Figure 2
Figure 2. CX3CR1 signaling inhibits HLA-C–induced KIR phosphorylation and regulates DCNK-IS organization. (A) Autologous resting NK cells were exposed to mDCs (10:1 ratio) in the absence or presence of the indicated mAbs, during 24 hours. Anti-MHC class I mAb does not influence IFN-γ production in mDC and NK culture. In contrast, anti-MHC class I mAb prevents the effects of CX3CL1 neutralization. (B) HLA-Cw4 mDCs were cocultured for 24 hours in 96-well plates with the KIR-negative NK cell line YTS stably transfected with KIR2DL1 (YTSKIR2DL1) or with KIR2DL1(1-250) lacking the KIR intracytoplasmic domain (YTSKIR2DL1(1-250)). The levels of IFN-γ released in coculture supernatants were measured by ELISA. Data represent means of triplicates plus or minus SE. Five independent experiments were performed with similar results. (C) KIR2DL1 distribution in HLA-Cw4 mDC/autologous NK cocultures was studied by confocal imaging. HLA-Cw4 mDCs were admixed with autologous resting NK cells, spread onto a lysine-coated slide, and cultured 25 minutes at 37°C. After fixation, cells were permeabilized and stained with anti-KIR2DL1 mAb. Normaski (left panel) and fluorescence (right panel) images are shown. Fifty mDCs forming tight conjugates with KIR2DL1-expressing autologous NK cells were analyzed in each condition. (D) YTSKIR2DL1 were incubated with mDCs (ratio 2:1) during 0, 3, and 10 minutes at 37°C and immunoprecipitated with protein G beads coated with anti-KIR2DL1 mAb (T-20 clone; Santa Cruz). The immunoprecipitate was immunoblotted with anti-SHP-1 mAb (top panel), anti-phosphotyrosine mAb (middle panel), and with anti-KIR2DL1 mAb (clone 2F9, Abcam; bottom panel). (E) Lipid rafts containing ganglioside GM1 were stained using FITC-labeled cholera toxin and analyzed by confocal microscopy. Lipid rafts became clustered at the HLA-Cw4 mDC and YTSKIR2DL1 cell interface after conjugate formation. CX3CL1 neutralization prevented lipid raft clustering at the DCNK-IS. Representative pictures of 3 independent experiments are reported. Scale bar, 10 μm. (F) YTSKIR2DL1/WIP-FLAG were mixed with HLA-Cw4 mDCs for the indicated times at 37°C. Cell lysates were immunoprecipitated with anti-FLAG mAb. The formation of a multiprotein complex with or without CX3CL1 mAb blocking was studied as described.20 Immunoprecipitated proteins were visualized using anti-FLAG, anti-myosin IIA, and anti-actin.

CX3CR1 signaling inhibits HLA-C–induced KIR phosphorylation and regulates DCNK-IS organization. (A) Autologous resting NK cells were exposed to mDCs (10:1 ratio) in the absence or presence of the indicated mAbs, during 24 hours. Anti-MHC class I mAb does not influence IFN-γ production in mDC and NK culture. In contrast, anti-MHC class I mAb prevents the effects of CX3CL1 neutralization. (B) HLA-Cw4 mDCs were cocultured for 24 hours in 96-well plates with the KIR-negative NK cell line YTS stably transfected with KIR2DL1 (YTSKIR2DL1) or with KIR2DL1(1-250) lacking the KIR intracytoplasmic domain (YTSKIR2DL1(1-250)). The levels of IFN-γ released in coculture supernatants were measured by ELISA. Data represent means of triplicates plus or minus SE. Five independent experiments were performed with similar results. (C) KIR2DL1 distribution in HLA-Cw4 mDC/autologous NK cocultures was studied by confocal imaging. HLA-Cw4 mDCs were admixed with autologous resting NK cells, spread onto a lysine-coated slide, and cultured 25 minutes at 37°C. After fixation, cells were permeabilized and stained with anti-KIR2DL1 mAb. Normaski (left panel) and fluorescence (right panel) images are shown. Fifty mDCs forming tight conjugates with KIR2DL1-expressing autologous NK cells were analyzed in each condition. (D) YTSKIR2DL1 were incubated with mDCs (ratio 2:1) during 0, 3, and 10 minutes at 37°C and immunoprecipitated with protein G beads coated with anti-KIR2DL1 mAb (T-20 clone; Santa Cruz). The immunoprecipitate was immunoblotted with anti-SHP-1 mAb (top panel), anti-phosphotyrosine mAb (middle panel), and with anti-KIR2DL1 mAb (clone 2F9, Abcam; bottom panel). (E) Lipid rafts containing ganglioside GM1 were stained using FITC-labeled cholera toxin and analyzed by confocal microscopy. Lipid rafts became clustered at the HLA-Cw4 mDC and YTSKIR2DL1 cell interface after conjugate formation. CX3CL1 neutralization prevented lipid raft clustering at the DCNK-IS. Representative pictures of 3 independent experiments are reported. Scale bar, 10 μm. (F) YTSKIR2DL1/WIP-FLAG were mixed with HLA-Cw4 mDCs for the indicated times at 37°C. Cell lysates were immunoprecipitated with anti-FLAG mAb. The formation of a multiprotein complex with or without CX3CL1 mAb blocking was studied as described.20  Immunoprecipitated proteins were visualized using anti-FLAG, anti-myosin IIA, and anti-actin.

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