Our research group has been investigating the T-cell-driven immune response to infused FVIII with the aim of identifying additional T-cell epitopes. One goal of these studies is to facilitate the ‘rational design’ of less immunogenic FVIII proteins. Herein, some novel T-cell assays that

our laboratory has adopted to assess FVIII immunogenicity are described. In addition, detailed phenotypes of FVIII-specific T cells are explored for possible clues as to tolerogenic mechanisms that guide clinical response to ITI. The MHC class II molecule binds to peptides ~11–20 amino acids in length, with binding determined by four pockets in the MHC groove. Binding motifs for many common MHC class II molecules have been identified [29]. An individual’s HLA class PD0332991 supplier II type determines whether no peptides or some specific peptides are presented on the surface of the MHC class II receptor. At present, the number and type of FVIII

peptides recognized and presented on a class II molecule in patients with haemophilia is an area of active research. Recombinant MHC class II molecules that bind FVIII-derived peptides are proving useful in the characterization of T-cell responses to FVIII [27]. MHC class II-peptide complexes are biotinylated at a specific site and streptavidin is used to cross-link the soluble molecules to form tetramers [30]. These fluorescently labelled molecules are able to detect antigen-specific CD4+ T cells by binding to a T-cell receptor capable of recognizing the MHC class II-peptide complex [30]. Midostaurin datasheet For several years our group has been investigating the number and characteristics of T-cell epitopes in haemophilia A patients with inhibitors. As FVIII is a large protein with many peptides, initial

studies have employed a systematic strategy known as Tetramer Guided Epitope Mapping (TGEM) (Fig. 6) [31]. In this protocol, soluble extracellular domains of MHC molecules are loaded with overlapping FVIII peptides and divided into pools each having 5 to 10 peptides. These pooled-peptide tetramers are used as reagents to analyse patient-derived CD4+ selleck cells previously stimulated with FVIII. The pool(s) with positive tetramer staining are identified by flow cytometry, with the tetramer indicated on the y-axis and CD4+ cells on the x axis. Peptides from a tetramer-positive pool are then loaded individually onto MHC molecules and the analyses are repeated in a process known as decoding. Decoding results (typically one, occasionally two) that resemble the original pooled result identify the specific peptide/s containing the epitope. T cells positive for tetramer staining can be sorted by flow cytometry, thus providing a rapid means of isolating and/or cloning these T cells. TGEM has been used to identify T-cell epitopes in patients with mild haemophilia A. An early study involved two brothers who had the same missense substitution FVIII-A2201P and shared the DRB1*01:01 HLA allele [32, 33].