Modulation of factor VIII-specific memory B cells.

The development of inhibitory antibodies against factor VIII (FVIII) is the major complication in patients with haemophilia A who are treated with FVIII products. Memory B cells play an essential role in maintaining established antibody responses. Upon re-exposure to the same antigen, they are rapidly re-stimulated to proliferate and differentiate into antibody-secreting plasma cells (ASC) that secrete high-affinity antibodies. It is, therefore, reasonable to believe that memory B cells have to be eradicated or inactivated for immune tolerance induction therapy to be successful in patients with haemophilia A and FVIII inhibitors. The aim of our studies was the development of strategies to prevent FVIII-specific memory B cells from becoming re-stimulated. We established a 6-day in vitro culture system that enabled us to study the regulation of FVIII-specific murine memory-B-cell re-stimulation. We tested the impact of the blockade of co-stimulatory interactions, of different concentrations of FVIII and of ligands for toll-like receptors (TLR). The blockade of B7-CD28 and CD40-CD40 ligand interactions prevented FVIII-specific murine memory B cells from becoming re-stimulated by FVIII in vitro and in vivo. Furthermore, high concentrations of FVIII blocked re-stimulation of FVIII-specific murine memory B cells. Triggering of TLR7 amplified re-stimulation by low concentrations of FVIII and prevented blockade by high concentrations of FVIII. We conclude that we defined modulators that either amplify or inhibit the re-stimulation of FVIII-specific murine memory B cells. Currently, we are investigating whether the same modulators operate in patients with haemophilia A and FVIII inhibitors.

Opportunities and limitations of mouse models humanized for HLA class II antigens.

MHC class II molecules are essential for shaping the CD4+ T-cell repertoire in the thymus and for selecting antigenic peptides that are presented to CD4+ T cells in the periphery. A range of different mouse models humanized for HLA class II antigens have been developed to study the regulation of MHC-class II restricted immune responses. These mouse models have been used to identify immunodominant peptides that trigger diseases and to characterize the interactions of T-cell receptors with disease-associated peptides and MHC class II molecules. Peptides presented to CD4+ T cells in these mouse models were shown to be similar to peptides presented to CD4+ T cells in patients who carry the same MHC class II haplotype. Opportunities and limitations associated with these mouse models will be discussed and the potential application of these models for understanding the regulation of antibody responses against factor VIII in hemophilia A will be indicated.

Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance.

Patients with severe hemophilia A frequently develop neutralizing anti-factor VIII antibodies after replacement therapy with factor VIII (FVIII). In a search for new strategies to induce immune tolerance against FVIII in these patients, we used a murine model of hemophilia A to investigate the importance of CD40/CD40 ligand (CD40L) interactions for the initiation of the anti-FVIII immune response. We focused our attention in particular on the induction of neutralizing anti-FVIII antibodies and the Th1/Th2 polarization of FVIII-specific T cells. The development of anti-FVIII antibodies was analyzed by ELISA systems (detection of total anti-FVIII antibodies) and Bethesda assays (determination of neutralizing anti-FVIII antibodies). Factor VIII-specific T cells were characterized by multiparameter flow cytometry and cytokine ELISAs for the detection of cytokine production in splenic CD4+ T cells after in vitro restimulation with FVIII. Hemophilic mice received four doses of FVIII and anti-CD40L antibody MR1 (24 h before FVIII). Subsequently mice received four doses of FVIII only. The induction of neutralizing anti-FVIII antibodies in hemophilic mice after treatment with human FVIII could be prevented completely by a blockade of CD40/CD40L interactions using MR1. Furthermore, FVIII-specific T-cell responses that included both Th1 and Th2 cells were suppressed when mice were treated with FVIII and MR1. The initial blockade of CD40/CD40L interactions was, however, not sufficient to induce a lasting immune tolerance against FVIII. The immune suppression was abolished and both neutralizing anti-FVIII antibodies and FVIII-specific T cells developed when treatment with FVIII was continued after the omission of MR1. In addition, there were no alterations in the Th1/Th2 polarization induced by the initial blockade of CD40/CD40L interactions.

Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A.

To investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy. knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.

Anti-inflammatory properties of human serum IgA: induction of IL-1 receptor antagonist and Fc alpha R (CD89)-mediated down-regulation of tumour necrosis factor-alpha (TNF-alpha) and IL-6 in human monocytes.

A deregulated expression and/or release of large amounts of inflammatory cytokines such as IL-1 and TNF-alpha accounts for most pathophysiological events in a variety of systemic inflammatory diseases, the effect being mediated by the interaction of these cytokines with their respective receptors. IL-1 receptor antagonist (IL-1Ra), mainly produced by monocytes/macrophages, is an inhibitor of IL-1 activity. The present study shows that human serum IgA induces significant IL-1Ra release in human peripheral blood mononuclear cells and adherent monocytes. IgA induced higher levels of IL-1Ra than Haemophilus influenzae type b (Hib) expressing lipopolysaccharide (LPS), purified LPS or phorbol myristate acetate (PMA), without induction of IL-1 beta release, and even inhibited LPS-induced IL-1 beta release. Induction of IL-1Ra by IgA could be detected both at the mRNA and protein levels in resting and activated monocytes. Ligation of Fc alpha R with MoAb My-43 or treatment with human serum IgA induced protein tyrosine phosphorylation in human monocytes, and herbimycin A, a specific inhibitor of protein tyrosine kinase activity, inhibited IgA-induced IL-1Ra production, suggesting that Fc alpha R-mediated induction of tyrosine phosphorylation is required for the IgA-induced stimulation of IL-1Ra release. In addition, triggering of Fc alpha R with MoAb specifically down-regulated TNF-alpha and IL-6 release in human monocytes activated with Hib. By the induction of IL-1Ra and down-regulation of the release of inflammatory cytokines such as IL-1 beta, TNF-alpha and IL-6, interaction of IgA with human monocytes may actively contribute to the regulation of the inflammatory response.