During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.

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.

Development of inhibitory antibodies against factor VIII (FVIII) is a severe complication of replacement therapy in haemophilia A. Patients with inhibitors are treated with high FVIII doses in the context of immune tolerance therapy (ITT). Data from haemophilia A mouse model suggest that high FVIII concentrations prevent the formation of antibody secreting cells (ASCs) from memory B cells (MBCs) by inducing apoptosis. Fc gamma receptor 2B (CD32) is an important regulator of B cell function, mediating inhibitory signals after cross-linking with the B cell receptor. Here, the role of CD32 in the regulation of FVIII-specific MBCs was investigated using F8-/- and F8-/-CD32-/- knockout mice and monoclonal antibodies (mAbs). The initial immune response was similar between F8-/- and F8-/-CD32-/- mice, including concentration of anti-FVIII antibodies and number of FVIII-specific ASCs in spleen and bone marrow. In contrast, formation of ASCs from MBCs upon rhFVIII re-stimulation in vitro was abolished in F8-/-CD32-/- mice, whereas FVIII/anti-FVIII immune complexes significantly enhanced ASC formation in F8-/- mice. Inhibition of CD32 by mAbs or F(ab)2 fragments prevented ASC formation in a dose-dependent manner. Transfer of B cell-depleted splenocytes using CD45R (B220) depletion from CD32-competent mice did not restore ASC formation in F8-/-CD32-/- cells confirming that CD32 is required on B cells. We conclude that CD32 is a crucial regulator of FVIII-specific B cells and is required for the differentiation of MBCs into ASCs. Inhibition of CD32 could potentially improve the efficacy of FVIII in the context of ITT.

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Development of neutralizing antibodies against factor VIII (FVIII) is a severe complication of replacement therapy in hemophilia A. Long-term application of high doses of FVIII can induce tolerance in the context of immune tolerance therapy (ITT). Very high concentrations of FVIII have been shown to prevent the development of FVIII-specific antibody-secreting cells (ASCs) from memory B cells by inducing apoptosis. We have previously demonstrated that ASC differentiation from memory B cells is also abolished when CD32, an inhibitory Fc-gamma receptor expressed on B cells and dendritic cells, was genetically deleted or blocked by monoclonal antibodies (mAb). Here, we addressed the question how CD32 inhibition prevented ASC development by studying the FVIII-specific T cell response in the absence or presence of CD32 inhibition. Hemophilia A mice (B6;129S4-F8tmKaz/J) were immunized with recombinant human FVIII (rhFVIII) for 4 weeks, and spleen cells were re-stimulated with rhFVIII (0, 0.5, 1 or 10 IU/ml) ex vivo in the presence or absence of anti-CD16/CD32 antibody (2.4G2) to inhibit CD32. Formation of FVIII-specific ASCs was assessed on day 6 by ELISPOT. IFN-γ, IL-2, IL-4, IL-6, IL-10, and TNF-α were detected in culture supernatant using a cytometric bead-based multiplex assay on days 1 to 6. In line with previous findings, very high doses of rhFVIII (10 IU/ml) or blockade of CD32 with mAb 2.4G2 (at high or low doses of rhFVIII) inhibited the differentiation of FVIII-specific ASCs. We observed a FVIII-dose dependent increase in the secretion of IFN-γ, IL-2, IL-4, IL-6, and IL-10. Very high doses of rhFVIII (10 IU/ml) suppressed ACS formation, but not the formation of these cytokines indicating an intact FVIII-specific T cell response. Secretion of TNF-α appeared not to be FVIII-dose dependent and was also observed in the cultures without rhFVIII. Inhibition of CD32 with mAb 2.4G2 in the presence of ASC stimulating FVIII concentrations (e.g. 1 IU/ml) prevented the development of ASC, but also diminished the formation of IFN-γ (334.2 ± 58.0 pg/ml versus 14.9 ± 1.4 pg/ml) and significantly reduced IL-10 (297.7 ± 78.5 pg/ml versus 131.3 ± 26.8 pg/ml). IL-6 was only slightly reduced, whereas IL-4 remained unchanged. IL-2 was even increased at later time points during cell culture (day 4: 16.1 ± 3.4 pg/ml versus 24.0 ± 1.4 pg/ml, day 6: 3.1 ± 0.6 pg/ml versus 21.8 ± 3.6 pg/ml). These results indicate that very high doses of FVIII prevented ASC formation, but not FVIII-specific T cell stimulation. In contrast, inhibition of CD32 prevented ASC formation, but also changed the secretion of T cell dependent cytokines. The lack of IFN-γ and IL-10 production after re-stimulation with various doses of rhFVIII indicates a reduced stimulation of Th1 and Th2 helper cells. Similar effects have been described previously, when B7-1 co-stimulation of CD4+ T cells was prevented by anti-CD80 mAb. In conclusion, inhibition of FVIII-specific ASC formation by means of very high doses of FVIII or inhibition of CD32 appears to occur differently. High doses of FVIII induce apoptosis in FVIII-specific memory B cells, but do not prevent FVIII-specific T cell responses. In contrast, inhibition of the Fcγ receptor CD32 on B cells and dendritic cells interferes with the FVIII-specific T cell response indicating a defective antigen presentation. Both high dose FVIII treatment and CD32 blockade, alone or in combination, should be further investigated to specifically address the FVIII-specific immune response in hemophilia A, and to evaluate a potential improvement of ITT. Disclosures: Vollack: Biotest AG: Research Funding. Trummer:Biotest AG: Research Funding. Tiede:CSL Behring: Consultancy, Honoraria, Research Funding; Biogen Idec: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Biotest: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Baxter: Consultancy, Honoraria, Research Funding. Werwitzke:Biotest AG: Honoraria, Research Funding.

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