2003; baxter - Supplements - Haematologica

IV International Workshop on Immune Tolerance in Hemophilia65by a more restricted number of subjects. Crypticepitopes are not recognized when presentationstarts from the whole protein. However, crypticepitopes can be activated by peptides. As the tuningof the T-cell repertoire in the thymus is carriedout by deletion-selection on proteins, T-cells recognizingcryptic epitopes can be found in theperiphery.The quest for pathologically relevant T-cellsFrom the above, it appears that the identificationof T-cells of pathologic relevance, that is tosay T-cells with the capacity to drive the productionof inhibitor antibodies, could require furtherscrutiny. Nature offers us three situations withregards to FVIII. Healthy individuals with normallevels of FVIII have — presumably — had a chanceto eliminate strongly reactive T-cells from theirrepertoire during thymus maturation. T-cells toimmunodominant epitopes should, therefore, beabsent in the periphery. On the other hand, insevere hemophilia A, the absence of FVIII precludesthe negative selection of T-cells in thethymus: therefore in the periphery all T-cellsreacting to FVIII should be present. There is, however,an intermediate situation, in which patientssuffering from a mild degree of hemophilia A produceinhibitor antibodies towards normal wildtype FVIII, while remaining tolerant to their ownFVIII. Under such circumstances, the immunesystem should be purged from strongly reactive T-cells as for healthy individuals, except for these T-cells reacting against the site where the aminoacid substitution is located. Starting from suchpatients could offer a good opportunity to restrictthe population of T-cells greatly.T-cells were therefore cloned for the first timefrom T-cells of a mild hemophilia A patient carryinga mutation in the carboxy-terminal end ofthe C1 domain. 4 This patient produced inhibitorantibodies upon administration of wild-type FVIII,given to help the patient undergo a surgical intervention.T-cell cloning and characterizationA preparation of dendritic cells was made fromperipheral blood monocytes of the same patientfrom which T-cells had to be cloned. The latterwere selected by surface marker to obtain a pureCD4 + population. After several cycles of stimulationT-cells were cloned and expanded. Once theclones had been obtained, the search for specificitywas initiated by showing absence of proliferationand/or cytokine production in the presenceof autologous (mutated) FVIII, while strongactivation could be obtained by presentation ofwild-type FVIII.Synthetic peptides of various lengths centeredon the mutation site in the C1 domain were constructedand tested in the activation assay system.Three T-cell clones reacted towards the sameregion with subtle differences in the actual epitoperecognized. Recombinant FVIII moleculescarrying mutations within the carboxy-terminalend of the C1 domain were then tested for reactivitywith T-cell clones, some of which reactedstrongly.FVIII-specific T-cells: perspectivesWith such material at hand, it is now possibleto evaluate the MHC class II restriction of theFVIII recognition by T-cells, and perhaps identifyspecific alleles associated with activation of suchcells. This, as described above, could offer a markerpredisposing to the development of inhibitoryantibodies.A number of strategies aiming at making specificT-cells ignorant or unresponsive, or even todeleting such T-cells have been described in animalmodels in alternative research fields. It ishoped that the information gained in animalmodels will also hold true for FVIII. This is currentlybeing tested in mouse models of hemophiliaA. It can be expected that specific immunotherapyfor FVIII inhibitors targeting specificT-cells will become available in the near future.References1. Qian J, Collins M, Sharpe AH, Hoyer LW. Preventionand treatment of factor VIII inhibitors inmurine hemophilia A. Blood 2000; 95:1324-9.2. Bray GL, Kroner BL, Arkin S, Aledort LW, HilgartnerMW, Eyster ME, et al. Loss of high-responderinhibitors in patients with severe hemophilia A andhuman immunodeficiency virus type 1 infection: areport from the Multi-Center Hemophilia CohortStudy. Am J Hematol 1993; 42:375-9.3. Reding MT, Wu H, Krampf M, Okita DK, Diethelm-Okita BM, Christie BA, et al. Sensitization of CD4 +T cells to coagulation factor VIII: response in congenitaland acquired hemophilia patients and inhealthy subjects. Thromb Haemost 2000; 84:643-52.4. Jacquemin M, Burny W, Vantomme V, Chaux P,Lavend’homme R, Gilles JG, et al. High levels ofIFNγ production by FVIII-specific T cells from amild/moderate hemophilia A patient with inhibitor.42 nd Annual Meeting of the American Society forHematology, San Francisco, CA, USA, December2000.haematologica vol. 88(supplement n. 12):september 2003