Prevention Of Murine FVIII-Specific Memory B Cells Due To FcγR2B (CD32) Blockade Is Associated With Distinctive Alterations In The FVIII-Specific Cytokine Profile
Authors: Vollack Nadine , Trummer Arne , Tiede Andreas , Werwitzke Sonja 
Published: October 2019

Abstract
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.