The development of antibodies that interfere with factor VIII (FVIII) pro-coagulant activity, often referred to as “inhibitors”, can complicate the treatment of hemophilia A. These alloimmune responses, as well as the rare development of autoimmune FVIII inhibitors, are associated with significant morbidity and mortality. The production of anti-FVIII antibodies follows stimulation of helper T cells by epitopes in FVIII. An immunodominant HLA-DRB1*0101-restricted T-cell epitope was recognized by CD4+ T cells from a mild hemophilia A inhibitor subject and from his brother, who had a sub-clinical inhibitor (James et al., J Thromb Haemost 5: 2399-2407, 2007). Their CD4+ T cells recognized overlapping synthetic peptides with sequences corresponding to FVIII residues 2186-2205, 2187-2205 and 2194-2213. Nineteen T-cell clones recognizing this epitope were isolated, with phenotypes representing four distinct T-cell lineages.
(1) to evaluate the promiscuity/immunodominance of an HLA-DRB1*0101-restricted T-cell epitope in FVIII; (2) to introduce amino acid substitutions that will prevent presentation of this epitope to the immune system by DR0101 and by other DR alleles.
The minimal epitope and MHC Class II (DR0101) “anchor” residues were determined using a competition assay measuring displacement of a labeled peptide having high affinity for recombinant DR0101 by a series of FVIII peptides. Peptide concentrations at which 50% inhibition of the labeled peptide binding occurred (IC50) were obtained by regression analysis. Binding of the peptides to five additional DR alleles was evaluated directly using recombinant proteins; predicted binding of peptides to additional DR alleles was evaluated using the program ProPred. Proliferation and cytokine production by the clones in response to wild-type and modified peptides were measured, and the concentrations at which half-maximal T-cell responses (EC50) to the FVIII peptides occurred were determined.
Binding of truncated peptides to DR0101 identified FVIII2194-2205 as the minimal epitope. Binding of FVIII2194-2205 peptides with single Arg substitutions identified F2196, M2199, A2201 and S2204 as anchor residues at positions 1, 4, 6 and 9, respectively, corresponding to peptide-binding pockets seen in the crystal structure of a DR0101-peptide complex. The relative binding of Ala-substituted peptides confirmed that F2196 and M2199 are anchor residues. T-cell stimulation requires recognition of peptides by both the Class II receptor and the T-cell receptor (TCR). Sequences of TCR variable regions (TCRBVs) expressed by the clones were identified as TCRBV20-1*01 (3 VDJ combinations), TCRBV6-6*01, TCRBV5-1*01, and TCRBV6-1*01, indicating at least six different T-cell progenitors recognized this epitope. The clones were next stimulated with peptides having modified epitopes. Strikingly, none proliferated or secreted cytokines when stimulated by FVIII2194-2205, F2196A, which also showed an IC50 > 10 μM when tested for binding to DR0101, DR0301, DR0401, DR1101, DR1104, and DR1501. Substitutions at other anchor positions affected binding to some but not all of the DR proteins. Predicted binding of the F2196A variant to 51 DR alleles was analyzed using ProPred; none bound at a threshold stringency of 10% (low stringency, thus the predicted epitopes included those with lower calculated affinities). In preparation for directly testing the immunogenicity of additional substitutions, all possible amino acid substitutions at position 2196 were evaluated using ProPred. 13 of 19 possible substitutions were predicted to prevent FVIII2194-2205 binding to all 51 DR alleles included in the algorithm (with a 3% threshold = intermediate stringency).
MHC class II anchor residues and TCR contact sites for an immunodominant HLA-DRB1*0101-restricted T-cell epitope have been mapped precisely. Both measured and predicted effects of amino acid substitutions indicated that this F2196 is essential for effective presentation of this epitope by multiple DR alleles. Effects of various sequence modifications on FVIII function, conformation and immunogenicity are currently being evaluated using recombinant FVIII and FVIII C2 domain proteins to indicate their possible therapeutic potential.
Pratt:CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxter: Honoraria; Grifols: Honoraria.
Asterisk with author names denotes non-ASH members.