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Protein kinase C-η controls CTLA-4–mediated regulatory T cell function

Nature Immunology volume 15, pages 465–472 (2014)Cite this article

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Abstract

Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η–deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4–PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η–deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4–PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.

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Figure 1: IS recruitment and CTLA-4 interaction of PKC-η in Treg cells.
Figure 2: Development of Foxp3+ Treg cells is independent of PKC-η.
Figure 3: Contact-dependent suppression by Treg cells depends on PKC-η.
Figure 4: Cytokine production and serum antibody concentrations of Prkch+/+ (WT) versus Prkch−/− mice.
Figure 5: Mapping and biological importance of the CTLA-4–PKC-η interaction sites.
Figure 6: CTLA-4–PKC-η recruits GIT2-αPIX-PAK complex and modulates Treg cell–APC interaction.

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  • 02 May 2014

    In the version of this article initially published, the second affiliation for Nicholas R.J. Gascoigne was incorrect. The correct affiliation is The Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We acknowledge H. Cheroutre and Y.-C. Liu for helpful discussions, the excellent services provided by members of the Flow Cytometry Core Unit at the La Jolla Institute for Allergy and Immunology, as well as the Animal Husbandry Units at LIAI and the Scripps Research Institute. This is publication number 1552 from the La Jolla Institute for Allergy and Immunology and 21923 from The Scripps Research Institute. This work was supported by US National Institutes of Health grants CA35299 (A.A.), GM065230 (N.R.J.G.) and P01AI089624 (M.K.). K.-F.K. was supported by LIAI-T1D-CRF 2012 Fellowship and Young Investigator Award #270056 from the Melanoma Research Alliance.

Author information

Author notes

  1. Nicholas R J Gascoigne

    Present address: Present address: Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,

  2. Kok-Fai Kong and Guo Fu: These authors contributed equally to this work.

  3. Nicholas R J Gascoigne and Amnon Altman: 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

    Kok-Fai Kong, Ann J Canonigo-Balancio, Stephane Becart & Amnon Altman

  2. Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA

    Guo Fu, Javier Casas & Nicholas R J Gascoigne

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

    Yaoyang Zhang & John R Yates III

  4. RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Tadashi Yokosuka & Takashi Saito

  5. PRESTO, Japan Science and Technology Agency, Saitama, Japan

    Tadashi Yokosuka

  6. Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Gisen Kim & Mitchell Kronenberg

  7. Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Nicholas R J Gascoigne

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Contributions

K.-F.K. and G.F. designed experiments, collected data, performed analyses and wrote the paper; J.C., T.Y. and T.S. performed microscopy experiment; A.J.C.-B. was involved in experiments and data collection; S.B. performed and assisted in melanoma studies; Y.Z. and J.R.Y. did the phosphoproteomic experiments and analyses; G.K. and M.K. provided critical reagents and were involved in study design; N.R.J.G. and A.A. designed the study, analyzed data and wrote the paper.

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Correspondence to Kok-Fai Kong, Nicholas R J Gascoigne or Amnon Altman.

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Integrated supplementary information

Supplementary Figure 1 Lack of CTLA-4 interaction with PKC isoforms other than PKC-η, and the expression of PKC-η and CTLA-4 in Treg vs. Teff cells.

(a) MCC-specific T hybridoma cells were left unstimulated (-) or stimulated (+) for 5 min with crosslinked anti-CD3 plus -CTLA-4 mAbs. Cell lysates (WCL) or CTLA-4 immunoprecipitates (2 left lanes in each panel) were resolved by SDS-PAGE, and immunoblotted with the indicated PKC-specific or anti-CTLA-4 Abs. (b) CD4+ T cells were purified from spleens of FIG mice, and FACS-sorted into GFP+ (Treg) and GFP− (Teff) cells. Equal number of sorted cells were subjected to RNA purification, reverse transcription, and quantitative PCR to determine the mRNA levels of Prkch and Ctla4 (left panel). Intracellular staining of PKC-η and CTLA-4 was performed to determine their respective protein levels (right panel).

Supplementary Figure 2 Frequency of Treg cell populations.

(a) The CD4+Foxp3+ cell population from thymi, spleens, peripheral lymph nodes (pLN) and mesenteric lymph nodes (mLN) of 8- to 12-week-old mice were determined by intracellular staining of Foxp3. (b) Number of GFP+ Treg cells recovered from mice undergoing homeostatic expansion (see Fig. 3b-d). Naïve T cells from allotypically marked CD45.1+ B6.SJL mice were transferred alone (None), or cotransferred with FACS-sorted CD4+GFP+ Treg cells from WT or Prkch−/− FIG mice into Rag1−/− mice. The numbers of GFP+ cells in spleens, pLN and mLN were determined. Each data point represents a single mouse.

Supplementary Figure 3 Prkch−/− Treg cells protect mice in a T cell transfer model of colitis.

(a) Sorted CD4+CD62L+ naïve T cells (Teff) in the absence or presence of WT or Prkch−/− GFP+ Treg cells were cotransferred into Rag1−/− mice and weight was monitored over time as indicated. Mice were sacrificed 10 weeks post-transfer. (b,c) The infiltrating T cell populations in spleens, peripheral lymph nodes (pLN) and mesenteric lymph nodes (mLN) were analyzed by flow cytometry and enumerated. **P < 0.05.

Supplementary Figure 4 Conservation of the membrane-proximal basic CTLA-4 motif and its importance in PKC-h association.

(a) Evolutionary conservation of the positively charged proximal motif (underlined, with basic residues in bold) in the cytoplamic tail of CTLA-4. Protein sequence of putative CTLA-4 proteins from the indicated organisms were aligned with human CTLA-4. The consensus sequence was generated using Weblogo (www.weblogo.berkeley.edu). (b) . Analysis of “tailess” CTLA-4 mutants for their interaction with PKC-η. WT or truncated CTLA-4 were cotransfected with Xpress-tagged WT PKC-η into JTAg cells. Cells were stimulated with anti-CD3 mAbs + CD86-Fc recombinant protein for 5 min and immunoprecipitated with an anti-CTLA-4 mAb prior to immunoblotting. The right panel shows a schematic representation of mouse CTLA-4 and its cytoplasmic tail. The CTLA-4 tail was partially (Δ192-223) or fully (Δ182-223) truncated.

Supplementary Figure 5 Effect of Prkch deletion on LFA-1 function.

Purified CD4+ cells from WT or Prkch-/- FIG mice were stimulated with anti-CD3 plus anti-CTLA-4 Abs for the indicated times. The function of LFA-1 was measured by its ability to bind to ICAM1-Fc. Cells were stained with fluorophore-conjugated anti-CD4 and anti-Fc antibodies. Shown are representative data gated on GFP+ cells (top panel) and cumulative data of 2 independent experiments (bottom panel).

Supplementary Figure 6 Phosphoproteome analysis of CD3- plus CTLA-4-costimulated Treg cells and signal requirements for recruitment of the GIT-PIX-PAK complex to CTLA-4.

(a) Purified CD4+ T cells from WT or Prkch−/− FIG mice were differentiated for 6 days into iTregs in the presence of TGF-β and IL-2 in standard SILAC media. The cells were stimulated with anti-CD3 plus anti-CTLA-4 mAbs for 5 min before cell lysis and sample preparation for phosphoproteomic analysis. Shown are representative hypophosphorylated proteins in Prkch−/− Tregs as compared to WT Tregs with a fold-change of >1.5. (b) Recruitment of GIT-PIX-PAK complex to CTLA-4-PKC-h complex is dependent on CTLA-4, but not CD28. MCC-specific T hybridoma cells were left unstimulated or stimulated with anti-CD3 and anti-B7, anti-CD3 and anti-CD28, or anti-CD3 and anti-CTLA-4 for 5 min prior to CTLA-4 immunoprecipitation. Immunoblotting was carried out with indicated antibodies.

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Kong, KF., Fu, G., Zhang, Y. et al. Protein kinase C-η controls CTLA-4–mediated regulatory T cell function. Nat Immunol 15, 465–472 (2014). https://doi.org/10.1038/ni.2866

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