2003; baxter - Supplements - Haematologica

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60B. M. Reipert et al.Table 1. Median anti-factor VIII antibody titers of differentIgG subclasses in hemophilic E-17 mice (n=3).Anti-factor VIII antibody titersIgG1 IgG2a IgG2b IgG3One dose FVIII n.d. n.d. n.d. n.d.Two doses FVIII 1280 2560 2560 120Three doses FVIII 40960 40960 15360 640Four doses FVIII 122700 122700 61440 512022 weeks after the 20480 81920 5120 640fourth dosen.d. – not detectable; IgG subclasses of anti-FVIII antibodies were analyzed asdescribed in Hausl et al. 17 Briefly, PolySorp multiwell plates were coated with FVIII(free from albumin). Plates were incubated with serial dilutions of plasma samplesfrom mice treated with FVIII. IgG subclasses of anti-FVIII antibodies bound to theimmobilized FVIII were detected by incubation with isotype-specific, horseradish-peroxidase-labeledsecondary monoclonal antibodies (Southern Biotechnologies, Birmingham,AL, USA) and subsequent substrate development. Antibody titers wereexpressed as the highest dilution of plasma samples showing a positive result (opticaldensity >0.3) in the assay. From Hausl et al. 17 with permission.developed within 7 to 14 days of intraportalinjection of adeno-associated virus (AAV) carryinghuman FVIII. Bethesda titers of anti-FVIIIantibodies (>100 BU/mL) persisted relativelyunchanged for 9 to 10 months. At 10 monthsafter injection of the virus, FVIII inhibitors disappearedand FVIII protein became detectable inthe blood. These results suggest that immune toleranceto human FVIII can be induced by sustainedexpression of human FVIII in a mousemodel.SCID mice and SCID/Factor VIII knockoutmiceMice with severe combined immunodeficiency(SCID) lack functional B and T cells 38 due toan arrest of B and T lineage committed cells inearly development. This arrest is caused by a genemutation that impairs the recombination ofantigen receptor genes. The SCID-related geneencodes a DNA-dependent protein kinase catalyticsubunit and is located in chromosome16. 39 The SCID mice are unable to produce antibodiesor to reject allogeneic skin grafts 38 and,therefore, do not develop a functionally activeimmune system. Furthermore, spleen cells ofSCID mice fail to show a proliferative response toB-cell and T-cell mitogens. 38 Other hematopoieticcells such as macrophages and NK cellsdevelop and function normally. Because of theirsevere immune deficiency, SCID mice are able toaccept xenotransplants and can, therefore, bereconstituted with peripheral blood mononuclearcells isolated from human blood donors. 38 Themajor advantage of such a model is the possibilityof studying at least part of the humanimmune system in a mouse model. The majordisadvantages are the lack of functional lymphoidorgans and difficulty in eliciting primaryimmune responses.Laulan et al. 40 grafted SCID mice with peripheralblood mononuclear cells from healthydonors or donors with hemophilia A. They injectedreconstituted mice intraperitoneally with 100U (10 mg) of human FVIII. A specific response toFVIII only developed in mice that received bloodcells from patients with hemophilia who hadFVIII inhibitors and not in those that receivedcells from healthy donors or patients with hemophiliabut no FVIII inhibitors. These results suggestit is possible to induce secondary but not primaryimmune responses against human FVIII inreconstituted SCID mice. The mice received,however, only one FVIII injection and this was ata dose (100 U per mouse equals about 4000U/kg) which is not comparable to that used inpatients with hemophilia A. In similar experimentsby Vanzieleghem et al. 41 SCID mice werereconstituted with peripheral blood mononuclearcells from healthy blood donors and treated withone intraperitoneal injection of 50 U (5 mg)human FVIII followed by three of 25 U (2.5 mg).All reconstituted mice spontaneously producedanti-FVIII antibodies in the absence of any treatmentwith FVIII. These antibodies were onlydetectable after affinity purification. Treatingmice with up to four doses of FVIII did not inducean increase in antibody titers detectable afteraffinity purification. Furthermore, treatmentwith FVIII did not stimulate antibodies detectablewithout affinity purification. When SCID micewere reconstituted with peripheral blood mononuclearcells from patients with hemophilia Awho had FVIII inhibitors, treating the mice withFVIII induced a detectable secondary immuneresponse. 42 In an attempt to improve the SCIDmouse model, Gilles et al. created a SCID/FVIIIknockout mouse by cross-breeding SCID micewith FVIII knockout mice. 42 Future studies willreveal the advantages and limitations of this newmodel.To summarize the results obtained with SCIDmice, this model might be suitable for investigatingspecific questions of secondary anti-FVIIIimmune responses in an environment that containsparts of the human immune system.DiscussionAn animal model suitable for developing newapproaches for inducing immune tolerance toFVIII should encompass features that as much aspossible resemble those found in patients withhemophilia A while still accommodating sophisticatedanalysis and manipulation of the ongoingimmune response. Hemophilia A is a hereditaryX-linked bleeding disorder caused by theincomplete function of circulating clotting FVIIIor its total absence due to a mutation in the FVIIIgene. The consequent coagulation deficiency ishaematologica vol. 88(supplement n. 12):september <strong>2003</strong>
IV International Workshop on Immune Tolerance in Hemophilia61Figure 4. Kinetic of FVIII-specific T cells producing IL-2, IFN- γ, IL-4 or IL-10 in the spleen of hemophilic E-17 mice after treatmentwith one, two or four doses of human FVIII. Dot plots show intracellular cytokine staining in CD4 + T cells after in vitrorestimulation of splenic T cells with FVIII. Results shown in the upper right-hand corner of each dot plot represent the percentageof CD4 + T cells that stained positive for the cytokine indicated. The negative cell population (lower left-hand corner) wasdefined by using cells that were stained with appropriate isotype-matched negative control antibodies. T cells were isolated,restimulated and stained as described. 16 From Sasgary et al. 16 with permission.haematologica vol. 88(supplement n. 12):september <strong>2003</strong>
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Index of authorsAhmad, R.U., 56Albe
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