2003; baxter - Supplements - Haematologica

56B. M. Reipert et al.genetic of the immune response is extremely wellcharacterized have potential advantages and meetmany of the above requirements.Factor VIII knockout miceIn 1995, Bi et al. described two murine modelsof hemophilia A in which a targeted gene disruptionin exon 16 (E-16) or exon 17 (E-16) ofthe FVIII gene resulted in a complete deficiencyof FVIII. 8 These mice express a typical phenotypeof hemophilia A 9,10 which can be corrected byhuman FVIII. 11 Qian et al. showed that E-16 andE-17 knockout mice develop anti-FVIII antibodiesafter intravenous injection of human FVIII intherapeutic doses and that this immune responseis dependent on the induction of FVIII-specific T-lymphocytes. 12 Human FVIII is certainly moreforeign to hemophilic mice than to humans withhemophilia and can be expected to induce astronger immune response in mice than murineFVIII would do. The lack of a convenient sourceof murine FVIII limits its use but human FVIIIcan be used instead because it interacts with themurine proteins of the coagulation system 13 dueto its sequence homology with the murineFVIII. 14 As mentioned above, this interaction inan environment of a severe bleeding syndromemight be important for regulating the immuneresponse. Therefore, human FVIII can be considereda suitable model antigen in the search fornew strategies to induce immune tolerance.We used the E-17 model in a series of differentstudies. In our hands, all E-17 mice developeddetectable anti-FVIII antibodies after two doses ofhuman FVIII (200 ng recombinant FVIII, freefrom albumin) that increased in titer after subsequentdoses. 15, 16 Titers of total anti-FVIII antibodiesanalyzed by ELISA correlated with titers ofneutralizing anti-FVIII antibodies measured byBethesda assays, 16 (Figure 1). Anti-FVIII antibodysecreting cells (ASC) first appeared in the spleenwhere they were detectable after two doses ofFVIII, 17 (Figure 2a). Their appearance correlatedwith that of anti-FVIII antibodies in blood plasma(Figure 2b). Anti-FVIII ASC in bone marrowwere detectable after three doses of FVIII (Figure2a). These cells had probably formed initially inthe spleen and then migrated to the bone marrow.We did not see any formation of anti-FVIIIASC in lymph nodes confirming that the spleenis the major development location for immuneresponses against blood-borne antigens. The IgGsubclassdistribution of anti-FVIII ASC was similarin spleen and bone marrow and matched thesubclasses of anti-FVIII antibodies in blood plasma(Figure 3, Table 1), indicating that bothorgans contribute to circulating antibodies in theblood. The IgG1 and IgG2a subclasses dominatedthe anti-FVIII antibody response. After FVIIItreatment had terminated, anti-FVIII antibodiespersisted for at least 22 weeks (Figure 2b). Thepersistence of antibodies correlated with thelong-term persistence of anti-FVIII ASC (Figure2a). These ASC could be either long-living ASC asFigure 1. Relation of total anti-FVIII antibody titers (ELISAtiter) to titers of FVIII-neutralizing antibodies (Bethesdatiter) in plasma obtained from hemophilic mice after onedose (), two doses () or four doses () of FVIII. Eachpoint represents values for an individual mouse. Blood sampleswere obtained 1 week after each dose. ELISA titers andBethesda titers were analyzed as described (16). From Sasgaryet al. 16 with permission.described by Slifka et al. 18 and Manz et al. 19 orcells continuously formed by antigen-driven differentiationof memory B cells as described byOchsenbein et al. 20 Future studies using celltransfer experiments should be able to showwhich model is best for explaining the maintenanceof high titers of anti-FVIII antibodies inhemophilic mice, and possibly also in patients.The outcome of such studies could have considerableimplications for creating new strategiesaimed at inducing immune tolerance to FVIII.The development of anti-FVIII antibodies in E-17mice correlated with the appearance of FVIII-specificCD4 + T cells. Of these, the most prominenttype that could be detected were CD4 + T cells producingIFN-γ, followed by T cells producingIL10 16 (Figure 4).The CD40/CD40L interaction is a key event inthe initiation of humoral immune responsesagainst T-cell-dependent antigens. 21 Previousstudies have shown that a blockade of CD40/CD40L interactions can achieve prolonged survivalof allografts in rodents and monkeys 22,23and prevent graft-versus-host disease 24,25 andautoimmunity in rodent models. 26,27 These effectsare probably due to tolerance induction in theCD4 + T-cell population 28 and tempt the speculationthat anti-CD40L antibodies can induce lastingT-cell tolerance to FVIII in hemophilia A. Asanti-FVIII antibody formation is T-cell dependent,inducing FVIII-specific T-cell toleranceshould prevent their formation. Using the E-17mouse model we could show that the blockade ofCD40-CD40 ligand interactions prevents theinduction of an anti-FVIII immune response, 29haematologica vol. 88(supplement n. 12):september 2003