We report the molecular cloning and characterization of the first leukocyte-associated Ig-like receptor 1 (LAIR-1) homologue in mice that we have named mouse LAIR-1 (mLAIR-1). The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region syntenic with human chromosome 19q13.4 where the leukocyte receptor cluster is located. The protein shares 40% sequence identity with human LAIR-1, has a single Ig-like domain, and contains two immunoreceptor tyrosine-based inhibitory motif-like structures in its cytoplasmic tail. Mouse LAIR-1 is broadly expressed on various immune cells, and cross-linking of the molecule on stably transfected RBL-2H3 and YT.2C2 cells results in strong inhibition of their degranulation and cytotoxic activities, respectively. Upon pervanadate stimulation, the mLAIR-1 cytoplasmic tail becomes phosphorylated, thereby recruiting Src homology region 2-containing tyrosine phosphatase-2. Interestingly, unlike human LAIR-1, Src homology region 2-containing tyrosine phosphatase-1 is not recruited to the mLAIR-1 cytoplasmic tail. Screening human and mouse cell lines for mLAIR-1 and human LAIR-1 binding partners identified several lines expressing putative ligand(s) for both receptors.

Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (V H ) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the V H 1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from V H gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10-, DP-14-, and DP-88-encoded antibodies. In contrast, binding of the DP-5-encoded antibodies required residues in both the amino-and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5-encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from V H gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VIII. IntroductionHemophilia A is an X-linked bleeding disorder that is characterized by the absence or dysfunction of blood coagulation factor VIII. Current treatment of hemophilia A consists of infusion of therapeutic amounts of factor VIII that can evoke an immune response in some patients. The presence of neutralizing antibodies to factor VIII, commonly termed factor VIII inhibitors, presents a serious complication of hemophilia care. 1 The biochemical properties of factor VIII inhibitors have been extensively studied with emphasis on the epitope specificity and mode of action of these antibodies. Binding sites for inhibitors have been identified within the A2, A3, and C2 domains of factor VIII. [2][3][4][5][6] In general, heterogeneous mixtures of anti-factor VIII antibodies are present in plasma from patients with factor VIII inhibitors, and in more than 80% of inhibitor plasmas antibodies directed against the C2 domain are present. 7 Epitope mapping of anti-C2 domain antibodies revealed the presence of an inhibitor binding site comprising amino acid residues Val2248-Ser2312 of the C2 domain. 3 A second inhibitor epitope in the C2 domain has been attributed to region Glu2181-Val2243. 6 Antibodies reactive with the C2 domain prevent factor VIII from binding to phospholipid surfaces and von Willebrand factor. 8,9 Both findings are in agreement with the presence of binding sites for phospholipids and von Willebrand factor in the C2 domain. 10,11 An add...

We evaluated inhibitor formation in a group of patients with mild haemophilia A caused by an Arg593 to Cys mutation. A remarkably high cumulative inhibitor incidence of 14% over 22 years was observed. Three of 49 patients developed transient, low-titre inhibitors, which remained below 2.0 BU mL(-1). Four patients with an Arg593 to Cys mutation developed high-titre inhibitors (>5.0 BU mL(-1)). Three of these patients have been described previously. In this study, we characterized inhibitory antibodies in a fourth patient with high-titre inhibitors. Epitope mapping studies revealed that antibodies were predominantly directed to the A2 domain of factor VIII. We addressed the role of human leucocyte antigen (HLA) class II alleles in inhibitor development in patients with an Arg593 to Cys mutation by HLA genotyping. In the group of inhibitor patients raised frequencies of HLA-DRB1*01 and HLA-DQB1*05 were observed that did not reached statistical significance. Our data suggest that inhibitor development in mild haemophilia A patients with an Arg593 to Cys mutation is not linked to HLA class II profile.

After treatment with factor (F) VIII concentrate a significant number of patients with hemophilia A develop inhibitory antibodies that neutralize FVIII. Epitope mapping revealed that antibodies bind to selected regions within the A2, A3, and C2 domains of FVIII. Anti-A2 and anti-A3 antibodies interfere with assembly of FVIIIa with FIXa, whereas anti-C2 antibodies impede the interaction of FVIII with phospholipids. The immunologic mechanisms underlying inhibitor development in hemophilia A have not been fully elucidated. FVIII is recognized by the immune system as a foreign antigenic substance that evokes the T cell-dependent formation of high-affinity antibodies. Clonal analysis of B cell responses in hemophilia A patients has given further insight into the epitope specificity and molecular characteristics of FVIII inhibitors. Costimulatory blockade of FVIII-reactive T cells in a mouse model for hemophilia A has suggested new approaches for treatment of inhibitor patients. In this article, recent studies on the immunobiology of FVIII inhibitors are summarized and discussed with reference to their potential impact on treatment and prevention of immune responses in patients with hemophilia.

Summary. We characterized anti-factor VIII antibodies in a mild haemophilia A patient with an Arg 593 fiCys mutation in the A2 domain, using V gene phage-display technology. All isolated single-chain variable-domain antibody fragments were directed against residues Arg 484 -Ile 508 , a binding site for factor VIII inhibitors in the A2 domain. After a further period of replacement therapy, a transient rise in inhibitor titre was observed. These antibodies were directed against the A2 domain. Activation of a pre-existing pool of B cells, which express antibodies against residues Arg 484 -Ile 508 , could explain the rapid anamnestic response.Keywords: factor VIII inhibitors, haemophilia A, phage display, B lymphocyte, immunoglobulin repertoire.A serious complication in haemophilia A treatment is the development of antibodies that neutralize factor VIII activity (inhibitors). The prevalence of factor VIII inhibitors in patients with severe haemophilia A is approximately 25% (Mannucci & Tuddenham, 2001). Factor VIII inhibitors are infrequently observed in patients with mild or moderate haemophilia A (Sultan, 1992). The lower risk of inhibitor formation in this group of patients can be explained by the presence of tolerizing amounts of circulating endogenous factor VIII. Inhibitor development in these patients commonly arises after intensive factor VIII replacement therapy and is usually a transient event (Hay et al, 1998). Interestingly, some genetic defects are frequently observed in patients with mild haemophilia A who developed inhibitors. Amino acid substitutions located at the junction between the C1 and C2 domain or located in the A2 domain of factor VIII may predispose towards the development of factor VIII inhibitors (Hay et al, 1998). We have previously reported on the presence of human alloantibodies in a patient with an Arg 593 fiCys mutation (Fijnvandraat et al, 1997). In this patient, the anti-factor VIII antibodies were directed to wild-type A2 domain and not to the A2 domain containing the Arg 593 fiCys mutation when analysed at a late stage of inhibitor formation. These findings indicate that the missense mutation Arg 593 fiCys was related to antibody development.In the present study, phage display technology was used for the characterization of anti-factor VIII antibodies in the patient. Our findings showed that peripheral IgG + B cells can express human antibodies reactive with residues Arg 484 -Ile 508 . These antibodies are distinct from the alloantibodies found in plasma. Following a period of intensive treatment with FVIII concentrate after a surgical intervention, the patient developed a transient low-titre inhibitor that cross-reacted with endogenous factor VIII. We propose that activation of pre-existing factor VIII-specific B cells underlies the rapid appearance of cross-reactive antibodies. MATERIALS AND METHODSExpression, metabolic labelling and immunoprecipitation of A2 domain variants. The wild-type factor VIII A2 domain, the A2 domain containing the Arg 593 fiCys mutation (A2-R593C) and...