CD16/32-specific biotinylated 2.4G2 single-chain Fv complexed with avidin-FITC enhances FITC-specific humoral immune response in vivo in a CD16-dependent manner

Abstract

Fcgamma receptors (FcgammaRs) play an essential role in the regulation of immune response due to their ability to bind immune complexes. Activating FcgammaRs may facilitate antigen presentation and dendritic-cell maturation, while in the late phase of the immune response, the inhibitory FcgammaRIIb may down-regulate B-cell activation upon cross-linking with activating receptors. In this study, we investigated the in vivo role of FcgammaRs on the modulation of humoral immune response. In order to get well-defined immune complexes that can bind to both the activating and the inhibitory FcgammaRs, we designed a mono-biotinylated single-chain fragment variable construct from the rat anti-mouse CD16/32 clone 2.4G2, linked to avidin-FITC, and tested its effect on the FITC-hapten-specific T-independent type 2 (TI-2) and T-dependent (TD) immune response. When injected intravenously in mice, the complex bound to a small portion of B220+, CD11b(high) and CD11c(high) cells and was localized in the spleen on marginal zone macrophages 15 min after treatment. When applied as a booster following primary immunization with TI-2 (FITC-dextran) or TD (FITC-keyhole limpet haemocyanin) antigens, the complex elevated the number of hapten-specific IgM/IgG-producing B cells. This effect was diminished in CD16KO mice, suggesting that the activating-type FcgammaRIII might be a key mediator of this mechanism.

Hypoxia-induced complement dysregulation is associated with microvascular impairments in mouse tracheal transplants

“…To isolate CD55 expressing vascular endothelial cells, collagenase digestion of harvested graft was performed according to the standard protocol with some modifications [38]. Briefly, each freshly harvested graft was minced in cold HEPES buffer, and incubated with 2.5 mg/ ml of collagenase D (11088858001 Roche, USA) for 1 h. at room temperature in 50RPM shaking bath.…”

Modulation of immune response by combined targeting of complement receptors and low-affinity Fcγ receptors

“…They were generated and characterized as previously described [17,33]. Sequences of the variable regions of 7g6 and 2.4g2 have been deposited at the GenBank (AF214704, AF220555, FJ888349 and FJ888350).…”

Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production

“…None of the constructs induced any sign of disease when administered into DBA/1 mice that were not immunized previously with collagen (data not shown). We assumed that the elevation of arthritic scores of collagen primed mice by FcγRII/III targeted CII-peptide and 2.4G2 scFv tetramers might be the result of targeting FcγRII/III positive cells,24,25 including dendritic cells (DC) and macrophages.…”

“…2.4G2 scFv was prepared and purified as previously described 24,30. 2.4G2 recognizes both FcγRII (CD32) and FcγRIII (CD16) 31.…”

“…Our aim was to investigate how FcγRII/III targeting of this CII peptide by single chain Fv fragment (scFv) of the monoclonal antibody, 2.4G2 may modulate the immunopathogenesis of CIA. Previously we and others have shown that antibody response against model antigens can be substantially improved by FcγRII/III targeting 24,25. Extravidin-linked molecular constructs of the biotinylated CII-peptide and its complex with mono-biotinylated 2.4G2 scFv were injected intravenously into CII pre-immunized DBA/1 mice, and the in vivo effect on several parameters of CIA, including arthritic score, antibody response to CII and CII-peptide, as well as cytokine/chemokine secretion were monitored.…”