Mechanisms of NKT cell anergy induction involve Cbl-b-promoted monoubiquitination of CARMA1

Abstract

Repeated injection of alpha-galactosylceramide, an agonistic ligand for natural killer T (NKT) cells, results in long-term unresponsiveness or anergy, which severely limits its clinical application. However, the molecular mechanisms leading to NKT anergy induction remain unclear. We show here that the decreased IFN-gamma production and failed tumor rejection observed in anergized NKT cells are rescued by Cbl-b deficiency. Cbl-b E3 ligase activity is critical for the anergy induction, as revealed by the similarity between Cbl-b(-/-) and its RING finger mutant NKT cells. Cbl-b binds and promotes monoubiquitination to CARMA1, a critical signaling molecule in NFkappaB activation. Ubiquitin conjugation to CARMA1 disrupts its complex formation with Bcl10 without affecting its protein stability. In addition, CARMA1(-/-) NKT cells are defective in IFN-gamma production. The study identifies an important signaling pathway linking Cbl-b-induced monoubiquitination to NFkappaB activation in NKT cell anergy induction, which may help design approaches for human cancer therapy.

Fig. 1.

Cbl-b in NKT cell anergy induction. (A) Separated splenic NKT cells after vehicle- or α-GalCer-pretreatment were lysed, and Cbl-b expression was assessed by SDS-PAGE and immunoblotting with antibodies to the indicated proteins. A representative of two separate experiments is shown. (B) Cbl-b mRNA expression after anti-CD3 and anti-CD28 stimulation in vehicle- or α-GalCer-pretreated NKT cells was measured by real-time PCR and normalized to HPRT expression. A representative of two separate experiments is shown. (C) Production of IFN-γ and IL-4 in WT and Cbl-b^−/− mice after vehicle- or α-GalCer-pretreatment. Mice were injected three times with 2 μg α-GalCer or vehicle as pretreatment. Seven days after last injection, splenocytes (2 × 10⁵ per well) were cultured with graded doses of α-GalCer. After 72 h, IFN-γ and IL-4 concentrations were measured by ELISA. A representative of four separate experiments is shown. (D) Liver mononuclear cells from vehicle- or α-GalCer pretreated WT and Cbl-b^−/− mice were labeled with CFSE. Cells (1 × 10⁶) were then cultured with α-GalCer (100 ng/mL) for 96 h. Cells were then harvested, stained with α-GalCer-loaded CD1d DimerXI and anti-TCRβ antibody, and analyzed by flow cytometry. A representative of two separate experiments is shown. (E) IFN-γ and IL-4 production from NKT cells were measured in c-Cbl^−/− mice as described in C. Results in C and E are means ± SEM, and asterisk indicates P < 0.05 compared with WT (Mann–Whitney U test).

Fig. 2.

Cbl-b regulates NKT-mediated in vivo functions. (A) In vivo recovery of IFN-γ production in α-GalCer pretreated Cbl-b^−/− mice. Vehicle- or α-GalCer-pretreated WT and Cbl-b^−/− mice were injected with α-GalCer. Mice were bled at the indicated time points, and serum IFN-γ and IL-4 concentrations were evaluated by ELISA. n.d., not detected. (B and C) Transactivation of other cell population after α-GalCer injection. Representative FACS profile of CD69 on NK cell, CD8+, CD4⁺ T cell, and IFN-γ in NK cells from vehicle- or α-GalCer-pretreated WT and Cbl-b^−/− mice were obtained 8 h after additional α-GalCer injection. Results in A are means ± SEM, and asterisk indicates P < 0.05 compared with WT or littermate control (Mann–Whitney U test).

Fig. 3.

Regulation of tumor metastasis by Cbl-b E3 ligase. (A) Determination of B16F10 melanoma lung metastasis formation. WT and Cbl-b^−/− were treated with α-GalCer or vehicle, and mice were challenged i.v. with 5 × 10⁵ B16F10 melanoma cells and treated with α-GalCer (2 μg/injection) or vehicle at 0, 4, and 8 days after the tumor challenge. Mice were killed after 2 weeks of challenge, and the number of metastatic tumor nodules was counted in the lungs. A representative of three separate experiments is shown. (B and C) IFN-γ expression in vehicle- or α-GalCer-pretreated Cbl-b C373A mice and its littermate control (Litt) after additional stimulation with α-GalCer in vitro (B) and in vivo (C). IFN-γ concentration was measured by ELISA. A representative of three separate experiments is shown. (D) α-GalCer-mediated inhibition of tumor lung metastasis was evaluated in Cbl-b C373A knockin mice and its littermates after vehicle- or α-GalCer-pretreatment. A representative of two separate experiments is shown. Results are means ± SEM, and asterisk indicates P < 0.05 compared with WT or littermate control (Mann–Whitney U test).

Fig. 4.

Suppressed NFκB pathway signaling in anergized NKT cells. (A and B) Human NKT cell line was left untreated or treated with ionomycin (Iono) for 16 h. Cells were activated with anti-CD3 and anti-CD28 for indicated time points, and cell lysates were blotted with Abs as indicated. Results shown are representative of three individual experiments. (C) Human NKT cell line was transfected with indicated siRNA and treated with ionomycin as described in A. Cell lysates were blotted with the indicated antibodies. Results shown are representative of two individual experiments. (D) Liver mononuclear cells from WT and Cbl-b^−/− mice with vehicle- or α-GalCer-pretreatment were stimulated with anti-CD3, anti-CD28, and anti-NK1.1, and phosphorylation of p65 and Erk1/2 were detected in NK1.1⁺ T cells (NKT cells) by intracellular phospho-protein staining. Results shown are representative of two individual experiments. (E) Splenocytes (2 × 10⁵ per well) were cultured with α-GalCer (100 ng/mL) for 72 h in the presence of indicated doses of PS-1145. IFN-γ and IL-4 concentrations were measured by ELISA. Results shown are representative of three individual experiments. Results in E are means ± SEM.

Fig. 5.

Cbl-b regulates CARMA1 ubiquitination and its complex formation. (A) Interaction between CARMA1 and Cbl-b. CARMA1 and/or Cbl-b were expressed in 293T cells, and cell lysates were immunoprecipitated with anti-FLAG antibody. Samples were blotted with indicated antibodies. Results shown are representative of five individual experiments. (B) Jurkat TAg cells were stimulated with anti-CD3 and anti-CD28, and endogenous Cbl-b was immunoprecipitated from lysate of Jurkat TAg cells and blotted as indicated antibodies. Results shown are representative of two individual experiments. (C) Detection of CARMA1 ubiquitination in transfected 293T cells. The ubiquitinated CARMA1 was detected by immunoprecipitation, followed by blotting with indicated antibodies. (D) Endogenous monoubiquitination of CARMA1 in human NKT cell line. Cells were left untreated or treated with ionomycin (Iono) for 16 h and activated with anti-CD3 and anti-CD28 for indicated time points. Ubiquitinated CARMA1 was detected by immunoprecipitation, followed by blotting with anti-ubiquitin. Knock-down of Cbl-b was done by transfection of Cbl-b siRNA. Results shown are representative of two individual experiments. The molecular weight markers are labeled in C and D on the left. (E) Detection of CARMA1-Bcl10 complex formation in human NKT cell line. Human NKT cell line was treated with ionomycin and restimulated. Cell lysates were immunoprecipitated with anti-Bcl10 antibody, and samples were blotted with indicated antibodies. (F) Splenocytes (2 × 10⁵ per well) from CARMA1^−/− mice were stimulated with α-GalCer (100 ng/mL), and cytokine production was measured. Results in F are means ± SEM, and asterisk indicates P < 0.05 compared with WT or littermate control (Mann–Whitney U test).