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Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions - PubMed

. 2005 Oct;115(10):2914-23.

doi: 10.1172/JCI24772. Epub 2005 Sep 15.

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

Adam M Boruchov et al. J Clin Invest. 2005 Oct.

Abstract

Human monocyte-derived DCs (moDCs) and circulating conventional DCs coexpress activating (CD32a) and inhibitory (CD32b) isoforms of IgG Fcgamma receptor (FcgammaR) II (CD32). The balance between these divergent receptors establishes a threshold of DC activation and enables immune complexes to mediate opposing effects on DC maturation and function. IFN-gamma most potently favors CD32a expression on immature DCs, whereas soluble antiinflammatory concentrations of monomeric IgG have the opposite effect. Ligation of CD32a leads to DC maturation, increased stimulation of allogeneic T cells, and enhanced secretion of inflammatory cytokines, with the exception of IL-12p70. Coligation of CD32b limits activation through CD32a and hence reduces the immunogenicity of moDCs even for a strong stimulus like alloantigen. Targeting CD32b alone does not mature or activate DCs but rather maintains an immature state. Coexpression of activating and inhibitory FcgammaRs by DCs reveals a homeostatic checkpoint for inducing tolerance or immunity by immune complexes. These findings have important implications for understanding the pathophysiology of immune complex diseases and for optimizing the efficacy of therapeutic mAbs. The data also suggest novel strategies for targeting antigens to the activating or inhibitory FcgammaRs on human DCs to generate either antigen-specific immunity or tolerance.

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Figures

Figure 1
Figure 1

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.

Figure 2
Figure 2

Monocytes, circulating conventional DCs, and cytokine-induced moDCs all express a range of FcγRs, whereas freshly isolated plasmacytoid DCs lack detectable surface expression of all FcγRs. Freshly isolated PBMCs were labeled with fluorochrome-conjugated mAbs. (A) After gating on HLA-DRbright PBMCs that were lineage marker negative, CD32a (left) and CD32b (right) were detected on CD123low conventional DCs (conv. DCs) but not on CD123bright plasmacytoid DCs (pDCs). (B) Monocytes were identified as CD14+ PBMCs. moDCs were studied as immature cells, gated according to characteristic phenotype (48) but lacking the surface CD83 expression of mature moDCs. Open histograms correspond to isotype controls, and filled histograms represent staining of the indicated FcγR. Most often, CD32a and CD32b were coexpressed on the same subpopulation of moDCs, as shown by a representative sample in C.

Figure 3
Figure 3

Various stimuli modulate the balanced expression of CD32a and CD32b on immature moDCs. The indicated reagents were added to cultures of immature moDCs from day 3 to day 6. Expression of FcγRs was measured by flow cytometry (CD16 and CD64 not shown). Analyzed cells were immature or specifically gated for the absence of CD83 in cultures where there was a small amount of maturation (PGE2 and TNF-α). The mean fold changes (± SD) in the frequency of cells expressing a given FcγR induced by each reagent, compared with untreated cells, are shown in A. Density was calculated on the FcγR+ cells as the number of anti-FcγR antibodies bound per cell using a commercially available kit. The mean fold changes (±SD) in FcγR density induced by the reagents in 5 independent experiments, compared with the averaged FcγR densities on untreated/control moDCs, are shown in B. Sample histograms for untreated immature moDCs and IFN-γ–treated immature moDCs are shown in C. Open histograms correspond to isotype controls, and filled histograms show staining by the indicated anti-FcγR mAbs.

Figure 4
Figure 4

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 1.

Figure 5
Figure 5

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).

Figure 6
Figure 6

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.

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