Journal: BMC Immunology

Article Title: C-reactive protein (CRP) but not the related pentraxins serum amyloid P and PTX3 inhibits the proliferation and induces apoptosis of the leukemia cell line Mono Mac 6

doi: 10.1186/s12865-017-0230-z

Figure Lengend Snippet: CRP receptor expression on 5 leukemia cell lines. CD16, CD32, CD64, CD89, and CD209 expression on the indicated cell lines was examined by flow cytometry. Top row: Blue trace is CD64, red is CD89. Middle row: Blue is CD16, red is CD209. Bottom row: Blue is CD32-Fun2, Red is CD32-IV3. Black traces are control IgGs. Plots are representative of 3 independent experiments

Article Snippet: Mouse monoclonal antibodies to CD16 (GRM1 and 3G8, mouse IgG1 BioLegend), CD32 (Fun2; AT10 mouse IgG1, BioRad; and 6C4, mouse IgG1, eBioscience, Rockford, IL), CD64 (clone 10.1), and CD89 (clone A59), mouse IgG1, IgG2a, and IgG2b (all from BioLegend), and human Fc blocker (BD Biosciences) or human Fc receptor binding inhibitor (eBioscience) diluted to a final concentration of 10 μg/ml were added to Mono Mac 6 cells in the presence or absence of CRP.

Techniques: Expressing, Flow Cytometry, Control

Journal: BMC Immunology

Article Title: C-reactive protein (CRP) but not the related pentraxins serum amyloid P and PTX3 inhibits the proliferation and induces apoptosis of the leukemia cell line Mono Mac 6

doi: 10.1186/s12865-017-0230-z

Figure Lengend Snippet: Anti-CD64 antibodies attenuate the CRP effect on Mono Mac 6 cells. Anti-CD32, anti-CD64, anti-CD89 antibodies, mouse IgG isotypes, or 2 Fc blockers were added to Mono Mac 6 cells for 2 h before 75 μg/ml CRP was added to the cells. Cell densities at 48 h were normalized to the density with CRP alone. Values are mean ± SEM, n = 3 independent experiments. ** indicates p < 0.01 (t-test)

Article Snippet: Mouse monoclonal antibodies to CD16 (GRM1 and 3G8, mouse IgG1 BioLegend), CD32 (Fun2; AT10 mouse IgG1, BioRad; and 6C4, mouse IgG1, eBioscience, Rockford, IL), CD64 (clone 10.1), and CD89 (clone A59), mouse IgG1, IgG2a, and IgG2b (all from BioLegend), and human Fc blocker (BD Biosciences) or human Fc receptor binding inhibitor (eBioscience) diluted to a final concentration of 10 μg/ml were added to Mono Mac 6 cells in the presence or absence of CRP.

Techniques:

Journal: BMC Immunology

Article Title: C-reactive protein (CRP) but not the related pentraxins serum amyloid P and PTX3 inhibits the proliferation and induces apoptosis of the leukemia cell line Mono Mac 6

doi: 10.1186/s12865-017-0230-z

Figure Lengend Snippet: CRP regulates receptor expression on Mono Mac 6 cells. a Flow cytometry analysis of CD64 and CD89 expression on Mono Mac 6 cells in the presence or absence of CRP at 48 h. b Quantification of mouse IgG isotype, CD32, CD64, and CD89 antibody binding to Mono Mac 6 cells after incubation with or without CRP for 48 h. Values are mean ± SEM, n = 4 independent experiments. ** indicates p < 0.01 (t-test)

Article Snippet: Mouse monoclonal antibodies to CD16 (GRM1 and 3G8, mouse IgG1 BioLegend), CD32 (Fun2; AT10 mouse IgG1, BioRad; and 6C4, mouse IgG1, eBioscience, Rockford, IL), CD64 (clone 10.1), and CD89 (clone A59), mouse IgG1, IgG2a, and IgG2b (all from BioLegend), and human Fc blocker (BD Biosciences) or human Fc receptor binding inhibitor (eBioscience) diluted to a final concentration of 10 μg/ml were added to Mono Mac 6 cells in the presence or absence of CRP.

Techniques: Expressing, Flow Cytometry, Binding Assay, Incubation

Journal: Cancer cell

Article Title: Dendritic cell paucity leads to dysfunctional immune surveillance in pancreatic cancer

doi: 10.1016/j.ccell.2020.02.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-human CD32 (FUN-2) , Fluidigm , #3169020B.

Techniques: Recombinant, Cell Stimulation, Enzyme-linked Immunosorbent Assay, CyQUANT Assay, LDH Cytotoxicity Assay, Expressing, Staining, Derivative Assay, Software

Journal:

Article Title: Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions

doi: 10.1172/JCI24772

Figure Lengend Snippet: 2B6 is a novel mAb that specifically detects an extracellular domain of CD32b. Neutrophils and PBMCs were isolated from peripheral blood samples. Cells were stained with various anti-CD32 mAbs and counterstained with anti-CD66b to define neutrophils (N) or anti-CD20 to define B cells (B). (A) mAb 2B6 detected CD32b on B cells but not CD32a on neutrophils. (B) mAb FL18.26 detected CD32a or CD32b, and it stained neutrophils as well as B cells. (C) In contrast, mAb IV.3 (Fab) detected CD32a on neutrophils but not CD32b on B cells. (D) Some mAbs were able to distinguish between the common polymorphic variants of CD32a. mAbs FL18.26 (B) and IV.3 (C) bind both the R131 and H131 subtypes of CD32a and stain neutrophils from HH homozygotes, RR homozygotes, and HR heterozygotes equally. mAbs 3D3 and 41H16 recognize only the R131 subtype (16), shown staining neutrophils from CD32a131RR individuals but not from CD32a131HH individuals, with intermediate staining of neutrophils from heterozygous (CD32a131HR [HR]) individuals.

Article Snippet: Anti-human CD32 mAbs included clone 41H16 (kind gift of Jan G.J. van de Winkel, University Medical Center, Utrecht, The Netherlands); clone IV.3 (HB-217; ATCC), with Fab fragments produced by the MSKCC Monoclonal Antibody Core Facility; clone 2B6 [described below; unconjugated and FITC-conjugated mouse IgG1 and FITC-conjugated chimeric F(ab′) 2 ; MacroGenics]; PE-conjugated clone CIKm5 (CALTAG Laboratories); and FITC-conjugated clones FL18.26 and 3D3 (BD Biosciences — Pharmingen).

Techniques: Isolation, Staining

Journal:

Article Title: Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions

doi: 10.1172/JCI24772

Figure Lengend Snippet: Ligation of CD32a or CD32b on immature moDCs has opposing effects on maturation phenotype. (A–E) MoDCs were cultured on plates with immobilized (Imm.) human IgG to ligate FcγRs (filled histograms). CD32a (A) or CD32b (B) was specifically ligated by first incubating moDCs with blocking antibodies against either CD32b or CD32a, respectively. CD32a and CD32b were ligated simultaneously (C) by preincubating moDCs without blocking antibodies. DCs with or without blocking antibodies were also cultured on untreated plates as negative controls (open histograms). Cells were harvested at 48 hours, and DC phenotype was assessed by flow cytometry. Histograms from 1 representative experiment of 8 that used CD32a131HH or -HR samples are shown in A–C. Immature IFN-γ–treated moDCs (D) and soluble IgG–treated moDCs (E) were washed to remove these factors and recultured with (filled histograms) or without (open histograms) immobilized human IgG. Cells were harvested at 24–48 hours and phenotype was assessed by flow cytometry. Representative histograms from 1 of 5 separate experiments are shown. (F) In contrast to results obtained from CD32a131HH or -HR samples (C, filled histograms), CD32a131RR samples were not matured to the same extent after coculture with immobilized human IgG (F, filled histograms; n = 4 experiments). Immobilized mouse IgG1 (F, open histograms), which ligates CD32a but not CD32b in CD32a131RR individuals (10), led to maturation that was similar to conditions specifically targeting CD32a on CD32a131HH or -HR samples (A, filled histograms). Averaged changes in CD83 and CD86 expression are summarized in Table ​Table11.

Article Snippet: Anti-human CD32 mAbs included clone 41H16 (kind gift of Jan G.J. van de Winkel, University Medical Center, Utrecht, The Netherlands); clone IV.3 (HB-217; ATCC), with Fab fragments produced by the MSKCC Monoclonal Antibody Core Facility; clone 2B6 [described below; unconjugated and FITC-conjugated mouse IgG1 and FITC-conjugated chimeric F(ab′) 2 ; MacroGenics]; PE-conjugated clone CIKm5 (CALTAG Laboratories); and FITC-conjugated clones FL18.26 and 3D3 (BD Biosciences — Pharmingen).

Techniques: Ligation, Cell Culture, Blocking Assay, Flow Cytometry, Expressing

Journal:

Article Title: Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions

doi: 10.1172/JCI24772

Figure Lengend Snippet: Coligation of CD32b limits CD32a-mediated cytokine release. Immature moDCs were cocultured with immobilized IgG to target FcγRs. After 2 days, supernatants were collected and cytokines measured using a flow cytometry–based multiplexed bead assay. Mean cytokine levels (picograms per milliliter) are plotted on the y axis. (A) For samples derived from CD32a131HH or -HR donors, CD32a, CD32b, or both were ligated on immature moDCs after first blocking or not with the mAb to the other isoform (n = 6 independent experiments). P values for each cytokine reflect differences between CD32a-targeted DCs and DCs on which CD32a and CD32b were targeted simultaneously. (B) IFN-γ– and soluble IgG–mediated shifts in the expression of CD32a and CD32b were also assessed (n = 4 independent experiments). P values for IFN-γ–treated DCs (top) and IgG-treated DCs (bottom) reflect differences between DCs from respective conditions cocultured with or without immobilized IgG. (C) Immobilized mouse IgG was used as the ligand for FcγRs from CD32a131RR samples (n = 3 independent experiments).

Article Snippet: Anti-human CD32 mAbs included clone 41H16 (kind gift of Jan G.J. van de Winkel, University Medical Center, Utrecht, The Netherlands); clone IV.3 (HB-217; ATCC), with Fab fragments produced by the MSKCC Monoclonal Antibody Core Facility; clone 2B6 [described below; unconjugated and FITC-conjugated mouse IgG1 and FITC-conjugated chimeric F(ab′) 2 ; MacroGenics]; PE-conjugated clone CIKm5 (CALTAG Laboratories); and FITC-conjugated clones FL18.26 and 3D3 (BD Biosciences — Pharmingen).

Techniques: Flow Cytometry, Derivative Assay, Blocking Assay, Expressing

Journal:

Article Title: Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions

doi: 10.1172/JCI24772

Figure Lengend Snippet: Targeting CD32a or CD32b affects DC allostimulatory capacity in the MLR. Two days after ligating CD32a, CD32b, both, or neither on immature moDCs, the moDCs were harvested and washed. These moDCs were then recultured at varying doses with 105 allogeneic T cells in triplicate round-bottomed 96-microwell plates without additional cytokines. DC doses ranged from 3,000 to 300 cells per well, yielding DC:T cell ratios from 1:30 to 1:300. [3H]TdR uptake by proliferating allogeneic T cells over the last 12 hours of a 4–5 day culture was measured as an index of DC immunogenicity. (A) The averaged triplicate values (mean ± SEM) for [3H]TdR incorporation by T cells stimulated in 4 independent experiments using samples derived from CD32a131HH or -HR donors are depicted logarithmically (log2) on the y-axis. (B) Experiments using samples from CD32a131RR donors were performed in parallel, with immobilized mouse or human IgG ligating FcγRs on the immature moDCs. The averaged triplicate values (mean ± SEM) from 3 independent experiments are depicted logarithmically (log2) on the y axis. Differences between conditions in A and B were tested using a stratified (by DC:T cell ratios) permutation t test.

Article Snippet: Anti-human CD32 mAbs included clone 41H16 (kind gift of Jan G.J. van de Winkel, University Medical Center, Utrecht, The Netherlands); clone IV.3 (HB-217; ATCC), with Fab fragments produced by the MSKCC Monoclonal Antibody Core Facility; clone 2B6 [described below; unconjugated and FITC-conjugated mouse IgG1 and FITC-conjugated chimeric F(ab′) 2 ; MacroGenics]; PE-conjugated clone CIKm5 (CALTAG Laboratories); and FITC-conjugated clones FL18.26 and 3D3 (BD Biosciences — Pharmingen).

Techniques: Derivative Assay