Antigen-induced oligomerization of the B cell receptor is an early target of Fc gamma RIIB inhibition.

Mice, cells, Abs, and Ags Primary B cells were isolated from spleens of IgH B1-8/B1-8 Ig κ −/− transgenic mice (provided by M. Shlomchik, Yale University, New Haven, CT) by negative selection using MACS sorting as described previously ( 18 ). Isolated B cells were cultured overnight with CpG and LPS (Calbiochem, San Diego, CA) and experimented next day. The J558L B cells stably expressing B1-8-γ-cyan fluorescent protein (CFP) and Igα-yellow fluorescent protein (YFP) (γCαY) were established and characterized as previously described ( 21 ). Daudi B cell line stably expressing Lyn16-CFP-YFP fusion protein and CH27 B cell line stably expressing the lipid raft probe Lyn16-CFP, the nonlipid raft probe CFP-Ger, or the full length LynFL-CFP were established and characterized as previously reported ( 19 ). ST486, a human B cell line and A20II1.6, a mouse B cell line, both negative for endogenous FcγRIIB expression, were purchased from the American Type Culture Collection (Manassas, VA). PT67 cell line, a virus packaging cell line, was purchased from BD Clontech (Palo Alto, CA). Cy3- and Cy5-conjugated Fab goat Abs specific for mouse IgM and IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Cy3-conjugated Fab rat mAb specific for mouse IgM (clone no. II/41) was purchased from Rockland (Gilbertsville, PA). Rabbit IgG Abs specific for BSA (rabbit anti-BSA) were purchased from Bethyl Laboratories (Montgomery, TX) and F(ab′) 2 rabbit anti-BSA were prepared as described ( 20 ). PE-conjugated and biotin-conjugated rat mAb specific for mouse FcγRIIB (clone no. 2.4G2) and APC-conjugated mouse mAb specific for human FcγRIIB (clone no. FLI8.26) were purchased from BD Pharmingen (San Diego, CA). Biotin-conjugated mouse mAb specific for human FcγRIIB (clone no. AT10) was purchased from AbD Serotec (Raleigh, NC). Biotin-conjugated F(ab′) 2 goat Abs specific for mouse IgG and IgM, biotin-conjugated F(ab′) 2 rabbit Abs specific for human IgM, and streptavidin were purchased from Jackson ImmunoResearch Laboratories. Rat mAb specific for mouse FcγRIIB (clone no. 190907) was purchased from R&D Systems (Minneapolis, MN) and Fabs mAb 190907 were prepared using an immobilized papain kit (Pierce, Rockford, IL) following the manufacturer’s protocol. Conjugations of Abs with Alexa514, 568, or 647 were performed using Alexa Fluor mAb labeling kits (Molecular Probes, Eugene, OR) following manufacturer’s protocols. BSA conjugated 1:14 with 4-hydroxy-5-iodo-3-nitrophenyl acetyl (NIP) (NIP 14 -BSA) and BSA conjugated 1:16 with phosphorylcholine (PC 16 -BSA) were purchased from Biosearch Technologies (Novato, CA). NIP 14 -BSA and PC 16 -BSA were conjugated to a Cys-containing peptide terminated with a His 12 tag (ASTGTASACTSGASSTGSH 12 ) using succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce) following manufacturer’s protocols. ICs were formed by mixing 10 nM His 12 tagged NIP 14 -BSA or PC 16 -BSA with 20 nM rabbit anti-BSA (for IgG-IC) or F (ab′) 2 rabbit anti-BSA [for F(ab′) 2 -IC]. Recombinant ICAM-1 with a His 12 tag was a gift of J. Huppa (Stanford University, Palo Alto, CA). The mouse ICAM-1/huFc chimera protein with a His 12 tag was purchased from R&D Systems. Conjugation of His 12 -tagged NIP 14 -BSA and PC 16 -BSA to Cy5 and His 12 -tagged ICAM-1 to AlexaFluor488 were performed following manufacturer’s protocols ( 19 ).

Plasmid constructs and transfections Plasmids expressing

Lyn16-YFP or Lyn16-CFP and containing monomeric CFP or yellow fluorescent protein (YFP) were constructed as described ( 19 ). The pMSCV series plasmids were purchased from Clontech. Plasmids of pCDNA3.1 and pCDNA6 series were purchased from Invitrogen (Carlsbad, CA). Plasmids of pECFP-N1 or pEYFP-N1 were purchased from BD Clontech, and the GFP gene in these two plasmids was modified using a QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) to reduce the dimerization of GFP as suggested ( 22 ). LynFL-CFP plasmid expressing full length Lyn kinase and CFP-Ger plasmid expressing the geranylgenylated CFP were constructed as described ( 19 , 23 ).

Plasmids of pBLUEScript containing mouse

FcγRIIB1 and pMSCVpuro plasmids containing human FcγRIIB WT or I232T loss of function mutant were kindly provided by Z. I. Honda (Tokyo University, Tokyo, Japan) ( 24 ). Using these plasmids as templates, the genes encoding mouse or human FcγRIIB were amplified by PCR and inserted into pECFP-N1, pEYFP-N1, or pCDNA6/Bsd via the restriction enzyme sites of SacII and HindIII. To acquire constructs of FcγRIIB fused with monomeric YFP in pMSCV plasmid, the released fragments containing genes of FcγRIIB and YFP from plasmid pEYFP-N1-FcγRIIB by BglII and NotI double digestion were subcloned into the multiple cloning site of pMSCV plasmid with a NotI restriction enzyme site placed into its multiple cloning site ( 23 ). In the case of mouse FcγRIIB, the BglII restriction site AGA T CT carried by mouse FcγRIIB itself was synonymously mutated to AGA C CT using a QuikChange Mutagenesis Kit (Stratagene). The Y309F mutation in mouse FcγRIIB ITIM motif, and the CD314 mutant deleting the C-terminal 16aa residues in mouse FcγRIIB was made using a QuikChange mutagenesis kit (Stratagene). A J558L B cell line expressing the fluorescence resonance energy transfer (FRET) pair B1-8-γ-CFP and γCαY was generated and maintained in the laboratory as described ( 21 ).

Mouse FcγRIIB WT or Y309F and human FcγRIIB

WT or I232T were transfected into γCαY B cells in pCDNA6 by electroporation. Cell lines stably expressing the plasmid were acquired by blasticidin selection and cell sorting. PT67 packaging cells expressing murine stem cell virus (MSCV) containing FcγRIIB-YFP were prepared as described ( 18 , 19 ). Briefly, the PT67 cell line was transfected with a pMSCV plasmid containing mouse FcγRIIB-YFP construct by spin infection and the virus in the supernatant was used to infect a CH27 B cell line stably expressing Lyn16-CFP. Stable cell lines expressing Lyn16-CFP and FcγRIIB-YFP were acquired by puromycin selection and cell sorting. Transient transfection was performed using Amaxa transfection kits and the transfected B cells were imaged after overnight culture.

Preparation of planar lipid bilayers containing ICAM-1 and ICs

Planar fluid lipid bilayers were prepared as previously detailed ( 25 – 27 ). Briefly, Ni-NTA-containing lipid bilayers were prepared using the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid and 1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl) iminodiacetic acid]-succinyl (nickel salt) (DOGS-Ni-NTA; Avanti Polar Lipids, Alabaster, AL) with 90% DOPC and 10% DOGS-Ni-NTA. ICs were formed by mixing 10 nM His 12 tagged NIP 14 -BSA or PC 16 -BSA with 20 nM rabbit BSA-specific (for IgG-IC) or F(ab′) 2 rabbit BSA-specific [for F(ab′) 2 -IC]. F(ab′) 2 -IC or IgG-IC was further mixed with 10 nM His 12 tagged ICAM-1, and then incubated on Ni-NTA–containing lipid bilayers for 20 min for binding. After washing, the F(ab′) 2 -IC or IgG-IC containing lipid bilayers were ready to be used in total internal reflection fluorescence (TIRF) imaging. ICAM-1, IgG-IC and F(ab′) 2 -IC were mobile on planar lipid bilayer determined as described previously ( 19 , 20 ). Biotin-containing planar lipid bilayers were prepared with 99% DOPC and 1% 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine-cap-biotin (DOPE-cap-biotin) (Avanti Polar Lipids) to which biotinylated ICAM-1 and Abs were attached through streptavidin as previously reported ( 19 ). Briefly, 50 nM streptavidin was incubated with biotin-containing lipid bilayer for 10 min. After washing, 10 nM biotinylated ICAM-1 and 10 nM biotinylated F(ab′) 2 Abs specific for either mouse or human Igand/or10 nM biotinylated rat Abs specific for either mouse or human FcγRIIB were bound to the planar lipid bilayer. Live cell imaging by total internal reflection fluorescence microscopy and image processing TIRF images were acquired using an Olympus IX-81 microscope (Melville, NY) equipped with a TIRF port, Cascade II 512 × 512 electron-multiplying CCD camera (Roper Scientific, Tucson, AZ), Olympus 100 × 1.45 N.A. or Zeiss 100 × 1.4 objective lens (Carl Zeiss, Salem, MA). A 442-nm diode pump solid state laser, a 488-nm/514-nm argon gas laser and a 568-nm/647-nm red krypton/argon gas laser were equipped and used as indicated. All images were acquired at 37°C on a heating stage. The acquisition was controlled by Metamorph (Molecular Devices, Sunnyvale, CA) and the exposure time was 100 ms for 512 × 512 pixels image, unless specially indicated. The acquired images were analyzed and processed with Image Pro Plus (Media Cybernetics, Silver Spring, MD), Image J (National Institutes of Health) or Matlab (The MathWorks, Natick, MA) software as indicated. Before analysis, images were split, aligned, background subtracted, and corrected for spectral bleed-through using Image Pro Plus or Matlab software depending on needs. Characterization of the B cell clustering was the protocol reported by Batista et al. ( 12 ). Briefly, the B1-8 primary B cells were labeled with 200 nM Fab IgM-specific Cy3-conjugated rat mAb (clone II/41). The labeled cells were washed twice and then loaded to the lipid bilayer containing F(ab′) 2 -IC or IgG-IC. TIRF images were acquired every 2 s immediately after the loading of the B cells to the chamber. Cy3 was excited by 514 nm laser, and Cy3 fluorescence was collected through a 550/40 ET BP emission filter. The spreading area, the mean fluorescence intensity, and number of the BCR microclusters were measured using Image J software. The number of BCR microclusters per cell at maximal spreading was manually counted after background filtering the TIRF image by Matlab software. The threshold efficiency used for the filtering was best estimated by comparing the mean fluorescence intensity (FI) of BCR microclusters with the mean FI of IgM-specific Cy3-conjugated monoclonal Fab fragment nonspecifically bound to the lipid bilayer, which was acquired with the same total internal reflection fluorescence microscopy (TIRFM) settings used for B cell image. Trajectories of individual microclusters were tracked automatically using a Matlab-supported code. The mean square displacement (MSD) for each BCR microcluster was calculated from positional coordinates as reported ( 28 , 29 ). A detailed description of the automatic tracking is given below in Single particle tracking and analysis . To sort the movement of individual BCR microcluster into different categories (random, directed, or confined movement), the MSD plot of each BCR microcluster was fitted with the three functions to define random, directed, and confined movement, respectively as below: (1) F ( t ) = a + 4 ∗ D ∗ t ; (2) F ( t ) = a + 4 ∗ D ∗ T + υ 2 ∗ ( T 2 ) ; (3) F ( t ) = a + ( L 2 / 3 ∗ [ 1 − exp ( − 12 ∗ D ( ) ∗ T / [ L 2 ] ) ] ) . For each fit, the square of correlation coefficient ( R 2 ) is acquired as a parameter to quantify the goodness of each fit. In this report, if the R 2 values of one certain BCR microcluster from all three fittings are