b cell specific grb2 ko  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: ( a ) Splenic mouse B cells deficient for Grb2 were retrovirally transfected with expression vectors encoding mouse γ2am (wt or YF) together with IRES-driven EGFP and Grb2 along with IRES-driven RFP. The resulting populations in the different gates (G1–G4) are shown on the right. The cells were stimulated with F(ab′) 2 fragments against IgG (α-IgG, indicated by an arrow, 20 μg ml −1 ) ( b – d ) or IgM (α-IgM, 20 μg ml −1 ) ( e ). ( b ) Ca 2+ mobilization kinetics of cells from all gates transfected with wild-type γ2am. ( c , d ) Ca 2+ mobilization kinetics of cells expressing either wild type (wt, blue curves) or YF-mutant (red curves) mIgG2a-BCRs from gate G4 ( c ) and G3 ( d ) on anti-IgG stimulation or G4 on anti-IgM stimulation ( e ). Ca 2+ mobilization was recorded in the presence of 1 mM extracellular CaCl 2 . Data are representative of three independent experiments.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Transfection, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: ( a ) Experimental outline. Grb2 wt/wt Cγ1 cre/+ ( n =3) and Grb2 fl/fl Cγ1 cre/+ mice ( n =5) were repeatedly immunized with purified gB with aluminium hydroxide (alum) at the indicated time points. Splenic B cells were purified 70 days after third immunization by complement-mediated T-cell lysis and anti-CD19 magnetic bead separation. CD19-positive cells containing 800 memory B cells each (identified as B220+, IgG1+, gB+) were transferred intravenously into Rag1 −/− recipient mice and challenged 6 days later by an intravenous injection of 2 μg virus-like particles (VLPs) of human CMV in PBS. ( b ) gB-specific IgG1 titres were measured by ELISA. ( c ) ELISPOT for gB-specific IgG1-secreting cells was performed at day 43 after VLP challenge. Error bars represent mean+s.d. of at least three analyses; Student’s t -test was used. * P <0.05 ** P <0.01.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Purification, Lysis, Injection, Virus, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: ( a ) Schematic illustration of chimeric receptor variants used in this study. Chimeric constructs consisting of mouse YF-mutant γ2am fused to the N-terminal ( b ) or C-terminal ( c ) SH3 domains of Grb2 were retrovirally expressed in DG75 B cells. The SH3 domains were either wild type (wt, blue curves) or inactivated by tryptophan to lysine (W36K and W193K, respectively, red curves) or a phenylalanine to alanine (F165A, orange curve) mutations. Cells were either stimulated with anti-IgG or anti-IgM F(ab′) 2 fragments (indicated by arrows) and Ca 2+ mobilization was recorded in the presence of 1 mM extracellular CaCl 2 . ( d ) Chimeric γ2am-YF-chimeras containing the N-terminal SH3 domain of either Grb2 (wt: dark blue line, W36K: dark red line) or GRAP (wt: light blue line, W36K: light red line) were retrovirally expressed in murine splenic B cells and analysed as before. Data are representative of four independent experiments.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Construct, Mutagenesis

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: DG75 B cells were stimulated via their BCR for 3 min to induce tyrosine phosphorylation of signalling proteins. Subsequently, lysates of these cells were used for affinity purifications with GST-coupled N-terminal SH3 domains of Grb2 and GRAP. Inactivated (W36K) variants were used as controls. Purified proteins were analysed by immunoblotting with anti-phosphotyrosine (α-p-Tyr) antibodies (upper panel) and antibodies to c-Cbl, Sos, SLP65 and Btk as indicated. To assess the efficiency of affinity purification, signals of lysates (equivalent to 2% of input lysates) are shown on the right. Data are representative of four independent experiments.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Phospho-proteomics, Purification, Western Blot, Affinity Purification

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: BTK -deficient DG75 B cells were retrovirally transduced to express chimeric γ2am-YF molecules containing either the N-terminal (blue lines) or C-terminal (red lines) SH3 domains of Grb2. Subsequently, the cells were retrovirally transfected with Citrine-tagged Btk, resulting in two populations that were either Btk-negative (Cit-neg) or Btk-positive (Cit-pos) ( a ). BCR-induced Ca 2+ mobilization was analysed in all populations on stimulation with polyclonal F(ab′) 2 fragments to IgG ( b ) or IgM ( c ). ( d ) Wild-type DG75 cells expressing chimeric γ2am-YF molecules containing either the N-terminal SH3 domain of GRAP (red line) or a variant thereof (XIII) having three amino-acid substitutions at positions three, four and five (S3A, V4I and L6K, blue line) were analysed as before. ( e ) The same chimeric receptors as in ( d ) were analysed in BTK-deficient DG75 cells. ( f ) BTK -deficient DG75 cells were retrovirally transfected with wild type (wt, blue line) or tyrosine to alanine mutant (YA, red line) γ2am-encoding expression vectors and stimulated as before. Data are representative of at least three independent experiments.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Transfection, Expressing, Variant Assay, Mutagenesis

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: On phosphorylation by ITAM-bound Syk, the ITT motifs (white boxes in cytoplasmic mIgG tails) provide docking sites for the ubiquitous adaptor protein Grb2. Grb2 in turn brings along Bruton’s tyrosine kinase (Btk) via a constitutive interaction that is mediated by the N-terminal SH3 domain of Grb2. The incorporation of Grb2/Btk into the BCR signalosome stabilizes the Ca 2+ initiation complex consisting of Btk, the adaptor protein SLP65 (which in addition interacts with a non-ITAM tyrosine-phosphorylation motif in Igα) and phospholipase C-γ2 (PLC-γ2) at the activated receptor, and thereby lowers the threshold for activation of PLC-γ2 by Btk. This active signal amplification loop is complemented by a passive signal amplification that is brought about by sequestration of ITT-bound Grb2 from negative regulators of Ca 2+ mobilization such as CD22 and Dok-3.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Phospho-proteomics, Activation Assay, Amplification