helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II–restricted CD4+ thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4+ T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4+ T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II–restricted CD4+ cytotoxic T lymphocytes.

At a glance

Figures

  1. Some mature CD4+ T cells do not maintain ThPOK expression in the periphery.
    Figure 1: Some mature CD4+ T cells do not maintain ThPOK expression in the periphery.

    (a) Frequency of GFP+ cells (numbers above bracketed lines) among gated CD45+TCRβ+ lymphocytes isolated from the spleen and mLNs of naive Thpok-GFP reporter mice (n = 4 or 5). (b) Frequency of GFP+ cells (numbers above bracketed lines) among gated CD45+TCRβ+ IELs isolated from the small intestine (sIEL) of naive Thpok-GFP reporter mice (n = 4 or 5). (c) Cell surface staining for CD8α and GFP on gated CD45+TCRβ+CD8βCD4+ IELs as in b. Numbers in quadrants indicate percent cells in each throughout. (d) Cell surface staining for CD8α and CD8β on gated CD45+TCRβ+CD4+ IELs as in b. (e) Cell surface staining for CD8α and intracellular for staining granzyme B (Gzmb) on or in gated CD45+TCRβ+CD8βCD4+ lymphocytes isolated from the spleen and mLNs and IELs from the small intestine of naive wild-type C57BL/6 mice (n = 4 or 5). (f) Mean fluorescence intensity (MFI) of granzyme B in CD4+, CD4+CD8α+ or CD8αα+ T cells among gated CD45+TCRβ+CD8β lymphocytes from the spleen and mLNs and IELs of naive wild type C57BL/6 mice (n = 4 or 5); results are presented relative to those of the CD4+CD8α+ subset, set as 100%. *P < 0.05 (analysis of variance (ANOVA) with Bonferroni's post-test). (g) Frequency of CD107+ cells (number in outlined area at right) among total gated CD45+TCRβ+CD8βCD4+ and CD45+TCRβ+CD8αβ+ IELs after stimulation with antibody to TCRβ. (h) Frequency of CD107+ cells among CD4+ splenocytes or TCRγδ+, CD8αα+, CD8αβ+, CD4+CD8α and CD4+CD8α+ IEL subsets after stimulation with antibody to TCRβ (n = 4 replicates). *P = 0.02 (nonparametric two-tailed Mann-Whitney test). (i) Cytotoxicity of lymphocyte subsets, assessed by lactate dehydrogenase–release assay. Data are representative of two (a,b), three (cf) or four (gi) independent experiments (error bars (f,h,i), s.e.m.).

  2. Mature ThPOK- CD4+ T cells are the progeny of ThPOK-expressing thymocytes.
    Figure 2: Mature ThPOK CD4+ T cells are the progeny of ThPOK-expressing thymocytes.

    (a) Endogenous Thpok (left) and the transgene encoding ThPOK-Cre (right) in thymocytes of the CD8 and CD4 lineages. STH, Thpok silencer; PTH, Thpok promoter; E4P4, Cd4 enhancer-promoter; IRES, internal ribosomal entry site; CD2, human gene encoding CD2; PA, poly(A) tail. (b) Staining for CD8α and CD4 (middle) and for human CD2 (hCD2) and YFP (left and right) in thymocytes gated on expression of CD4 and CD8α (gates 1–6 (middle), where 1 represents immature DP thymocytes and 5 and 6 represent mature CD4+ and CD8+ SP thymocytes, respectively), from Rosa26-YFP mice and Rosa26-YFP mice expressing ThPOK-Cre (with the transgene in a; Rosa26-YFP × ThPOK-Cre). Numbers above bracketed lines (left and right) indicate percent cells positive for human CD2 (ThPOK-Cre+; top rows) or YFP+ cells (bottom rows). (c) Staining for CD4 and CD8α (contour plots) and YFP (histograms) in cells from the peripheral lymph nodes (pLN) and IELs from the small intestine of mice as in b, gated on TCRβ+CD4+ lymphocytes. Numbers adjacent to outlined areas indicate percent cells in each. Data are representative of at least three independent experiments.

  3. ThPOK- CD4+ effector cells lost ThPOK as mature cells in the periphery.
    Figure 3: ThPOK CD4+ effector cells lost ThPOK as mature cells in the periphery.

    (a) Staining of CD8α and intracellular IL-17 in gated CD45+TCRβ+CD4+ IELs from the small intestine (sIEL) and large intestine (lIEL) of Rag1−/− recipient mice 8 weeks after adoptive transfer of naive CD45RBhiTCRβ+CD8αCD25CD4+ spleen T cells. (b) Staining of CD8α and CD4 in gated CD45+TCRβ+CD4+ T cells from the spleen and mLNs, lymphocytes from the lamina propria of the small intestine (sLPL) and large intestine (lLPL) and IELs from the small intestine and lage intestine of Rag1−/− recipient mice 8 weeks after adoptive transfer of naive CD45RBhiTCRβ+CD8αCD25CD4 spleen T cells. (c) Expression of Thpok-GFP in gated CD45+TCRβ+ CD4+ (SP) or CD4+CD8α+CD8β (DP) lymphocytes isolated from various tissues (as in a,b) of Rag1−/− recipient mice 8 weeks after transfer of sorted CD45RBhiTCRβ+CD8αCD25CD4+ spleen T cells from Thpok-GFP donor mice. Numbers above bracketed lines indicate percent GFP+ cells (mean ± s.e.m.). (d) Frequency of GFP+ cells among gated CD45+TCRβ+ CD4+ (SP) or CD4+CD8α+CD8β (DP) lymphocytes from spleen or mLNs or IELs of Rag1−/− recipients after transfer of sorted naive CD45RBhiTCRβ+CD8αGFP+CD25CD4+ spleen T cells from Thpok-GFP donor mice. *P < 0.001 (ANOVA and Bonferroni's post-test). (e) Surface staining for CD8α on retransferred sorted (>99.7% purity) CD45+TCRβ+CD8αCD4+ donor IELs isolated from mLNs (pooled from three recipient mice) and IELs of the small intestine another Rag1−/− recipient 8 weeks after the second transfer. Numbers adjacent to outlined areas indicate percent CD8α+ cells (mean ± s.e.m.). (f) ChIP with tiling array of CD4+ SP thymocytes from FH-ThPOK mice. I–V, Cd8 enhancers; green arrowhead indicates binding of ThPOK to the E8I region. (g) Frequency of CD8α+ cells (numbers adjacent to outlined areas) among CD45+TCRβ+CD8βCD4+ IELs isolated from a wild-type mouse (WT) and an E8I-deficient mouse (E8I-KO). (h) Frequency of CD8α+CD4+ IELs from Rag1−/− recipient mice 8 weeks after transfer of wild type or E8I-deficient CD45RBhiTCRβ+CD8αCD25CD4+ spleen T cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.001 (ANOVA and Bonferroni's post-test). Data are representative of three independent experiments (ad; error bars (d), s.e.m.), two independent experiments (e,f,h) or three independent experiments with four or five mice per genotype (g).

  4. Activated CD4+ helper T cells that lose ThPOK expression differentiate into CTLs.
    Figure 4: Activated CD4+ helper T cells that lose ThPOK expression differentiate into CTLs.

    (a) Intracellular staining for IL-17 and GFP in gated CD45+TCRβ+CD4+ IELs from Rag1−/− recipients of sorted naive CD45RBhiTCRβ+CD8αGFP+CD25CD4+ spleen T cells from Thpok-GFP donor mice, assessed 8 weeks after cell transfer. (b) Gene-expression microarray analysis of mRNA from sorted CD45+TCRβ+ ThPOK+CD8αCD4+, ThPOKCD8αCD4+ and ThPOKCD8α+CD4+ IELs from naive Thpok-GFP reporter mice; results are relative to the change in expression in the ThPOK+CD4+CD8α subset. (c) Gene-expression microarray analysis of mRNA from sorted CD45+TCRβ+ ThPOK+CD8αCD4+, ThPOKCD8αCD4+ and ThPOKCD8α+CD4+ IELs from Rag1−/− recipients of sorted naive TCRβ+CD8αGFP+CD45RBhiCD25CD4+ spleen T cells from Thpok-GFP donor mice (presented as in b). (d) Heat map of normalized expression of TH17 signature genes in donor CD4+ T cells as in c, determined by microarray analysis as in b. (e,f) Quantitative real-time PCR analysis of mRNA from TH17-associated genes in sorted T cell subsets as in c (e) or d (f); results are presented relative to those for Rpl32. (g,h) Quantitative real-time PCR analysis of mRNA from CTL-associated genes in sorted T cell subsets as in c (g) or d (h), presented as in e,f. (i) Expression of ThPOK-GFP and 2B4 in gated CD45+TCRβ+CD4+ IELs as in a (left), and quantitative real-time PCR analysis of mRNA encoding CD8α, granzyme B, CRTAM and ThPOK in IELs sorted for (CD45+TCRβ+CD4+) GFP+, GFPCD8α and GFPCD8α+ subsets isolated from Rag1−/− recipients as in a (n = 4; right). (j) Staining for CD4 and CD8α (left and middle) and 2B4 (right) in gated TCRβ+CD4+ IELs from Rag1−/− recipients (n = 3) of untransfected (GFP) or ThPOK-transfected (GFP+) TCRβ+CD8αCD45RBhiCD25CD4+ donor spleen T cells. Data are representative of three (a,ej) or two (bd) independent experiments (mean and s.e.m. in ei).

  5. The Thpok silencer forms the switch that terminates Thpok expression in mature CD4+ T cells.
    Figure 5: The Thpok silencer forms the switch that terminates Thpok expression in mature CD4+ T cells.

    (a) Staining for CD8α and CD4 on gated TCRβ+CD4+ IELs isolated from the small intestine of naive wild type, B2m−/− and ThpokSΔ/SΔB2m−/− mice (left; n = 2 or 3 mice per genotype). Right, frequency of CD8α+CD4+ cells among those TCRβ+CD4+ IELs (each symbol represents an individual mouse; small horizontal lines indicate the mean). *P < 0.0001 (two-tailed unpaired t-test). (b) Staining as in a of IELs isolated from the small intestine of Rag1−/− recipients 4–5 weeks after adoptive transfer of ThpokSfl/Sfl TCRβ+CD4+ cells transfected with an empty retroviral vector encoding GFP alone (EV) or retroviral vector encoding Cre-GFP (Cre), with gating on GFP+ or GFP cells (left). Right, frequency of CD4+CD8α+ cells among those TCRβ+CD4+ IELs (presented as in a). (c) Staining for CD8α and GFP on IELs isolated from the small intestine of Rag1−/− recipients after transfer of sorted naive TCRβ+CD8αGFP+CD45RBhiCD25CD4+ spleen T cells from wild-type donor mice (WT) or MAZR-deficient Thpok-GFP donor mice (MAZR-KO), gated as in a. (d) Frequency of CD8α+GFPCD4+ IELs as in c (presented as in a). *P = 0.03 (two-tailed unpaired t-test). Data are representative of three (a,c,d) or two (b) independent experiments.

  6. The ThPOK loss and reprogramming of CD4+ CTLs is an antigen-driven process in vivo.
    Figure 6: The ThPOK loss and reprogramming of CD4+ CTLs is an antigen-driven process in vivo.

    (a) Frequency of CD4+CD8α+ T cells among gated CD45+TCRβ+CD4+CD8β IELs from wild-type mice or mice deficient in the receptor for IL-7 (Il7ra−/−). Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P = 0.03 (nonparametric two-tailed Mann-Whitney test). (b) BrdU staining of IELs from the small intestine of naive wild type mice after intraperitoneal injection of 1 mg BrdU, followed by 6 d of BrdU (0.8 mg/ml) in the drinking water (left), and staining for Ki67 (middle) and CD69 (right) of CD8α+ or CD8α gated CD45+TCRβ+CD8βCD4+ IELs from naive wild-type mice. (c) Staining for CD8α and Foxp3 in gated CD45+TCRβ+CD8βCD4+ IELs (top) and lymphocytes from the lamia propria (bottom) of OT-II Rag1−/− mice (n = 4) after 4 weeks on an OVA-containing diet. (d) Frequency of CD8α+ and GFP+ cells among CD45+TCRβ+ CD4+ OT-II Thpok-GFP IELs isolated from Rag1−/− recipient mice after 4 weeks on an OVA-containing diet. (e) Frequency of CD107+, IFN-γ+ and TNF+ cells among OT-II Thpok-GFP ThPOK or ThPOK+ CD45+TCRβ+ CD4+ IELs as in d, analyzed at day 0 without IL-15 exposure (0) or after 3 d of in vitro culture with recombinant IL-15 (3) before restimulation with OVA(323–339). (f) Frequency of CD107+, IFN-γ+ and TNF+ cells among wild-type CD8α+ and CD8α CD45+TCRβ+CD8βCD4+ polyclonal IELs cultured without or with IL-15 as in e before restimulation with antibody to TCR-β. Data are representative of two (ac), five (d) or three (e,f) independent experiments.

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Author information

  1. These authors contributed equally to this work.

    • Daniel Mucida,
    • Mohammad Mushtaq Husain,
    • Sawako Muroi &
    • Femke van Wijk

Affiliations

  1. Division of Developmental Immunology, La Jolla Institute for Allergy & Immunology, La Jolla, California, USA.

    • Daniel Mucida,
    • Mohammad Mushtaq Husain,
    • Femke van Wijk,
    • Ryo Shinnakasu,
    • Yujun Huang,
    • Florence Lambolez,
    • Michael Docherty,
    • Antoine Attinger,
    • Jr-Wen Shui,
    • Gisen Kim,
    • Christopher J Lena,
    • Yunji Park,
    • Mitchell Kronenberg &
    • Hilde Cheroutre
  2. Laboratory for Transcriptional Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan.

    • Sawako Muroi,
    • Yoshinori Naoe,
    • Chizuko Miyamoto &
    • Ichiro Taniuchi
  3. Laboratory of Mucosal Immunology, The Rockefeller University, New York, New York, USA.

    • Bernardo Sgarbi Reis
  4. Institute of Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria.

    • Shinya Sakaguchi &
    • Wilfried Ellmeier
  5. Division of Vaccine Discovery, La Jolla Institute for Allergy & Immunology, La Jolla, California, USA.

    • Peng Wang
  6. Department of Immunology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

    • Koji Atarashi &
    • Kenya Honda
  7. Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

    • Toshinori Nakayama
  8. Present addresses: Laboratory of Mucosal Immunology, The Rockefeller University, New York, New York, USA (D.M.), Department of Pediatric Immunology, University Medical Center Utrecht, Wilhelmina Children's Hospital, Utrecht, The Netherlands (F.v.W.), RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan (R.S., K.A. and K.H.), Section of Immunology, Department of Mechanism of Aging, National Center for Geriatrics and Gerontology, Japan (Y.N.), Department of Gastroenterology, University of California San Diego Medical Center, San Diego, California, USA (M.D.), Merck-Serono, Versoix, Switzerland (A.A.), The Center for Nanomedicine, Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai, China (P.W.) and Division of Integrative Bioscience and Biotechnology, Pohang University of Science and Technology, Pohang, Korea (Y.P.).

    • Daniel Mucida,
    • Femke van Wijk,
    • Ryo Shinnakasu,
    • Yoshinori Naoe,
    • Michael Docherty,
    • Antoine Attinger,
    • Peng Wang,
    • Koji Atarashi,
    • Yunji Park &
    • Kenya Honda

Contributions

H.C., I.T., M.K., D.M. and M.M.H. conceived of the project; M.M.H., D.M. and F.v.W. generated the phenotypic and functional data; I.T., S.M., Y.N. and C.M. generated the data on fate mapping and deletion of the Thpok silencer and did the ChIP assays; R.S. transfected cells; Y.H. provided the data on IL-7R-deficient mice; B.S.R., M.D. and A.A. generated the gene arrays; G.K., F.L. and C.J.L. transferred cells and analyzed mice; J.-W.S. and D.M. infected mice with citrobacter; K.A. and K.H. reconstituted germ-free mice; S.S. generated the data on the role of MAZR; Y.P. analyzed Myd88−/− mice; P.W., D.M., F.v.W., B.S.R. and H.C. analyzed the gene-array data; T.N. and W.E. provided expertise; M.K. provided conceptual advice and helped with data analysis and writing of the manuscript; I.T. and H.C. generated concepts, designed experiments, analyzed data and wrote the manuscript; and all authors contributed to the writing of the manuscript and provided advice.

Competing financial interests

The authors declare no competing financial interests.

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