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

Abstract

While T cells have been clearly implicated in a number of disease processes including autoimmunity, graft rejection, and atypical immune responses, the precise Ags recognized by the pathogenic T cells have often been difficult to identify. This has particularly been true for MHC class II-restricted CD4+ T cells. Although such cells can be demonstrated to have undergone clonal expansion at sites of pathology, they are frequently difficult to establish as stable T cell clones. Furthermore, in general, larger peptides in higher concentrations are required to stimulate CD4+ T cells than CD8+ T cells, which makes some of the techniques developed to identify CD8+ T cell Ags impractical. To circumvent some of these problems, we developed a model system consisting of two parts. The first part involves the construction of an indicator T cell hybridoma expressing a chimeric TCR comprised of murine constant regions and human variable regions specific for influenza hemagglutinin 307–319 presented by DR4. The second part consists of a library of fibroblasts each expressing multiple peptides as amino terminal covalent extensions of the β-chain of HLA-DR4 (DRA1*0101, DRB1*0401). Using this model system, we screened ∼100,000 peptides and identified three novel peptides stimulatory for the HA1.7 TCR. While there is some convergence at residues known to be important for T cell recognition, all three peptides differ markedly from each other and bear little resemblance to wild-type hemagglutinin 307–319.

High-throughput engineering and analysis of peptide binding to class II MHC

“…Unlike common surface display systems such as phage display (23,31), animal cell display (35), baculovirus/insect cell display (36), or even classical yeast display (28), which were developed for identification of T cell receptor ligands, the yeast codisplay method eliminates the limitations of expressing single-chain derivatives of multimeric proteins and covalently attaching MHC-II with peptides or other proteins. In the codisplay approach, MHC-II/peptide binding takes place intracellularly between nontethered species, better mimicking peptide loading of MHC-II in APCs (3), and yet yeast codisplay retains the advantage of creating a genotype-phenotype link for high-throughput screening and easy information retrieval.…”

“…In the codisplay approach, MHC-II/peptide binding takes place intracellularly between nontethered species, better mimicking peptide loading of MHC-II in APCs (3), and yet yeast codisplay retains the advantage of creating a genotype-phenotype link for high-throughput screening and easy information retrieval. Adaptation of this approach for screening peptide libraries to identify T cell stimulatory ligands (28,35,36) should be straightforward. Furthermore, whereas we have focused on characterizing and engineering MHC-II/peptide recognition, the approach is amenable to extension to a host of other systems for quantitative characterization of protein-protein and proteinpeptide-binding specificities, complementing other methods for studying such interactions (37,38) and broadening the impact of the method.…”

Bi-specific MHC Heterodimers for Characterization of Cross-reactive T Cells*

“…The fraction of cross-reactive cells in the overall LCMV-responsive and VVresponsive populations varies between individual mice because of the "private" nature of the T cell response (5,9,31), and the fraction of cross-reactive T cells present after in vitro culture depends on the conditions used to expand the antigen-specific cell population. 3 The particular T cell line investigated here exhibited an unusually high degree of cross-reactivity, thus providing an opportunity to evaluate the novel MHC heterodimer staining strategy, and to characterize in detail the nature of the cross-reactive T cell population.…”

“…If MHC heterodimers were available, they would be expected to exhibit specific binding only to T cells carrying receptors able to bind to both component MHC-peptide complexes, as the affinity of monomeric MHCpeptide engagement by T cell receptors (ϳ10 M (28)) generally is too low to survive typical washing protocols. 3 Our strategy for production of a novel, bi-specific H-2K b heterodimer is shown in Fig. 3.…”

“…During the negative selection phase of T cell development, autoreactive T cells capable of binding self-MHC-peptide complexes at high affinity are deleted, and it has been suggested that negative selection removes so many TCR sequences that a high level of TCR cross-reactivity is required for the immune system to be able to recognize a sufficiently large set of foreign peptides (2). T cell cross-reactivity has been observed in many systems (3)(4)(5)(6) and the structural basis for the phenomenon is beginning to be clarified (7,8). T cell cross-reactivity appears to provide a molecular basis for T cell heterologous immunity, in which exposure to one pathogen provides protection against another (9).…”