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A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1

Nature Immunology volume 17, pages 825–833 (2016)Cite this article

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An Erratum to this article was published on 19 July 2016

This article has been updated

Abstract

Signaling via the inducible costimulator ICOS fuels the stepwise development of follicular helper T cells (TFH cells). However, a signaling pathway unique to ICOS has not been identified. We found here that the kinase TBK1 associated with ICOS via a conserved motif, IProx, that shares homology with the tumor-necrosis-factor receptor (TNFR)-associated factors TRAF2 and TRAF3. Disruption of this motif abolished the association of TBK1 with ICOS, TRAF2 and TRAF3, which identified a TBK1-binding consensus. Alteration of this motif in ICOS or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH cells and the development of GCs, interfered with B cell differentiation and disrupted the development of antibody responses, but the IProx motif and TBK1 were dispensable for the early differentiation of TFH cells. These results reveal a previously unknown ICOS-TBK1 signaling pathway that specifies the commitment of GC TFH cells.

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Figure 1: Importance of the newly identified ICOS signaling motif in TFH cell development.
Figure 2: ICOS-TBK1 interaction.
Figure 3: TBK1 is required for TFH cell differentiation.
Figure 4: The IProx ICOS motif is dispensable for the development of nascent TFH cells.
Figure 5: TBK1 is dispensable for the differentiation of nascent TFH cells.
Figure 6: Molecular basis of the ICOS-TBK1 interaction.

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Change history

  • 20 May 2016

    In the version of this article initially published online, in the keys in Figure 3i–l, the Icos-specific shRNA was incorrectly designated 'sh/cos'. The correct designation is 'shIcos'. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank members of the Altman and Crotty laboratories for discussions; the Flow Cytometry Core Unit, the Microscopy Unit and the Animal Husbandry Unit of the La Jolla Institute for Allergy and Immunology for services; P. Beemiller for imaging and bioinformatics services and assistance in computational analyses of immunohistochemistry with Matlab software; and the National Disease Resource Interchange for clinical samples. Supported by the US National Institutes of Health (CA35299 to A.A.; and AI109976, AI063107 and AI072543 to S.C.) and the Melanoma Research Alliance (Young Investigator Award 270056 to K.-F.K.). This is publication number 1794 from the La Jolla Institute for Allergy and Immunology.

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  1. Yaoyang Zhang & Youn Soo Choi

    Present address: Present addresses: Interdisciplinary Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China (Y.Z.), and Department of Medicine, Transplantation Research Institute, Seoul National University College of Medicine, Seoul, South Korea (Y.S.C.).,

  2. Amnon Altman, Shane Crotty and Kok-Fai Kong: These authors jointly directed this work.

Authors and Affiliations

  1. Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Christophe Pedros, Ann J Canonigo-Balancio, Amnon Altman & Kok-Fai Kong

  2. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA

    Yaoyang Zhang & John R Yates III

  3. Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Joyce K Hu, Youn Soo Choi & Shane Crotty

  4. Division of Infectious Diseases, School of Medicine, University of California San Diego, La Jolla, California, USA

    Shane Crotty

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Contributions

C.P. and A.J.C.-B. designed experiments, collected data and performed analyses; Y.Z. and J.R.Y. did the proteomics experiments and analyses; J.K.H. did the immunofluorescence and microscopy; Y.S.C. provided reagents and was involved in study design; and A.A., S.C. and K.-F.K. designed the study, analyzed data and wrote the paper.

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Correspondence to Shane Crotty or Kok-Fai Kong.

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Integrated supplementary information

Supplementary Figure 1 Evolutionary conservation of the ICOS cytoplamic tail, and of the ‘serine tongs’ of TRAF2 and TRAF3.

Amino acid sequences of the cytoplamic tail of putative ICOS orthologs from the indicated organisms are shown in a. The conserved proximal (IProx, in red), PI3K-binding YxxM (in blue) and distal (in brown) motifs are indicated. (b,c) Protein sequences of putative TRAF2 (b) and TRAF3 (c) orthologs from indicated organisms were aligned with the IProx motif of human ICOS. Conserved amino acid residues between IProx and TRAFs are indicated in red.

Supplementary Figure 2 Surface expression of reconstituted ICOS and efficiency of the in vivo knockdown of Icos and Tbk1.

(a) Histogram of GFP+ CD4+ T cells from host B6 mice 7 d after adoptive transfer of Icos−/– SMARTA CD4+ T cells transduced with RV encoding empty vector (EV) or wild-type ICOS (WT) or mIProx, YF or TL mutant and infected with LCMV Armstrong strain. Cumulative data for a (b) from two independent experiments. (c) Schematic representation of mouse Tbk1 transcript of 2750 bp along with the open reading frame (blue arrow). Regions targeted by shTbk1-1 and shTbk1-2 are indicated with short red lines. Diagram not drawn to scale. (d – g) Quantitation of Icos (d, e) and Tbk1 (f, g) transcripts in SMARTA CD4+ T cells from host B6 mice 3 d (d, f) or 7 d (e, g) after adoptive transfer of SMARTA CD4+ T cells transduced with shRNA targeting the Tbk1 (shTbk1-1 and shTbk1-2), Icos or control genes and infected with LCMV Armstrong strain. Shown are the fold-change (mean ± SEM) from two independent experiments. Each data point represents a single mouse. *P < 0.05; NS, not significant; ANOVA with post-hoc Tukey’s corrections analysis.

Supplementary Figure 3 Quantification of SMARTA CD4+ T cells in the spleen.

(a) Number of reconstituted Icos−/– SMARTA CD4+ T cells in host B6 mice 7 d after adoptive transfer of Icos−/– SMARTA CD4+ T cells transduced with RV encoding empty vector (EV) or wild-type ICOS (WT) or mIProx, YF or TL mutant ICOS and infected with LCMV Armstrong strain. (b) Number of SMARTA CD4+ T cells in host B6 mice 7 d after adoptive transfer of SMARTA CD4+ T cells transduced with shRNA targeting the Tbk1 (shTbk1-1 and shTbk1-2), Icos or control genes and infected with LCMV Armstrong strain. Shown are cumulative data (mean ± SEM) from two independent experiments. Each data point represents a single mouse. *P < 0.01; NS, not significant; ANOVA with post-hoc Tukey’s corrections analysis.

Supplementary Figure 4 The ICOS IProx motif and TBK1 affect the development of CXCR5+PD1+ GC TFH cells in Bcl6fl/flCD4-Cre mice following immunization with peptide.

(a) Flow cytometry of cells from host CD4-Cre x Bcl6fl/fl mice 10 d after adoptive transfer of Icos−/– SMARTA CD4+ T cells transduced with RV encoding empty vector (EV) or wild-type ICOS (WT) or mIProx, YF or TL mutant and immunized with KLH-gp61 plus adjuvant. (b) Flow cytometry of cells from host CD4-Cre x Bcl6fl/fl mice 10 d after adoptive transfer of SMARTA CD4+ T cells transduced with shRNA targeting the Tbk1 (shTbk1-1 and shTbk1-2), Icos or control genes and immunized with KLH-gp61 plus adjuvant. Numbers adjacent to outlined areas indicate percent CXCR5+PD1hi GC TFH cells. Cumulative data for a (c), or b (d) from two independent experiments. Each data point represents a single mouse. Shown is mean ± SEM; *p < 0.05; ANOVA with post-hoc Tukey’s corrections.

Supplementary Figure 5 TBK1 is dispensable for the differentiation of nascent TFH cells.

(a) Flow cytometry of cells from host B6 mice 3 d after adoptive transfer of SMARTA CD4+ T cells transduced with shRNA targeting the Tbk1 (shTbk1-1 and shTbk1-2), Icos or control genes and infected with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent CXCR5+SLAMlo TFH cells. Cumulative data for a (b) from two independent experiments. (c - f) Mean fluorescent intensity of CXCR5 (c), TCRβ (d), CD28 (e) and CD40L (f) protein expressions in CD4+GFP+ T cells. Each data point represents a single mouse. Shown are mean ± SEM; *P < 0.01; NS, not significant; ANOVA with post-hoc Tukey’s corrections.

Supplementary Figure 6 ICOS signalosome is independent of TRAF molecules and IKKɛ.

ICOS immunoprecipitations (IPs) from mouse primary CD4+ T cells activated in vitro with anti-CD3 plus anti-CD28 and rested in IL-2. Cells were left unstimulated (–) or restimulated with anti-CD3 plus anti-ICOS (+) for 2 minutes. IPs or whole cell lysates (WCL) were immunoblotted with the indicated Abs. 5 % WCL was used as input to control for immunoprecipitation.

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Pedros, C., Zhang, Y., Hu, J. et al. A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1. Nat Immunol 17, 825–833 (2016). https://doi.org/10.1038/ni.3463

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