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Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo

Nature Immunology volume 16, pages 267–275 (2015)Cite this article

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Abstract

The quality of the adaptive immune response depends on the differentiation of distinct CD4+ helper T cell subsets, and the magnitude of an immune response is controlled by CD4+Foxp3+ regulatory T cells (Treg cells). However, how a tissue- and cell type–specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell–specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the β-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3+ Treg cell subpopulation that was unable to control the maturation of IRF4+PD-L2+ dendritic cells required for the development of TH2 responses in vivo.

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Figure 1: Mouse and human Treg cells display high activity of CK2.
Figure 2: Ablation of Csnk2b in Treg cells has no effect on their thymic development.
Figure 3: Ablation of Csnk2b in Treg cells results in TH2-type lymphoproliferative disease in the lungs.
Figure 4: Treg cell–specific deficiency in CK2β leads to the spontaneous development of TH2 cells.
Figure 5: Ablation of Csnk2b in Treg cells has no effect on lung-homing ability.
Figure 6: Massive asthma-like airway pathology in Csnk2bfl/flFoxp3-Cre mice without provocation.
Figure 7: CK2 regulates the development of ILT3+ Treg cells.
Figure 8: ILT3+ Treg cells show attenuated TCR signaling.
Figure 9: ILT3+ Treg cells are unable to control IRF4 expression in DCs.

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Acknowledgements

We thank S. Sakaguchi (Osaka University) for BALB/c Foxp3-IRES-Cre mice; B. Boldyreff (University of Southern Denmark) for Csnk2bfl mice on the C57BL/6 background; T. Sparwasser (TWINCORE) for C57BL/6 DEREG mice; K.A. Hogquist (University of Minnesota) for Nur77GFP mice; M. Lohoff (Philipps University Marburg) for C57BL/6 Irf4−/− mice; H.C. Probst (University Medical Center Mainz) for Ly5.1+ C57BL/6 mice; A. Waisman (University Medical Center Mainz) for CD90.1+ C57BL/6 mice; A. Nikolaev and S. Fischer for technical help, and D. O'Neill for critical reading of the manuscript. Supported by Deutsche Forschungsgemeinschaft (DFG BO 3306/1-1, SCHM 1014/7-1, SCHM 1014/5-1, SFB TR128 projects B4 (T.Bop. and F.Z.), A7 (A.W.) and A9 (H.J.), SFB 1066 projects B1 (E.S.) and B8 (T.Bop.), and BR 3754/2-1 (I.H. and M.B.)), International Graduate School of Immunotherapy (GRK 1043 project C4 (E.S. and T.Bop.)) and Forschungszentrum Immunologie of the University Medical Center of the Johannes Gutenberg-University Mainz (E.S. and T.Bop.).

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Author notes

  1. Edgar Schmitt and Tobias Bopp: These authors jointly directed this work.

Authors and Affiliations

  1. Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany

    Alexander Ulges, Matthias Klein, Bastian Gerlitzki, Markus Hoffmann, Nadine Grebe, Valérie Staudt, Natascha Stergiou, Toszka Bohn, Till-Julius Brühl, Sabine Muth, Hajime Yurugi, Krishnaraj Rajalingam, Hans-Christian Probst, Hansjörg Schild, Edgar Schmitt & Tobias Bopp

  2. Department of Pulmonary Medicine, III. Medical Clinic of the University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany

    Sebastian Reuter

  3. Dermatology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany

    Iris Bellinghausen, Andrea Tuettenberg, Susanne Hahn & Helmut Jonuleit

  4. Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany

    Sonja Reißig & Ari Waisman

  5. Department of Immunology and Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

    Irma Haben & Minka Breloer

  6. Department of Neurology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany

    Frauke Zipp

  7. Experimentelle Stammzelltransplantation der Medizinische Klinik und Poliklinik II, Zentrum für Experimentelle Molekulare Medizin, Julius-Maximilians-Universität Würzburg, Würzburg, Germany

    Andreas Beilhack

  8. INSERM, U823, Université Joseph Fourier-Grenoble1, Institut Albert Boniot, Faculté de Médecine, Domaine de la Merci, La Tronche, France

    Thierry Buchou

  9. INSERM U1036, Institute de Recherches en Technologies et Sciences pour le Vivant/Biologie du Cancer et de l'Infection, Commissariat à l'Énergie Atomique et aux Énerigies Alternatives Grenoble, Grenoble, France

    Odile Filhol-Cochet

  10. KinaseDetect, Aarslev, Denmark

    Brigitte Boldyreff

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  1. Alexander Ulges
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Contributions

A.U. performed and analyzed most experiments; M.K., B.G., M.H., N.G., V.S., N.S., T.Boh., T.-J.B., S.M., H.Y., K.R. and H.-C.P. helped design and perform some experiments; S.Reu. helped to perform and analyze mouse asthma experiments; M.K. conducted RNA-seq experiments; I.B., A.T., S.H., H.J. performed experiments involving human T cells; S.Rei. performed and analyzed adoptive transfer colitis experiments; I.H. and M.B. performed and analyzed nematode infection experiments; F.Z., A.W., A.B., T.Bu., O.F.-C., B.B. and H.S. helped design, analyze and interpret experiments; E.S. and T.Bop. supervised the project, designed experiments and wrote the manuscript; and all authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Tobias Bopp.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 CK2β deficiency does not alter the number of peripheral Treg cells.

Percentage of Foxp3+ Treg cells among splenic CD4+ T cells in 8-14 week old Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice (n = 6 per group from 2 independent experiments). Data represent mean +/- SD.

Supplementary Figure 2 Schematic representation of experimental setup for asthma experiments.

At day 0 Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice were either sensitized with 20µg Ovalbumin (OVA) emulsified in 2mg aluminium hydroxide (Alum) (OVA in alum) or, as a control group, received PBS via i.p. injection (PBS). On day 15 and 16 following sensitization all animals were challenged with 1% OVA by ultrasonic nebulization via the airways. At day 18 following sensitization animals were sacrificed and analyzed for several experimental readouts as outlined in online methods. (n = 5 mice per group) Data are representative for two independent experiments.

Supplementary Figure 3 Elevated eosinophilia in Csnk2bfl/flFoxp3-Cre mice.

Total cell count of macrophages (MΦ), lymphocytes (lym), neutrophils (neut) and eosinophils (eo), in bronchoalveolar lavage (BAL) of sensitized (OVA in alum) and respective control (PBS) Csnk2bfl/fl as well as Csnk2bfl/flFoxp3-Cre mice after challenge with Ovalbumin (OVA). (n = 5 mice per group). Data represent mean +/- SD and are representative for two independent experiments.

Supplementary Figure 4 Csnk2b-deficient Treg cells show an unaltered ability to home to the lungs.

(a) Flow cytometric analysis of Foxp3 expression in CD4+ T cells from lungs of Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice upon OVA-challenge. Representative flow cytometry plots are shown for each group. (b) Percentage of Foxp3+ Treg cells among CD4+ T cells in lungs of Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice upon OVA-challenge. (n = 5 mice per group). Data represent mean +/- SD and are representative for two independent experiments. Samples were excluded from analysis, if <500 counts of CD4+Foxp3+ cells could be recorded.

Supplementary Figure 5 Unaltered expression of molecules with a presumed role in Treg cell–mediated suppression in the absence of CK2β.

(a) Mean fluorescence intensity (MFI) of the indicated molecules known to be involved in Treg cell-mediated suppression in CD4+Foxp3+ Treg cells of Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice. MFIs are normalized to the average MFI of Treg cells from Csnk2bfl/fl for each experiment (n = 6 mice per group pooled from 2 independent experiments). Data represent mean +/- SD. (b) Percentage of CD4+Foxp3+ Treg cells from Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice expressing the indicated Molecules involved in Treg cell-mediated suppression (CTLA-4 (surface expression and intracellular expression), Granzyme B, CD39 and CD73). Gating was performed according to isotype control staining (n = 6 mice per group pooled from 2 independent experiments). Data represent mean +/- SD. (c) Percentage of CD4+Foxp3+ Treg cells from Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice expressing components of the IL2-receptor complex (CD25, CD122, common γ chain (γc)). (n = 6 mice per group from 2 independent experiments). Data represent mean +/- SD. (d) Naïve CD4+ T cells (Teff) from C57BL/6 mice were labeled with CFSE and stimulated in coculture with unlabeled Csnk2b-deficient (Csnk2b-/-) or Csnk2b-sufficient (Csnk2b+/+) Treg cells in indicated ratios, as outlined in online methods. On day 4, CFSE fluorescence was measured by FACS analyses. Representatives of three independent experiments are shown.

Supplementary Figure 6 ILT3-expressing Treg cells represent a distinct Treg cell subset that is elevated in TH2 cell–mediated inflammation.

(a) Flow cytometric analysis of ILT3 expression on CD4+Foxp3+ Treg cells from different tissues of Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice. Representative flow cytometry plots and percentages of ILT3+ among CD4+Foxp3+ Treg cells in thymus, mesenteric lymph nodes (mLN), spleen and inguinal lymph nodes (iLN) are shown (n = 11 mice per group for thymus and n = 9 per group for mLN, spleen and iLN, combined from 2 independent experiments). Data show mean +/- SD. (b) Flow cytometric analysis of ILT3 expression in CD4+Foxp3+ Treg cells, stimulated for 3 days with plate-bound anti-CD3 and anti-CD28 in the presence of 300ng/ml mrIL-2 and indicated concentration of CK2 inhibitors (DMAT and CX4945). Treg cells were isolated at day 0 by CD25+ magnetic activated cell sorting from spleens of Csnk2b sufficient C57BL/6 mice. Data are representative for three independent experiments. (c) Flow cytometric analysis of ILT3 expression in CD4+Foxp3+ Treg cells from lungs of non-immunized (PBS) or OVA/Alum immunized C57BL/6 mice upon OVA-challenge. Immunization was performed as described in online methods. Representative flow cytometry plots as well as the percentage of ILT3+ among CD4+Foxp3+ Treg cells are shown. (n = 7 for PBS and n = 8 for OVA/alum treated mice pooled from 2 independent experiments). Data represent mean +/- SD. (d) Flow cytometric analysis of ILT3 expression in splenic CD4+Foxp3+ Treg cells from Non-infected and L. sigmodontis infected BALB/c mice at day 76 after infection. Representative FACS plots as well as the percentage of ILT3+ among CD4+Foxp3+ Treg cells are shown. (n = 10 for non-infected and n = 11 for L. sigmodontis infected group pooled from 2 independent experiments). Data represent mean +/- SD. (e) Total cell count in bronchoalveolar lavage fluid (BAL) of mixed bone marrow chimeras (Csnk2bfl/fl + Csnk2bfl/flFoxp3-Cre BMC) (n = 9) as well as from control Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice (n = 3). Csnk2bfl/fl + Csnk2bfl/flFoxp3-Cre BMC were generated as described in online methods. BMCs were analyzed 11 weeks post transfer. Data represent mean +/- SD from one single experiment. (f) Total cell count of eosinophils in bronchoalveolar lavage fluid (BAL) of Csnk2bfl/fl + Csnk2bfl/flFoxp3-Cre BMC as well as from control Csnk2bfl/fl and Csnk2bfl/flFoxp3-Cre mice. Data represent mean +/- SD from one single experiment. (g) Flow cytometric analysis of activated CD62L-CD4+ T cells in the lungs of Csnk2bfl/fl + Csnk2bfl/flFoxp3-Cre BMC, control Csnk2bfl/fl, and Csnk2bfl/flFoxp3-Cre mice. Percentage of CD62L- among CD4+Foxp3- T cells is shown. Data represent mean +/- SD from one single experiment. (h) Flow cytometric analysis of the origin of ILT3+ Treg cells in Csnk2bfl/fl + Csnk2bfl/flFoxp3-Cre BMC. Given is the percentage of ILT3+ among CD4+Foxp3+ Treg cells derived either from CD90.1+ Csnk2b-competent C57BL/6 wildtype (Csnk2bfl/fl BMC) or CD90.2+ Csnk2b-deficient Csnk2bfl/flFoxp3-Cre bone marrow (Csnk2bfl/flFoxp3-Cre BMC). Individual data points form Csnk2bfl/fl BMC or Csnk2bfl/flFoxp3-Cre BMC of respective BMC mice are shown. * = p<0.05; ** = p<0.01; *** = p<0.001. In case of non gausian distribution Mann-Whitney U test was used instead of students t-test.

Supplementary Figure 7 Csnk2b-deficient Treg cells are unable to suppress TH2 development.

Flow cytometric analysis of GATA-3 expression in Ly5.1+CD4+ T cells stimulated in absence or presence of either Ly5.2+ Csnk2b-competent (Csnk2b+/+) or Csnk2b-deficient (Csnk2b-/-) Treg cells for 96h as outlined in online methods. Shown are representative FACS-plots of GATA-3+ among CD45.1+CD4+ T cells (a) and the percentage of GATA-3+ among CD45.1+CD4+ T cells (b). Data are combined from 4 independent experiments and represent mean +/- SD. * = p<0.05; ** = p<0.001.

Supplementary Figure 8 ILT3+ Treg cells are able to suppress TH1 responses.

Analysis of IFN-γ-producing CD4+CD45.1+ T cells stimulated in vitro under TH1-polarizing conditions in the absence (Teff) or presence of different numbers of either Csnk2b-competent (Csnk2b+/+) or Csnk2b-deficient (Csnk2b-/-) Treg cells (a) and ILT3- or ILT3+ Treg cells from C57BL/6 wildtype mice (b). For each experiment the amount of IFN-γ producers in absence of Treg cells (Teff) was set to 100% and the percentages of IFN-γ-producing TH1 cells in co-cultures were calculated accordingly. Data show mean +/- SD combined from 4 independent experiments (a) and 3 independent experiments (b). (c) Flow cytometric analysis of T-bet expression in CD4+CD44+CD45.1+ T cells in mesenteric lymph nodes of colitis-bearing mice. Given is the percentage of T-bet+ T cells among CD4+CD44+CD45.1+ T cells (n = 4 for Teff, n = 6 for ILT3- Treg cells and n = 7 for ILT3+ Treg cells from one single experiment). Data represent mean +/- SD. (d) Bodyweight changes upon adoptive transfer of CD4+CD62L+ Teff in absence or presence of ILT3-, ILT3+ Treg cells isolated from C57BL/6 wildtype or Treg cells isolated from either Csnk2bfl/flFoxp3-Cre or Csnk2bfl/fl mice into lymphopenic Rag1-/- mice as described in online methods. (n = 18 for Teff, n = 14 for ILT3- Treg cells and ILT3+ Treg cells, n = 5 for Csnk2b-/- Treg cells and n = 7 for Csnk2b+/+ Treg cells from 2 independent experiments. * = p =0.0212; ** = p = 0.0181; *** = p = 0.0101.

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Ulges, A., Klein, M., Reuter, S. et al. Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo. Nat Immunol 16, 267–275 (2015). https://doi.org/10.1038/ni.3083

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