Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jul;121(3):392-404.
doi: 10.1111/j.1365-2567.2007.02588.x. Epub 2007 Mar 26.

Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization

Maria-Concetta Veri  1 , Sergey Gorlatov, Hua Li, Steve Burke, Syd Johnson, Jeffrey Stavenhagen, Kathryn E Stein, Ezio Bonvini, Scott Koenig
Affiliations

Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization

Maria-Concetta Veri et al. Immunology. 2007 Jul.

Abstract

Human CD32B (FcgammaRIIB), the low-affinity inhibitory Fcgamma receptor (FcgammaR), is highly homologous in its extracellular domain to CD32A (FcgammaRIIA), an activating FcgammaR. Available monoclonal antibodies (mAb) against the extracellular region of CD32B recognize both receptors. Through immunization of mice transgenic for human CD32A, we generated a set of antibodies specific for the extracellular region of CD32B with no cross-reactivity with CD32A, as determined by enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant CD32A and CD32B, and by fluorescence-activated cell sorting analysis of CD32 transfectants. A high-affinity mAb, 2B6, was used to explore the expression of CD32B by human peripheral blood leucocytes. While all B lymphocytes expressed CD32B, only a fraction of monocytes and almost no polymorphonuclear cells stained with 2B6. Likewise, natural killer cells, which express CD32C, a third CD32 variant, did not react with 2B6. Immune complexes co-engage the inhibitory receptor with activating Fcgamma receptors, a mechanism that limits cell responses. 2B6 competed for immune complex binding to CD32B as a monomeric Fab, suggesting that it directly recognizes the Fc-binding region of the receptor. Furthermore, when co-ligated with an activating receptor, 2B6 triggered CD32B-mediated inhibitory signalling, resulting in diminished release of inflammatory mediators by FcepsilonRI in an in vitro allergy model or decreased proliferation of human B cells induced by B-cell receptor stimulation. These antibodies form the basis for the development of investigational tools and therapeutics with multiple potential applications, ranging from adjuvants in FcgammaR-mediated responses to the treatment of allergy and autoimmunity.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Amino acid alignment of the extracellular domains of human CD32A and CD32B. Amino acid differences between the two receptors are marked with an asterisk (*). Residues 127–135 (underlined) correspond to the IgG Fc binding site. Residue 131, which is polymorphic in CD32A (H/R), is labelled (^). The entire 180-amino acid sequence of CD32B extracellular domain was used for immunization.
Figure 2
Figure 2
Anti-CD32B mAb binding to human CD32A and CD32B ELISA. Plates coated with either soluble CD32A-R131 (▴) or CD32B (♦) were used to analyse binding of the different antibodies. A pan-CD32 mAb, FLI8.26, and a CD32A-specific mAb, IV.3, were used as controls.
Figure 3
Figure 3
Binding of anti-CD32B mAbs to human CD32A and CD32B cell transfectants. 293H cells stably transfected with either full-length CD32A-R131 or CD32B were incubated with 5 μg/ml 1F2, 2D11, 3H7, 2E1, 1D5, 2D11, or FLI8.26 or 1 μg/ml 2B6 in the presence of 10% human serum. Binding was detected by F(ab′)2 fragments of Cy5-GAM and FACS analysis. Plots show relative level of staining as MFI (x-axis) versus the number of events recorded (y-axis). Staining by the anti-CD32 mAb (filled histograms) is overlaid with that of the corresponding isotype controls (solid line).
Figure 4
Figure 4
CD32 expression by peripheral blood leucocytes from healthy donors. (a) Representative 2B6 staining of peripheral blood leucocytes (PBL) from CD32A-H/H131 and CD32A-R/R131 homozygous normal donors. Immunofluorescence analysis of PBL was performed using CD20-FITC, CD3-FITC, CD56-FITC, CD14-FITC and CD16-FITC mAbs to define specific cell populations. Detection of 2B6 was performed by indirect immunofluorescence using Cy5-conjugated GAM F(ab′)2. (b) B lymphocytes (CD20-positive cells) and T lymphocytes (CD3-positive cells) from 19 healthy donors were stained with murine 2B6 by indirect immunofluorescence as described in (a). The scatter plot shows the percentages of CD32B-positive cells of each donor defined as the population above the threshold established by an isotype-matched control antibody. The black line indicates the median value. (c) CD32 and CD32B-specific staining of PBL from 48 healthy donors by direct immunofluorescence using KB61-FITC (top left panel) or 2B6-FITC (top right panel). In this analysis, PMN cells and monocytes were identified using forward and side scatter. B lymphocytes and NK cells were identified by staining with CD20-PE or CD56-PE, respectively. Scatter plots show the percentages of CD32 positive cells in each individual, defined as the population above the threshold established by isotype-matched control antibodies. The bottom panel shows a comparison of B-lymphocyte and monocyte MFI values for 2B6 staining. The MFI value shown is the value of the experimental sample after subtraction of the value of the isotype-matched control mAb in the same gate. In all panels, the black line indicates the median value. (d) FACS plots of four representative donors from the 48 donors summarized in (c). FACS histograms illustrate staining using mIgG1-FITC (filled) or 2B6-FITC (bold line).
Figure 5
Figure 5
Inhibition of immune complex binding by anti-CD32B antibodies. (a) Diagram illustrating the experimental set-up of the competition ELISA. (b,c) CD32B-specific mAb were tested for their ability to block IC binding to sCD32B-Fc in a competition ELISA. Each plot compares the binding to the corresponding isotype-matched control mAb. (d) Binding of 2B6, 3H7 or their corresponding Fab fragments to sCD32B-Fc in a direct binding ELISA. Bound antibodies were detected with a κ-chain-specific, horseradish peroxidase-conjugated GAM. (e) IC binding competition by 2B6, 3H7, or their corresponding Fab fragments. (f–h) CD32B-transduced CHO-K1 cells were preincubated with 2B6 (f), 3H7 (g) or 2H9 (h) followed by aggregated human IgG and the F(ab′)2 fragment of an FITC-conjugated GAH IgG. Murine isotype + GAH-FITC (dotted line), murine isotype + aggregated human IgG + GAH-FITC (thin line), and anti-CD32B mAb + aggregated human IgG + GAH-FITC (bold line). The level of binding of each of the mAbs to the CHO-CD32B cell line was determined by FACS analysis (insets).
Figure 6
Figure 6
Anti-CD32B-induced inhibition of both FcεRI activation and B-cell proliferation. (a) β-hexosaminidase release was measured in RBL-2H3-hCD32B cells. Cells were sensitized with mouse IgE, followed by incubation with 2B6, 1F2 or a murine IgG1 isotype control, and stimulated with GAM F(ab′)2. Binding of 2B6 and 1F2 to RBL-2H3-hCD32B by FACS analysis is shown in the inset. Results are representative of three independent experiments. (b) Purified B cells were activated using increasing concentrations of anti-human IgM mAb (x-axis) and 50 μg/ml of F(ab′)2 fragment of GAM IgG Fc specific in the presence of 5 μg/ml of either mouse IgG1 (speckled bars) or 2B6 mAb (black bars). The proliferation was assessed on day 3 by [3H]thymidine incorporation (y-axis), the reactions were performed in triplicate and standard deviations were calculated. Results are representative of three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ravetch JV, Bolland S. IgG Fc receptors. Annu Rev Immunol. 2001;19:275–90. - PubMed
    1. Daeron M, Latour S, Malbec O, et al. The same tyrosine-based inhibition motif, in the intracytoplasmic domain of Fc gamma RIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation. Immunity. 1995;3:635–46. - PubMed
    1. Hunter S, Indik ZK, Kim MK, Cauley MD, Park JG, Schreiber AD. Inhibition of Fcgamma receptor-mediated phagocytosis by a nonphagocytic Fcgamma receptor. Blood. 1998;91:1762–8. - PubMed
    1. Daeron M. Fc receptor biology. Annu Rev Immunol. 1997;15:203–34. - PubMed
    1. Zhu D, Kepley CL, Zhang M, Zhang K, Saxon A. A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation. Nat Med. 2002;8:518–21. - PMC - PubMed

LinkOut - more resources