An HLA-binding-motif-aided peptide epitope library: A novel library design for the screening of HLA-DR4-restricted antigenic peptides recognized by CD4+T cells

Abstract

Susceptibility to a series of autoimmune diseases is strongly associated with particular HLA class II alleles. Identification of T cell clones and antigenic epitopes bound by HLA class II molecules involved in autoimmune diseases is critical to understanding the etiology of these HLA class II-associated diseases. However, establishment of T cell clones in autoimmune diseases is difficult because the antigenic peptides are unknown. Peptide library methods which include all possible peptide sequences offer a potentially powerful tool for the detection of cross-reactive antigenic peptides recognized by T cells. Here, we reduced the number of peptides per mixture by utilizing the known binding motifs of peptides for the HLA-DRB1*0405 molecule and evaluated the effectiveness of this library design. Each library mixture evoked a strong proliferative response in the unprimed peripheral blood lymphocytes (PBL) from HLA-DRB1*0405-positive donors but little or no response in the PBL from HLA-DRB1*0405-negative donors. The library also detected antigenic peptides that activated three antigen-specific T cell lines restricted by HLA-DRB1*0405, with different specificities. The motif-based approach thus presents a powerful method for monitoring T cells in large, heterogeneous T cell populations and is useful for the identification of the mimic peptide epitopes of T cell lines and clones.

Identification of T Cell Ligands in a Library of Peptides Covalently Attached to HLA-DR4

“…However, the approach has worked for one class II-restricted myelin basic protein-specific clone that recognizes highly degenerate peptides (20). Synthetic peptide libraries that incorporate binding motifs for specific MHC molecules and are random at other positions have been developed and screened with Mycobacterium tuberculosis, GAD, and islet ␤ cell-reactive T cell clones (21)(22)(23). Escherichia coli expressed peptide libraries have likewise been constructed and successfully screened with class I-restricted T cell clones (24).…”

Use of BONSAI decision trees for the identification of potential MHC Class I peptide epitope motifs

“…Even among of peptides that bind to MHC with equally high affinities, there can be great variations in T cell responsiveness. The affect of single amino acid substitutions on responses by particular CTL clones have been well reported, but generalized sequence motif rules unrelated to binding affinity that influence or predict T cells have yet to be reported, although T cell responses to large synthesized peptide libraries indicate the existence of donor-independent activation motifs 3 . Identification of such motifs would be extremely useful for targeted vaccine development and epitope prediction in the investigation of immune responses in viral infection, cancer and autoimmune disease.…”

“…29 of 69 sequences negative for T cell reactivity did not contain the amino acid sequence [3,3]. Of the remaining sequences, 24 did not contain the motif [1,0,0].…”

“…Of the remaining sequences, 24 did not contain the motif [1,0,0]. This means that cumulatively, 76.8% of the negative peptides had the sequences [3,3] or [1,0,0]. Conversely, only 29.3% of the positive sequences contained either of these sequence motifs, indicating that the presence of these sequence combinations is inversely correlated with the potential for T cell reactivity.…”

“…Conversely, only 29.3% of the positive sequences contained either of these sequence motifs, indicating that the presence of these sequence combinations is inversely correlated with the potential for T cell reactivity. Furthermore, 121/174 (84%) of sequences positive for T cell reactivity did not possess the sequences [3,3] nor [1,0,3]. A peptide sequence that contains these "adverse motif" amino acid combinations is therefore much less likely to fall in the group of potential T cell epitopes than peptides without these sequences.…”