Volume 25, Issue 1, Pages 55-65 (July 2006) The B Cell Receptor Promotes B Cell Activation and Proliferation through a Non-ITAM Tyrosine in the Igα Cytoplasmic Domain  Heide Christine K. Patterson, Manfred Kraus, You-Me Kim, Hidde Ploegh, Klaus Rajewsky  Immunity  Volume 25, Issue 1, Pages 55-65 (July 2006) DOI: 10.1016/j.immuni.2006.04.014 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Generation of Igα Non-ITAM Tyrosine Mutant Mice (A) Amino acid sequence of the murine Igα cytoplasmic domain harboring non-ITAM tyrosine Igα Y204, highly conserved and phosphorylated upon BCR crosslinking (Kabak et al., 2002), and Igα Y176, conserved in human and mouse, but not chicken, in addition to its ITAM tyrosines. (B) IgαY204F/Y204F and IgαY176,204F/Y176,204F mice carry germline mutations in the Igα encoding mb-1 gene, resulting in expression of Igα protein with Y204 replaced by phenylalanine, and with both Y176 and Y204 replaced by phenylalanine, respectively. Immunity 2006 25, 55-65DOI: (10.1016/j.immuni.2006.04.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Reduction in B1 Cells, but Not Follicular and Marginal Zone B Cells in IgαY204F/Y204F Mice Flow cytometric analysis of bone marrow (A), spleen (B), and peritoneal lavage (C) from adult IgαY204F/Y204F mice. (A) Bone marrow B cell subsets shown as % of lymphocytes (top) and % of IgM− lymphocytes (middle) and bone marrow fractions A–F (Hardy and Hayakawa, 2001) as % of total bone marrow cells in 10-week-old mice (bottom, n = 6; fraction D: p = 0.02, fraction F: p = 0.00001). (B) Splenic B cells and B1 cells shown as % of lymphocytes (top), marginal zone B cells as % of CD19+ splenic B cells (second from top), and mature and transitional B cells in the spleen as % of lymphocytes (second from bottom). Absolute numbers of transitional B cells (B220+ AA4.1+ lymphocytes), follicular B cells (B220+ AA4.1− CD23+ lymphocytes), marginal zone B cells (CD19+ CD21++ CD23low CD1d++ lymphocytes), and B1 cells (CD19+ B220low CD5+ lymphocytes) in the spleen given for 10-week-old mice (bottom; n = 6; T1-T3: p = 0.09, B1: p = 0.00002). (C) Peritoneal B1a, B1b, and B2 cells as % of CD19+ lymphocytes (upper) and proportion of B2 cells (CD19+ CD43− CD5− lymphocytes), B1a cells (CD19+ CD43+ CD5+ lymphocytes), and B1b cells (CD19+ CD43+ CD5− lymphocytes) in the peritoneal lavage of 10- to 12-week-old mice given as % of total peritoneal cells (lower; n = 3–4, B1a: p = 0.00013). Closed circles denote results from individual mice; bars represent the mean of values obtained from several mice. p values were determined by Student's t test. Results were confirmed in two additional experiments with a total of 4–6 adult mice. Immunity 2006 25, 55-65DOI: (10.1016/j.immuni.2006.04.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Defective BCR-Induced B Cell Activation, Cell-Cycle Entry and Proliferation In Vitro, and Impaired T-Independent Plasmablast Production and Ig Secretion In Vivo in IgαY204F/Y204F Mice (A) B cell activation as judged by surface CD69 expression (top) and blast formation (bottom) after culture in the presence of 10 μg/ml anti-IgM for 16 and 36 hr, respectively. (B) Entry into S/G2/M phases of the cell cycle as determined by DNA staining of B cells with propidium iodide. Cells were cultured for 36 hr with and without 10 μg/ml (1) and 5 μg/ml (2) anti-IgM, and in the presence or absence of 10 μg/ml anti-IgM combined with BAFF (0.2 μg/ml), anti-CD40 (2 μg/ml), or IL-4 (12.5 U/ml), as well as with LPS (20 μg/ml) or CpG-DNA (0.1 μM) alone. (C) Cell divisions and recovery of live B cells after 72 hr of culture with 10 μg/ml anti-IgM (top) and as indicated (bottom), as suggested by the loss of CFSE and the apoptotic cell marker TO-PRO-3. (D) BCR-induced apoptosis and viability of purified IgαY204F/Y204F splenic B cells after culture for 24 hr with and without 10 μg/ml anti-IgM as determined by propidium iodide staining. (E) Generation of NP-specific IgM-secreting plasmablasts and NP-specific serum IgG3 titers in 8- to 12-week-old mice immunized with NP40-Ficoll. Spleens were analyzed for the presence of NP-specific IgM-secreting antibody-forming cells (AFCs) 4 days after immunization with 25 μg NP-Ficoll by ELIspot assay (top, day 4: p = 0.009, as determined by Student's t test). Closed circles represent means of NP-specific IgM-secreting cell numbers per 106 splenic cells of duplicate ELIspot wells for individual mice, and bars geometric means of values obtained from several mice (n = 4). Sera were analyzed by ELISA 7, 14, and 21 days after immunization with 10 μg NP-Ficoll for NP-specific IgG3 (bottom). Shown are results for individual mice (n = 6) and geometric means, with mice lacking a measurable response depicted at the detection limit of the assay (day 7, p = 0.008; day 14, p = 0.001; day 21, p = 0.002, as determined by Student's t test). All results shown are representative of two to four independent experiments. Immunity 2006 25, 55-65DOI: (10.1016/j.immuni.2006.04.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 No Role for Igα Y204 in Antigen Presentation, the T-Dependent Antibody Response, nor Cap Formation and BCR Internalization (A) Purified IgαY204F/Y204F IgHEL and IgHEL B cells were cultured in triplicates in the presence of HEL at indicated concentrations and a HEL peptide-specific TCR-expressing T cell line for 24 hr. IL-2 production by T cells was measured by ELISA. Closed circles represent means of triplicates performed for individual mice; bars represent the mean of results from two mice each. Results shown are representative of three independent experiments. (B) T-dependent antibody responses assessed by immunizing IgαY204F/Y204F and control mice with 5 and 50 μg NP-CGG, and determining NP-specific serum IgG1 levels by ELISA after 7, 14, and 21 days (n = 4–5). (C) Cap formation after BCR crosslinking with 10 μg/106 cells anti-IgM was examined by immunofluorescence microscopy. Examples of capped B cells are shown in the left panel, and the kinetics and percentage of B cells that polarized IgM to 1/3 to 1/4 of the cell circumference are depicted in the right panel. Results shown were confirmed in two additional independent experiments. (D) The kinetics of surface IgM downregulation was assessed by incubating splenic B cells with 10 μg/106 cells anti-IgM containing a fraction of biotinylated anti-IgM for the indicated times, followed by immediate fixation with nonpermeabilizing formaldehyde solution. Surface bound anti-IgM was visualized with a streptavidin conjugate. Results are representative of three independent experiments. Immunity 2006 25, 55-65DOI: (10.1016/j.immuni.2006.04.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Unimpaired Syk Phosphorylation, but Decreased BLNK Phosphorylation and Calcium Flux, and Distinct Protein Tyrosine Phosphorylation Pattern in IgαY204F/Y204F Splenic B Cells after BCR Crosslinking (A–D) Immunoblotting of whole-cell lysates prepared from purified splenic B cells stimulated with 10 μg/106 cells anti-IgM for indicated time points. Protein lysates were separated by 10% SDS-PAGE and blotted for total protein tyrosine phosphorylation (4G10 mAB), Igα and β-actin (A), Syk pY525 and 526 present in Syk fractions with lower electrophoretic mobility (arrows, [B]), BLNK pY72, pY84, pY96, and BLNK (C), and Btk pY223, Btk, PLCγ2 pY1217, and PLCγ2 (D). The tyrosine phosphorylated band of the size of Igα (arrow, [A]) corresponds to phosphorylated Igα as suggested by its selective near complete absence in whole-cell lysates of anti-IgM-stimulated Igα ITAM tyrosine mutant B cells (Kraus et al., 2001). Arrows (A) denote differentially tyrosine phosphorylated bands (compare Figure S3 for long exposure). (E) Calcium flux after addition of anti-IgM (20 μg/ml final concentration) to splenic B cells labeled with the calcium-sensitive dye Indo-1 and depicted as 2% probability contour plots of the 405/485 nm emission ratio. All results shown are representative of two to five independent experiments. Immunity 2006 25, 55-65DOI: (10.1016/j.immuni.2006.04.014) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Impaired IκBα Phosphorylation and Degradation and Decreased JNK and ERK Phosphorylation in BCR-Stimulated IgαY204F/Y204F B Cells Immunoblotting of whole-cell lysates prepared from purified splenic B cells stimulated with 10 μg/106 cells anti-IgM for indicated time points. Reduced protein lysates were separated by 10% SDS-PAGE and blotted for IκBα pS32 and 36, IκBα, and β-actin (A), JNK pT183 and pY185 and JNK, as well as ERK pT202 and pY204 and ERK (B). Results shown are representative of two to four independent experiments. Immunity 2006 25, 55-65DOI: (10.1016/j.immuni.2006.04.014) Copyright © 2006 Elsevier Inc. Terms and Conditions