The identification of CD4 ⁺ T cell epitopes with dedicated synthetic peptide libraries

Abstract

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4 ؉ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4 ؉ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the ␤ cell-reactive CD4 ؉ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4 ؉ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and͞or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.A large number of T cell-mediated diseases, exemplified by the autoimmune disease insulin-dependent diabetes mellitus (IDDM), are thought to be caused by the activation of CD4 ϩ T cells by antigen-derived peptides in the context of HLA class II (1, 2). For many T cell-mediated diseases, it is not known which antigen(s) cause(s) the activation of pathogenic CD4 ϩ T cells. Identification of antigens and epitopes in T cell-mediated diseases might enable immune intervention strategies with respect to treatment or prevention of disease. Even in the absence of an identified antigen source, peptides that stimulate pathogenic CD4 ϩ T cells may be useful in the development of primary immune intervention approaches via the induction of tolerance or the application of altered peptide ligands (3, 4).Various methods for the identification of T cell epitopes have been described. Most of them are based on identification of the source protein of an epitope by using molecular biology approaches (5-7) or peptide elution from HLA molecules followed by fractionation and sequence analysis (8, 9). These methods are time consuming or require large amounts of biological material. Alternative, fast synthetic peptide library approaches have been shown to be useful in the identification of T cell epitopes only for CD8 ϩ T cells (10, 11). Herein we show that CD4ϩ T cell epitopes can be identified by using a newly developed system for construction and screening synthetic peptide libraries. We applie...

Contribution of Individual Amino Acids Within MHC Molecule or Antigenic Peptide to TCR Ligand Potency

“…A few laboratories, including the authors', have successfully employed PS-SCL to define interactions within the trimolecular complex. This approach has allowed the identification of binding motifs for several MHC molecules (5, 6) as well as ligands for CD8 ϩ and CD4 ϩ T cells (7)(8)(9)(10)(11)(12)(13)(14)(15). However, it is not clear to what extent T cell clones (TCC) can be studied by the current technique, or exactly how PS-SCL allow the determination of T cell epitopes as compared with conventional epitope mapping approaches.…”

Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells

“…Synthesis and screening of an HLA DPw4 binding, onebead-one-peptide synthetic peptide library A 14-mer one-bead-one-peptide synthetic peptide library 19,23,23 consisting of about 8 Â 10 6 peptides was synthesized. The peptides in this library were enriched for potential HLA-DPw4 binding peptides by including amino acids at anchor positions that enhance binding of a peptide to HLA DPw4.…”

“…In previous studies, however, the synthetic epitopes formed valuable leads for identification of natural epitopes. 19,23,32 The 14-mer peptide library was synthesized by utilizing the known binding motifs of peptides presented by HLA DPw4, 16 to increase the effectiveness of the library. Out of 14 candidate epitopes of human, bacterial or viral origin, only the epitope encoded by human Syn 2 was recognized.…”

“…19 The purity of the peptides was determined by reversed-phase HPLC, and the integrity of the peptides was determined by matrixassisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy on a Lasermat mass spectrometer (Finnigan-MAT, Hemel Hempstead, UK). Individual peptides were tested at a final concentration of 0.1-1 mM in 0.01-0.1% DMSO (v/v).…”

A Focal Adhesion Kinase-Derived Peptide Binds the Src SH3 Domain in Two Orientations, As Demonstrated Using Paramagnetic Nuclear Magnetic Resonance

“…Peptides with the sequence RALPSI-PKLA were prepared by Fmoc chemistry using preloaded Tentagel resins, benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate/N-methylmorpholine (PyBop/ NMM) for in situ activation, and 20% piperidine in Nmethyl-2-pyrrolidone (NMP) for Fmoc removal. 32 Couplings were performed for 75 min. After final Fmoc removal, peptides were cleaved with a 19/1 trifluoroacetic acid (TFA)/H 2 O mixture and isolated by diethyl ether/pentane precipitation.…”