Recruitment of Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B cells

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1 SUPPLEMENTARY FIGURES Recruitment of Grb2 to surface IgG and IgE provides antigen receptor-intrinsic costimulation to class-switched B cells Niklas Engels, Lars Morten König, Christina Heemann, Johannes Lutz, Takeshi Tsubata, Sebastian Griep, Verena Schrader & Jürgen Wienands Figure S1 Tyrosine-phosphorylation of γ2am. (a) Ramos B cell transfectants expressing either wild-type (WT) or YF IgG-BCR were left untreated (0) or stimulated with anti-igg for 2 minutes (2). Following immunoprecipitation (IP) with anti-igg, obtained proteins were analyzed under either reducing (Red) or non-reducing (N-Red) conditions by immunoblotting with biotinylated anti-phosphotyrosine (mab 100) in combination with HRPO-linked streptavidin (top) or with anti-γ2a (bottom). (b) Surface IgG2a expression of Ramos B cell transfectants expressing either wild-type (WT) or mutant IgG-BCRs harboring a YF exchange in the γ2am cytoplasmic tail were analyzed by flow cytometry. IgG-negative Ramos parental cells served as negative control (open histogram). (c) The endogenously expressed IgG2a-BCR of IIA1.6 cells was left unligated (0) or activated with biotinylated anti-igg for 3 minutes (3). Lysates of these cells were incubated with a streptavidin matrix to selectively purify cell surface-exposed migg molecules that previously had been stimulated. Obtained material was size-separated under reducing (Red) or non-reducing conditions (N-Red). Following transfer to nitrocellulose membranes, proteins were analysed with monoclonal anti-phosphotyrosine (mab 100, top) or anti-γ2am (bottom). Relative molecular masses of marker proteins are indicated on the right in kda. 1

2 Figure S2 Expression of γ2am in primary mouse B cells. (a) LPS-activated splenic B cells retrovirally transfected with γ2am (WT or YF) were gated on EGFP-negative (gate G1) or EGFP-positive (gate G2) cells, respectively, during Ca 2+ flux analysis (see Figs. 3d and e). (b) Ca 2+ flux in EGFP-negative cells from G1. Figure S3 Expression of γ2am mutants in Ramos B cells. Lysates of parental Ramos B cells (Ramos) and transfectants expressing either wild-type or YF or Δtail IgG-BCR were analyzed with antibodies against γ2a (top) or Syk (bottom). Relative molecular masses of marker proteins are indicated on the right in kda. 2

3 Figure S4 Expression of chimeric IgM-G receptors in K46 cells. (a) Schematic illustration of chimeric IgM-G receptor variants used in this study. Representative clones were stained with FITC-conjugated anti-igm (b) or anti-λ (c) and analyzed by flow cytometry. 3

4 Figure S5 Affinity purification of γ2am-itt binding partners. Proteins that bind to the nonphosphorylated (γ2am-pep) and phosphorylated (p-γ2am-pep) γ2am peptides were affinitypurified from lysates of resting Ramos B cells and visualized by silver staining. A bare streptavidin matrix served as negative control. Relative molecular masses of marker proteins are indicated on the right in kda. Figure S6 Ca 2+ modulation by phospho-itt does not require PI3K activity. Ramos B cells expressing wild-type or YF mutant γ2am were left untreated (black and grey curves, respectively) or preincubated with 1 µm Wortmannin for 30 min (blue and red curves, respectively) and subsequently stimulated with anti-γ or anti-μ F(ab ) 2 fragments (left and right panel, respectively). The induced Ca 2+ responses were recorded by flow cytometry in the presence of 1 mm extracellular CaCl 2. BCR stimulation is indicated by arrows. Data are representative of three independent experiments. 4

5 Figure S7 Direct binding between phospho-γ2am and Grb2. Recombinant GST-Grb2-SH2 fusion protein (input) was incubated with either streptavidin-sepharose alone (SA-matrix) or streptavidin-sepharose plus 2 µm of either phosphorylated γ2am-peptide (p-γ2am-pep) or the non-phosphorylated γ2am-peptide (γ2am-pep). Recombinant GST served as negative control. After washing the SA-matrix with 1% NP40-containing lysis buffer, bound protein was subjected to SDS-PAGE and stained with coomassie brilliant blue. Relative molecular masses of marker proteins are indicated on the right in kda. Figure S8 γ2am-itt phosphorylation does not require Grb2 binding. (a) Ramos B cells expressing wild-type or NA mutant γ2am were left untreated (0) or stimulated with anti- IgG (2). Expression and tyrosine phosphorylation of γ2am chains was analyzed by anti-igg immunoprecipitation and subsequent immunoblotting with antibodies to γ2am (top) and phosphotyrosine mab 102 (bottom). Relative molecular masses of marker proteins are indicated on the right in kda. 5

6 Figure S9 CellVue-based proliferation assay of retrovirally infected splenic B cells. 24 hours after infection with EGFP-expressing retroviruses cells were stained with CellVue Claret (a, red curve) and compared with unstained cells (black curve). Subsequently, cells expressing wild-type IgG2a were left unstimulated (b and c, left panels) or were stimulated with 10 µg/ml anti-igg F(ab ) 2 fragments (right panels). 96 hours after stimulation the median CellVue fluorescence intensities of untransfected cells (b, G1) and EGFP-positive cells (G2) from the same wells was measured. (c) Histogram overlays of cells from G1 (black curve) and G2 (green curve) shown in (b). 6

7 Figure S10 The εm-itt amplifies Ca 2+ mobilization in primary B cells. Murine splenic B cells retrovirally transduced to express chimeric γ2a-εm carrying the wild-type or YF ε-itt were analyzed for Ca 2+ mobilization upon stimulation with anti-γ F(ab ) 2 (left) or anti-µ F(ab ) 2 fragments (right) in buffer containing 1 mm CaCl 2 (a). (b) The individual contribution of intracellular Ca 2+ release and influx of Ca 2+ across the plasma membrane, respectively, was analyzed using different buffer conditions. Cells were stimulated first in Ca 2+ -free buffer containing 0.5 mm EGTA followed by analysis in the presence of 1 mm extracellular CaCl 2. 7

8 Figure S11 Schematic illustration of the experimental set-up employed in experiments shown in Fig. 8. Ramos cells co-expressing a YF IgG-BCR and a fusionprotein consisting of the extracellular and transmembrane domains of CD8 coupled to the intracellular domain of εm in either wild-type (WT) or YF configuration were stimulated either with anti-cd8 alone or in combination with anti-igg F(ab ) 2 fragments that co-crosslink the anti-cd8 antibody and the IgG-BCR. 8