Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation of NK cell-mediated cytotoxicity by a SAP-independent receptor of the CD2 family.

doi: 10.4049/jimmunol.167.10.5517

Figure Lengend Snippet: FIGURE 4. Engagement of CRACC by mAb 162 enhances lysis of P815 by both normal and SAP-deficient NK cells. A, Anti-CRACC mAb activates lysis of P815 cells by NK92 in rADCC (}), and so does the anti-2B4 mAb (Œ), as compared with a control mouse IgG (F). On the contrary, an F(ab)2 of the anti-CRACC ({) induces no activation. B, Cy- totoxicity of an NK cell clonal population was tested against 51Cr-labeled P815 cells at 5:1 ratio in the presence of 2-fold dilutions of anti-CD16 (circles) or anti-NKp46 mAbs (squares) in combination with 10 g/ml of anti-CRACC mAb (filled symbols) or control mouse IgG (open symbols). The maximum concentration of anti-CD16 and anti-NKp46 was 50 g/ml. In the first point of the curves, anti-CD16 and anti-NKp46 are absent, showing the effect of anti-CRACC alone. The spontaneous lysis of P815 by the NK cell line was 1.6% (data not shown). C–F, NK cell polyclonal populations derived from normal controls (C and D) and XLPD patients (E and F) were tested in rADCC against 51Cr-labeled P815 cells in the pres- ence of anti-CRACC (}), anti-CRACC F(ab)2 ({), control IgG1 (F), anti-2B4 (Œ), and anti-NKp46 (I). Cytotoxicity was tested against 51Cr- labeled P815 cells at the indicated E:T ratios in the presence of 10 g/ml mAb. It is of note that the spontaneous lysis of P815 by different NK cell lines is variable and so is the increase of lysis mediated by anti-CRACC. Such variability is most likely due to the presence within different NK cell polyclonal populations of NK cell clones with different activating and in- hibitor receptor repertoires, each contributing to the total P815 lysis.

Article Snippet: In four-color flow cytometric analysis, PBMCs were sequentially incubated with PBS-20% human serum, anti-CRACC mAbs 162, PE-conjugated human-adsorbed goat anti-mouse IgG2b (Southern Biotechnology Associates, Birmingham, AL) and PBS-20% normal mouse serum.

Techniques: Lysis, Control, Activation Assay, Labeling, Concentration Assay, Derivative Assay, Clone Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation of NK cell-mediated cytotoxicity by a SAP-independent receptor of the CD2 family.

doi: 10.4049/jimmunol.167.10.5517

Figure Lengend Snippet: FIGURE 3. CRACC is an 66-kDa glycoprotein. Anti-CRACC mAb or control IgG1 (c) immunoprecipitates from surface biotinylated NK92 cells were treated with O-glycanase and N-glycanase F as indicated, and subsequently analyzed by Western blot analysis with streptavidin-HRP.

Article Snippet: In four-color flow cytometric analysis, PBMCs were sequentially incubated with PBS-20% human serum, anti-CRACC mAbs 162, PE-conjugated human-adsorbed goat anti-mouse IgG2b (Southern Biotechnology Associates, Birmingham, AL) and PBS-20% normal mouse serum.

Techniques: Control, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation of NK cell-mediated cytotoxicity by a SAP-independent receptor of the CD2 family.

doi: 10.4049/jimmunol.167.10.5517

Figure Lengend Snippet: FIGURE 5. CRACC associates with two phosphorylated proteins of 39 and 19 kDa upon stimulation with pervanadate, but not with SAP, EAT-2, SHP-1, SHP-2, or SHIP. Pervanadate-treated (left panels) or unstimulated (right panels) NK92 cells were subjected to immunoprecipitation with anti- CRACC mAb, anti-2B4 mAb, anti-NKG2A mAb, or control IgG (C). The precipitates were analyzed by anti-phosphotyrosine blot (A) or anti-SHP-1, anti-SHP-2, anti-SHIP, anti-SAP, or anti-EAT2 Western blot (B). Precip- itated proteins are indicated by arrows. Molecular mass markers are indi- cated. A, A control immunoprecipitation of 2B4 reveals 2B4 tyrosine phos- phorylation and association with 25- and 39-kDa proteins. No phosphorylation of CRACC or 2B4 was observed in unstimulated NK92 cells.

Article Snippet: In four-color flow cytometric analysis, PBMCs were sequentially incubated with PBS-20% human serum, anti-CRACC mAbs 162, PE-conjugated human-adsorbed goat anti-mouse IgG2b (Southern Biotechnology Associates, Birmingham, AL) and PBS-20% normal mouse serum.

Techniques: Immunoprecipitation, Control, Western Blot, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation of NK cell-mediated cytotoxicity by a SAP-independent receptor of the CD2 family.

doi: 10.4049/jimmunol.167.10.5517

Figure Lengend Snippet: FIGURE 6. CRACC mediates cytotoxicity by activating an ERK-de- pendent pathway. A, NK92 cells were stimulated with anti-CRACC mAb or control IgG mAb in the presence of a cross-linking Ab for the indicated time periods and examined by Western blot analysis using anti-phospho- ERK1/2 mAb (upper panel) and anti-ERK1/2 (lower panel) antiserum. B, MEK inhibitor PD98059 reduces CRACC-mediated lysis of P815. NK92 cells were preincubated for 1 h with DMSO (squares) or PD98059 (20 M) (circles) before coincubation with 51Cr-labeled P815 target cells in the presence of anti-CRACC mAb (I, E) or control IgG1 (F). C, PD98059 inhibits CRACC-mediated ERK phosphorylation at concentrations used in cytotoxicity experiments. Cells were stimulated with anti-CRACC mAb or control IgG mAb (c) in the presence of the indicated concentrations of PD98059 (Inh). Western blot analysis was performed as described in A. NK cell viability was not affected by PD98059, as assessed by propidium iodide-annexin V flow cytometric analysis, excluding the possibility that PD98059 might reduce NK cell function (data not shown).

Article Snippet: In four-color flow cytometric analysis, PBMCs were sequentially incubated with PBS-20% human serum, anti-CRACC mAbs 162, PE-conjugated human-adsorbed goat anti-mouse IgG2b (Southern Biotechnology Associates, Birmingham, AL) and PBS-20% normal mouse serum.

Techniques: Control, Western Blot, Lysis, Labeling, Phospho-proteomics, Cell Function Assay