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

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.

Figure 1

IS recruitment and CTLA-4 interaction of PKC-η in T_reg cells. (a) Immunoblot analysis of T hybridoma cells left unstimulated (-) or stimulated (+) with anti-CD3 plus CD86-Fc for 5 min. CTLA-4 IPs (left and center lanes) or whole cell lysates (WCL) were immunoblotted with the indicated Abs. Arrowheads mark the two PKC-η species. (b) Immunoblot analysis of IPs (left and center lanes) or WCL (right lane) from T hybridoma cells stimulated with anti-CD3 plus CD86-Fc for 5 min. CTLA-4 IPs were left untreated (-) or treated with alkaline phosphatase (AP; +) prior to immunoblotting. (c) Immunoblot analysis of cytosol or nuclear fractions from sorted CD4⁺CD62L⁺GFP^- (naïve) and CD4⁺GFP⁺ (T_reg) cells (2 × 10⁶ each) from wild-type FIG mice. Data in (a-c) are representative of at least four experiments. (d) Confocal imaging of PKC-η (top row) or PKC-θ (bottom row) and TCRβ localization in in vitro differentiated iT_reg cells from AND TCR-Tg Rag2^−/− mice, which were retrovirally transduced with eGFP-tagged mouse PKC-θ or PKC-η 1 d after anti-CD3 and -CD28 stimulation. Sorted GFP⁺ T cells (~90% FoxP3⁺ by intracellular staining; not shown) obtained on d 4 and stimulated for 5-10 min by conjugation with MCC-pulsed LPS-stimulated B cells were fixed and analyzed. eGFP-tagged PKC, TCR and nuclear DAPI staining are shown in green, red and blue, respectively. Images are representative of at least 100 cells collected from three independent experiments.

Figure 2

Development of Foxp3⁺ T_reg cells is independent of PKC-η. (a-d) Cell counts (Log₁₀) of CD4⁺Foxp3⁺ cells from thymi (a), spleens (b), peripheral lymph nodes (pLN; c) and mesenteric lymph nodes (mLN; d) of 8-12 week-old wild-type or Prkch^−/− mice determined by intracellular Foxp3 staining. Each data point represents a single mouse. ns, non significant; *P < 0.05. (e-j) Mean fluorescence intensity (MFI) of Foxp3 (e), TCRβ (f), CTLA-4 (g), CD25 (h), GITR (i) and CD44 (j) expression determined on gated CD4⁺Foxp3⁺-cells from wild-type or Prkch^−/− mice. (k) IL-10 production (measured by ELISA) by CD4⁺GFP⁺ T_reg cells from wild-type and Prkch^−/− FIG mice, which were stimulated with plate-bound anti-CD3 mAb and CD86-Fc in the presence of IL-2 overnight. For description of FIG mice, see Fig. 3 and the corresponding text.

Figure 3

Contact-dependent suppression by T_reg cells depends on PKC-η. (a) In vitro suppression assay measuring the proliferation of CellTrace Violet-labeled naïve B6 CD4⁺CD25^- T_eff cells cocultured in the absence (1:0) or presence of Foxp3⁺ T_reg cells from wild-type or Prkch^−/−FIG mice at the indicated T_eff/T_reg cell ratios, and stimulated with anti-CD3 mAb and splenic DCs for 3 d. Percentages of CellTrace Violet-diluting T_eff cells were calculated (left panel). The three right panels show representative FACS dot plots of dye-diluting T_eff cells cultured with T_reg cells at a 1:1 ratio. Data are representative of at least five independent experiments. (b-d) In vivo homeostatic proliferation assay showing the number (Log₁₀) of B6.SJL CD45.1⁺ naïve T cells recovered from spleens (b), pLN (c) or mLN (d) of recipient Rag1^−/− mice 7 d after i.v. transfer of naïve cells (2 × 10⁶) in the absence (none) or presence of FACS-sorted CD4⁺GFP⁺ T_reg cells (.5 × 10⁶) from FIG or Prkch^−/−-FIG mice. Each data point represents a single mouse. (e) Sequential measurements of B16-F10 melanoma growth in groups of Rag1^-/- mice (n ≥ 5), which received CD25-depleted B6 splenic cells (1.5 × 10⁷; a source of T_eff cells) without or with 0.5 × 10⁶ CD4⁺GFP⁺ T_reg cells from wild-type or Prkch^−/− FIG mice, and were inoculated one day later with tumor cells (2 × 10⁵). Tumor diameters along two perpendicular axes were measured 2-3 times/week. *P < 0.05; **P < 0.01; ***P < 0.001. This experiment is representative of at least three independent experiments.

Figure 4. Cytokine production and serum antibody levels of WT vs. Prkch^−/− mice

(a-d) Cytokine expression in culture supernatants of WT or Prkch^−/− FACS-sorted CD44^hi T cells, which were stimulated with anti-CD3 plus -CD28 mAbs for 24 h and assayed by an ELISA for levels of IL-2 (a), IFNγ (b), IL-4 (c) and IL-17A (d). (e-g) Serum levels of IgE (e), anti-double stranded DNA (f) or anti-histone (g) antibodies in 8-12 week-old wild-type and Prkch^−/− mice determined by an ELISA. Each data point represents a single mouse from two pooled experiments. *P < 0.01 **P < 0.001; ***P < 0.0001.

Figure 5

Mapping and biological significance of the CTLA-4-PKC-η interaction sites. (a) Immunoblot analysis of CTLA-4 IPs or WCL from JTAg cells, which were cotransfected with the indicated PKC-η vectors plus wild-type CTLA-4 and stimulated as in Fig. 1a. IgH, heavy chain of the precipitating antibody. Schematic representation of human PKC-η and its phosphorylation sites is shown on top. Data are representative of at leas five independent experiments. (b-d) In vivo homeostatic proliferation assay performed as described in Fig. 3b-d showing the number (Log₁₀) of B6.SJL CD45.1⁺ naïve T cells, which were recovered from spleens (b), pLN (c) or mLN (d) of recipient Rag1^−/− mice 10 d post i.v. transfer of naive cells (2 × 10⁶) in the absence (vector) or presence of FACS-sorted rCD2⁺CD4⁺GFP⁺ T_reg cells (.5 × 10⁶). T_reg cells were derived from Prkch^−/− BM mouse chimeras reconstituted with wild-type PKC-η or a CTLA-4 non-interacting PKC-η-S28A, S32A using retroviral pMIG-IRES-rCD2 vector. Each data point represents a single mouse. ns, non significant; *P < 0.05; **P < 0.01; ***P < 0.001. (e) Immunoblot analysis of CTLA-4 IPs or WCL from JTAg cells cotransfected with the indicated CTLA-4 vectors plus wild-type PKC-η, and processed as in (a). Schematic representation of mouse CTLA-4 is shown on top.

Figure 6

CTLA-4-PKC-η recruits GIT-PIX-PAK complex and modulates T_reg cell-APC interaction. (a) Immunoblot analysis of cytosolic (C) and nuclear (N) fractions of FACS-sorted wild-type or Prkch^−/− FIG CD4⁺GFP⁺ T_reg cells, which were stimulated with anti-CD3ε plus -CTLA-4 mAbs for 5 min. (b) Immunoblot analysis of CTLA-4 IPs or WCL of FACS-sorted GFP⁺ T_reg cells derived from FIG mice and left unstimulated (-) or stimulated (+) with ani-CD3 plus anti-CTLA-4 antibodies for 5 min. (c) Expression of phospho-PAK and total PAK2 in lysates of CD4⁺GFP⁺ T_reg cells from wild-type or Prkch^−/− FIG mice determined by immunoblotting with the corresponding antibodies. (d) Conjugation assay measuring formation of cell doublets between FACS-sorted CD4⁺GFP⁺ wild-type or Prkch^−/− FIG T_reg and CellTrace Violet-labeled splenic DCs at different times during a 3 d coculture period in the presence of anti-CD3 mAb and IL-2. Percentages of GFP⁺ and Violet⁺ double-positive doublets are shown. *P < 0.05; **P <0.001. (e) CD86 depletion from APCs cocultured in the absence (circles) or presence of FACS-sorted CD4⁺GFP⁺ wild-type (squares) or Prkch^−/− (triangles) FIG T_reg cells. A first set of CD45.2⁺ splenic DCs was cultured for 9 h in the presence of anti-CD3 mAb and IL-2 prior to the addition of a second set of CD45.1⁺ splenic DCs. The geometric mean fluorescence intensity of CD86 was enumerated on gated CD11c⁺ Annexin V⁻ CD45.2⁺ and CD11c⁺ Annexin V⁻ CD45.1⁺ cells, respectively. The t_1/2 values of CD86 decay curves were calculated using the GraphPad Prism™ program. This experiment is representative of three independent experiments. ns, non significant; *P < 0.05.