2003; baxter - Supplements - Haematologica

IV International Workshop on Immune Tolerance in Hemophilia 19the A2 domain of factor VIII. 9 Antibodies directedtoward residues Arg 484 -Ile 508 prevent the stimulatoryeffect of isolated A2 domain on the catalyticactivity of factor IXa. 10 The inhibitory propertiesof anti-A3 and anti-A2 antibodies are inagreement with our current model of the assemblyof the factor VIIIa-IXa complex. Initial highaffinity binding is mediated by interaction of thefactor VIII light chain with residues present in theEGF1-and EGF2 domains of factor IXa whereasstimulation of factor VIII cofactor activity resultsfrom interaction of the A2 domain with the proteasemoiety of factor IXa. 3 Apparently, factor VIIIinhibitory antibodies can block the interaction offactor VIIIa with factor IXa via two distinct mechanisms.Conflicting data have been reported on the epitopefor factor VIII inhibitors in the C2 domain.Recombinant factor VIII fragments expressed inEscherischia coli revealed the presence of a bindingsite within region Val 2248 -Ser 2312 . 11 Evaluationof the functional inhibition of a panel of anti-C2inhibitors against a series of human/porcinehybrids suggested that residues Glu 2181 -Val 2243 areinvolved in binding of factor VIII inhibitors. 12 Theapparent discrepancies between the two studiesmay be explained by the different approachesthat have been used to characterize factor VIIIinhibitors. Antibodies directed toward the C2domain interfere with binding of factor VIII tophospholipids. 11,13 Current data suggest that arestricted number of 3-4 major binding sites forinhibitory antibodies are present on factor VIII.At present it is not clear why only a limited numberof sites on factor VIII is targeted by theimmune system. This may be explained by theuse of only a selected proportion of the totalavailable immunoglobulin repertoire for the generationof antibodies that interact with immunodominantregions on factor VIII. To explore thisissue we analyzed the anti-factor VIII repertoireof a number of well-characterized inhibitorpatients by phage display.Assembly of human immunoglobulin repertoiresImmunoglobulins are composed of a light anda heavy chain that contain both variable andconstant regions. The variable or V-regions areencoded by two and three gene segments for thelight and heavy chain, respectively. The heavychain locus is present on chromosome 14 whereasloci encoding κ and λ light chains are presenton chromosome 2 and 22, respectively. The individualloci contain large numbers of differentgene segments that are assembled into functionalantibody molecules during maturation of Bcells. The heavy chain locus consists of over 120variable heavy chain gene segments, 27 diversity(D) segments and 6 joining (J) segments 14,15which are followed by gene segments that encodeconstant parts of immunoglobulin heavy chains(µ, δ, γ3, γ1, α1, γ2, γ4, ε, α2). The κ light chainlocus on chromosome 2 comprises 91 variablelight (VL) chain gene segments and 5 J segments.The λ light chain on chromosome 22 contains>45 variable light chain and four J segments. Notall variable heavy and light chain segments areused for assembly of human antibodies. Approximately50-75% of VH and VL segments are nonfunctional.This may be due to the absence of anopen reading frame or lack of recombinationsites flanking the variable segments. 16 Assemblyof immunoglobulin molecules proceeds in ahighly regulated manner. The first event thatoccurs is the joining of a D segment with a J segmentin the variable region of the immunoglobulinlocus. Subsequently, a VH gene segmentand a µ constant region is added giving riseto a functional heavy chain which is co-expressedtogether with a surrogate light chain on a B-cellprecursor in the bone marrow. 17 Next, a VL segmentis fused with a JL segment resulting in afunctional light chain segment that replaces thesurrogate light chain. Following negative selectionof self-reactive B-cells in the bone marrow,immature B-cells are transported to the peripherywhere they express both IgM and IgD. 17 Internalizationof antigen via surface immunoglobulinson B-cells is followed by proteolytic processingof antigens into peptides that are amenablefor presentation by MHC class II molecules onthe surface of B-cells. T helper cells specificallyrecognizing the presented peptide then help B-cells to proliferate. Proliferating B-cells migrate tothe lymph nodes where germinal centers arisethat provide an environment for adequate T-cellhelp for the generation of high affinity antibodies.Affinity maturation proceeds via amino acidreplacements in the variable parts of the antibody,a process termed somatic hypermutation.Additionally, class switching from IgM to IgA,IgG or IgE occurs at this stage. Following affinitymaturation, B-cells develop into plasma cellsthat produce antibodies that are present in plasma.Alternatively, B-cells develop into memoryB-cells, which are present for extended periods inthe periphery thereby facilitating secondaryresponses to incoming antigens.Analysis of factor VIII inhibitors by phagedisplay: general outlineUsing peripheral B lymphocytes as a source ofRNA we isolated immunoglobulin repertoires ofpatients with an inhibitor using a series of polymerasechain recations (PCR) that targeted thevariable domains of the immunoglobulin heavychain. 18 Several restrictions were applied to preferentiallyamplify the anti-factor VIII repertoireof these patients. The majority of factor VIIIinhibitors are of subclass IgG4: therefore, anIgG4 specific amplification step was introducedin our experimental protocol. The concentrationof IgG4 is approximately 5% of the total quantityof IgG in human serum. Although this numberdoes not necessarily correspond to the percentageof IgG4 positive B cells in the peripheralblood, it is anticipated that the resultinghaematologica vol. 88(supplement n. 12):september 2003