SummaryAntibodies (Ab) that inhibit factor VIII (fVIII) may develop in patients with hemophilia A and rarely in individuals without congenital fVIII deficiency (acquired hemophilia). Synthesis of fVIII inhibitors requires CD4+ T cells. We investigated the proliferative response of blood CD4+ cells from 11 patients with congenital or acquired hemophilia and 12 healthy subjects, to recombinant human fVIII, and to pools of overlapping synthetic peptides spanning the sequences of individual fVIII domains. All patients had CD4+ cells that responded to fVIII. The intensity of the responses fluctuated over time: several patients had brief periods when they did not respond to fVIII. All healthy subjects had transient CD4+ responses to fVIII, that were significantly lower than those of hemophilia patients. Also, healthy subjects responded to fVIII less frequently and for shorter periods than hemophilia patients. All patients and healthy subjects recognized several fVIII domains: the A3 domain was recognized most strongly and frequently. The transient sensitization of CD4+ cells to fVIII in healthy subjects suggests that inadequate tolerization of CD4+ cells to fVIII, due to lack of endogenous fVIII, is an important factor in the development of clinically significant anti-fVIII antibodies in hemophilia A.

Summary.  Approximately 25% of severe hemophilia A patients develop antibodies (Ab) that neutralize the procoagulant function of factor (F)VIII (inhibitors). Autoimmune FVIII inhibitors may develop in individuals without congenital FVIII deficiency and cause acquired hemophilia. Low titers of anti‐FVIII Ab may be present in hemophilia A patients without inhibitors and in healthy blood donors. FVIII‐specific CD4+ T‐cells drive the synthesis of anti‐FVIII Ab. We examined the epitope repertoire of CD4+ T‐cells from 15 healthy subjects, 10 hemophilia A patients without inhibitors, 11 hemophilia A patients with inhibitors, and six acquired hemophilia patients. Blood CD4+ T‐cells were challenged in proliferation assays with a panel 16 overlapping synthetic peptides, spanning the sequence of the FVIII C2 domain. The sequence region 2291–2330 contained the most frequently and strongly recognized peptides in each of the four subject groups. Crystallographic B factor data and the location of these peptides within the three‐dimensional structure of the C2 domain confirm that this region has a high degree of solvent exposure and flexibility within the peptide backbone, which are structural features typical of immunodominant universal CD4+ epitopes. Furthermore, this sequence region overlaps inhibitor‐binding sites, suggesting that CD4+ T‐cells recognizing peptide sequences within this region might be involved in inhibitor synthesis. The sequence regions 2191–2210 (recognized strongly by each study group except hemophilia A patients with inhibitors) and 2241–2290 (recognized primarily by acquired hemophilia patients and healthy subjects) share the same structural features, and also overlap inhibitor‐binding sites. Although similar, there appear to be important differences in the CD4+ epitope repertoires of congenital and acquired hemophilia patients.

SummaryMice genetically deficient in factor VIII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-γ and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-γ, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfVIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis. Abbreviations used: antibodies, Ab; antigen presenting cells, APC; Arbitrary Units, AU; enzyme-linked immunosorbant assay, ELISA; factor VIII, fVIII; human factor VIII, hf VIII; intravenous, i.v.; optical density, OD; polymerase chain reaction, PCR; phosphate buffered saline solution, PBS; PBS containing 3% bovine serum albumin, PBS/BSA; PBS containing 0.05% polyoxyethylene sorbitan monolaurate, PBS/Tween-20; phytohemoagglutinin, PHA; stimulation index, SI

Previous studies suggested that tetanus and diphtheria toxoids (TTD and DTD, respectively) contain "universal" epitopes for human CD4+ cells (residues 632-651 and 950-969 of TTD and 271-290, 321-350, 351-370, 411-430, and 431-450 of DTD). To investigate whether CD4+ cells of 100 randomly selected subjects recognized those sequences, the proliferation of CD4+ cell-enriched blood lymphocytes to TTD and DTD and individual synthetic universal epitopes was measured. CD4+ cells of 98 subjects recognized both toxoids, those of 1 subject only TTD, and those of 1 only DTD. The TTD peptides and DTD peptides 271-290 and 331-350 were recognized by >/=80% of the toxoid-sensitized subjects. The other DTD sequences were recognized by 63%-71% of subjects. DR-homozygous subjects recognized several universal epitopes less frequently than did DR-heterozygous subjects. The intensity of responses to the epitope peptides correlated with that to TTD or DTD, consistent with recognition of the peptides by CD4+ cells specific for the cognate toxoid.

Summary.   Severe hemophilia A patients treated with factor (F)VIII may develop antibodies (Ab) that block FVIII function (inhibitors). Autoimmune inhibitors may develop in subjects without congenital hemophilia, and cause acquired hemophilia. Hemophiliacs without inhibitors and healthy subjects may also have small amounts of antiFVIII Ab. FVIII‐specific CD4+ T cells induce antiFVIII Ab synthesis. Here, we have examined their epitope repertoire in hemophilia patients and healthy subjects. We used overlapping synthetic peptides, spanning the sequence of the FVIII A3 domain, to challenge blood CD4+ T cells in proliferation assays. The epitopes recognized in hemophilia A patients with or without inhibitors, acquired hemophilia patients, or healthy subjects overlapped, yet had characteristic differences. Most members of one or more study groups recognized the sequence regions 1691–1710, 1801–1820, 1831–1850, and 1941–60. In the proposed three‐dimensional structure of the A3 domain, these sequences are largely exposed to the solvent and flanked by flexible sequence loops: these are structural features characteristic of ‘universal’ CD4+ T epitopes. Hemophilia A patients with inhibitors recognized prominently only the sequence 1801–1820, which overlaps a known inhibitor binding site. This is consistent with the possibility that CD4+ T cells recognizing epitopes within residues 1801–1820 have a role in inducing inhibitor synthesis. In contrast, CD4+ T cells sensitized to sequences 1691–1710 and 1941–60, which are recognized by healthy subjects and hemophilia A patients without inhibitors, might curb inhibitor synthesis.