MHC class II/CD38/CD9: a lipid-raft–dependent signaling complex in human monocytes by Marie-Thérèse Zilber, Niclas Setterblad, Thierry Vasselon, Christelle Doliger, Dominique Charron, Nuala Mooney, and Catherine Gelin Blood Volume 106(9):3074-3081 November 1, 2005 ©2005 by American Society of Hematology

CD9 mAb inhibits Sag-induced T-cell proliferation. CD9 mAb inhibits Sag-induced T-cell proliferation. Purified T cells (4 × 104cells/well) were cultured with SEA in the presence of fixed monocytes (8 × 103 cells/well) with mAbs (soluble or precoated to monocytes) of the indicated specificity. [3H]thymidine incorporation measured at day 4, for T cells in the presence of uncoated fixed monocytes, was 40.500 ± 2.098 cpm. Results are representative of at least 3 independent experiments. Error bars indicate standard deviation. Marie-Thérèse Zilber et al. Blood 2005;106:3074-3081 ©2005 by American Society of Hematology

CD9 is associated with MHC class II and CD38 on human monocytes. CD9 is associated with MHC class II and CD38 on human monocytes. Monocytes were surface-labeled with biotin before lysis in NP-40 (A) or CHAPS (B-D). Immunoprecipitations were then performed with the HLA-DR (D1.12), CD38 (IB4), or isotype control mAbs. After electrophoresis under nonreducing conditions and transfer to a nitrocellulose membrane, the precipitated material was revealed by chemiluminescence. (C) The HLA-DR, CD38, and CD9 molecules were immunoprecipitated, from unlabeled monocytes, with the specific D1.12, IB4, and PHN 200 mAbs, respectively. Immunoprecipitates were analyzed by Western blot with biotinlabeled CD38 (Leu 17), CD9 (Syb.1), or anti–HLA-DR (DA6.147) mAbs. (D) Monocyte lysates were precleared twice in a period of 24 hours with CD38 (IB4) or isotype control mAb together with protein G-sepharose. HLA-DR and CD38 were then immunoprecipitated and Western blotted with biotin-labeled CD38 (Leu 17), CD9 (Syb.1), or anti–HLA-DR (DA6.147) mAbs. Supernatants of CD38 and HLA-DR precipitates obtained after Ig or CD38 preclearing were blotted with an antiactin mAb and show that equal quantities of proteins were used for immunoprecipitations (not shown). WB indicates Western blot; IP, immunoprecipitation. Marie-Thérèse Zilber et al. Blood 2005;106:3074-3081 ©2005 by American Society of Hematology

Co-capping of CD9 with MHC class II and CD38. Co-capping of CD9 with MHC class II and CD38. Purified monocytes untreated (A, C, E, G) or pretreated with 10 mM MβCD (B, D, F, H) were incubated with CD38 (A-D) or CD9 (E-H) mAbs (10 μg/mL) for 20 minutes on ice and subsequently with Texas red–conjugated goat anti–mouse Ig for 45 minutes at 22°C to allow complete capping. Cells were then stained with the indicated FITC-conjugated mAbs and analyzed. Confocal optic sections for the merged images of representative cells are shown. Marie-Thérèse Zilber et al. Blood 2005;106:3074-3081 ©2005 by American Society of Hematology

The CD38-CD9-MHC class II complex is present in membrane rafts. The CD38-CD9-MHC class II complex is present in membrane rafts. Purified monocytes were lysed in 0.5% Triton X-100 and subjected to sucrose density gradient ultracentrifugation. Raft (fractions 2-4) and nonraft (fractions 7-9) fractions were collected and lysed in CHAPS before immunoprecipitation. After electrophoresis under nonreducing conditions and transfer to a nitrocellulose membrane, Western blots were performed with biotin-labeled CD38 (Leu 17), CD9 (Syb.1), or anti–HLA-DR (DA6.147) mAbs. Marie-Thérèse Zilber et al. Blood 2005;106:3074-3081 ©2005 by American Society of Hematology

CD38 and HLA-DR–mediated intracellular calcium fluxes are dependent on raft integrity. CD38 and HLA-DR–mediated intracellular calcium fluxes are dependent on raft integrity. Purified monocytes were untreated (NT) or treated with 10 mM MβCD (30 minutes, 37°C), loaded with indo-1, and tested for calcium mobilization. After measurement of basal level, the following F(ab')2 preparations of mAbs were added: CD38 mAb (IB4), HLA-DR mAb (D1.12), and CD9 mAb (PHN 200). Results are representative of at least 3 independent experiments. Marie-Thérèse Zilber et al. Blood 2005;106:3074-3081 ©2005 by American Society of Hematology

CD9 ligation induces tyrosine phosphorylation of the tyrosine kinases Fgr and Hck. CD9 ligation induces tyrosine phosphorylation of the tyrosine kinases Fgr and Hck. Purified monocytes were either unstimulated or stimulated with the appropriate mAb. (A) Cells were lysed and subjected to SDS-PAGE before transferring to nitrocellulose membranes and immunoblotting with antiphosphotyrosine mAb. The same quantity of protein was loaded in each well. Molecular mass markers are indicated. (B-E) Cells were lysed and immunoprecipitates prepared using either an anti-Hck Ab (B, D) or an anti-Fgr Ab (C, E). After blotting, filters were probed with an antiphosphotyrosine mAb (B-C). The filter shown in B was stripped and reprobed with an anti-Hck Ab revealing identical amounts of Hck in all samples (D). The filter shown in C was stripped and reprobed with an anti-Fgr Ab revealing identical amounts of Fgr in all samples (E). Arrows indicate phosphorylated substrates; NS, no stimulation. Marie-Thérèse Zilber et al. Blood 2005;106:3074-3081 ©2005 by American Society of Hematology

A role for CD9 in the formation of antigen-dependent monocyte/T-cell conjugates. A role for CD9 in the formation of antigen-dependent monocyte/T-cell conjugates. CMFDA-labeled monocytes loaded or not with SEE were incubated for various periods of time with CMTMR-labeled Jurkat cells before flow cytometry. (A) Conjugate formation at t = 0 minute and t = 40 minutes of incubation between monocytes loaded with 10 μg/mL of SEE and Jurkat cells are detected as CMFDA+ CMTMR+ events. (B) Kinetic analysis of conjugate formation between CMFDA-labeled monocytes loaded or not with SEE (10 μg/mL) and CMTMR-labeled Jurkat cells. (C) CMFDA-labeled monocytes loaded or not with SEE (10 μg/mL) were incubated with CD9 (PHN200), CD38 (IB4), anti–HLA-DR (D1.12), CD11A (HI-111), or isotype control mAbs (5 μg/mL) before conjugate formation with Jurkat cells. The percent of SEE-specific conjugates are indicated for different times. (D) Formation of conjugates was analyzed for different concentrations of SEE and different concentrations of CD9 mAbs. Marie-Thérèse Zilber et al. Blood 2005;106:3074-3081 ©2005 by American Society of Hematology