Development of neutralising antibodies (inhibitors) against factor VIII (FVIII) is a frequent and severe complication of replacement therapy in haemophilia A. Previous data from haemophilia A mouse model demonstrates that both CD32 inhibition and high doses of rhFVIII prevent the differentiation of FVIII-specific memory B cells (MBCs) into antibody secreting cells (ASCs). Here, cellular targets responsible for the suppression of ASC formation by means of CD32 inhibition and high dose of rhFVIII were analysed. We investigated apoptosis on FVIII-specific MBCs using a pan caspases inhibitor, and screened for defects in rhFVIII presentation by analysing T cell release of Th1- and Th2-cytokines in vitro. Although high dose of rhFVIII suppressed ASC formation, cytokine response was not affected. Upon re-stimulation of splenocytes with high dose of rhFVIII, prevention of apoptosis fully restored the FVIII-specific recall response. In contrast, genetic deletion or inhibition of CD32 significantly altered Th1- and Th2-response. CD32 blockade and inhibition of apoptosis resulted in a partial rescue of FVIII-specific ASCs. Normal cytokine secretion could not be restored. In conclusion, suppression of FVIII-specific recall response by CD32 and high doses of rhFVIII is mediated by distinct mechanisms. High dose of rhFVIII induces apoptosis in FVIII-specific MBCs but does not influence FVIII-specific T cell response. CD32 blockade, however, may suppress the FVIII-specific recall response by two ways: i) increasing apoptosis of FVIII-specific MBCs and ii) disturbing FVIII-specific T cell response by modulating presentation of rhFVIII to CD4 T cells in vitro.
Fc gamma receptors (FcγRs) for IgG regulate adaptive immune responses by modulating activating and inhibitory signalling pathways within immune cells. Data from a haemophilia A mouse model demonstrate that genetic deletion or blockade of the inhibitory FcγR (CD32) suppresses the formation of antibody-secreting cells (ASCs) in vitro. Mechanisms preventing the FVIII-specific recall response, however, remain unclear. Here, the potential role of CD32 inhibition was studied by differentially modulating receptor activity with selected anti-CD32 monoclonal antibodies (mAbs). Splenocytes from immunized FVIII mice were restimulated with FVIII in the absence or presence of different anti-CD32 mAbs over 6 days. At day 6, cytokine release was quantified from cell culture supernatant and the formation of FVIII-specific ASCs assessed. Binding of FVIII-containing immune complexes (F8-ICs) to bone marrow-derived dendritic cells (BMdDCs) was also investigated. The antagonistic CD32 mAb AT128 suppressed the formation of FVIII-specific ASCs and reduced secretion of IFN-γ and IL-10. In contrast, the agonistic mAbs AT130-2 and AT130-5, and their F(ab') fragments, allowed the formation of FVIII-specific ASCs, even though the full IgG of AT130-2 reduced binding of F8-ICs to CD32. Data suggest that an inhibitory signal is transmitted when F8-ICs bind to CD32 and that this signal is required during memory B cell (MBC) activation to support formation of FVIII-specific ASCs. If the inhibitory signal is lacking due to CD32 deletion or blockade with antagonistic anti-CD32 mAbs, FVIII-specific T cell stimulation and ASC formation are suppressed, whereas agonistic stimulation of CD32 restores T cell stimulation and ASC formation.
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