Phage‐displayed libraries of peptide/major histocompatibility complexes

Abstract

Characterizing peptide epitopes targeted by major histocompatibility complex (MHC)-restricted T cells of unknown specificity would have broad implications. In this article we introduce and validate an original phage-displayed library of noncovalent complexes of peptide and MHC (P/MHC). We show that soluble MHC molecules associate with peptides presented by a phage, thereby resulting in the formation of multivalent P/MHC phages. Complex formation is stabilized by the interaction of the soluble partner (MHC) with two components, peptide and g 2-microglobulin, both of which are covalently linked to the phage. As proof of concept, we have used this strategy to express peptide libraries in the context of H-2K b . Using monoclonal antibody 25D (specific for ovalbumin/H-2K b ) as a template to screen the library, we were able to select a variant epitope functionally and structurally related to the wild-type peptide. Interaction studies between monoclonal antibody 25D and cells suggest that the variant peptide has been selected on the basis of a decreased dissociation rate between the peptide/H-2K b complex and its ligand. A weak agonist of the N15 TCR (vesicular stomatitis virus/H-2K b -specific) was also isolated from another P/MHC library. This strategy opens up new perspectives for antigen discovery and the manipulation of T cell responses.

Deciphering CD4+ T cell specificity using novel MHC–TCR chimeric receptors

“…To efficiently identify cells presenting T cell epitopes we constructed novel chimeric receptors (termed MCRs) by grafting the extracellular domains of the MHC carrying directly tethered peptides [4][5][6] on top of the TCR truncated at the membrane (see Methods). In contrast to chimeric antigen receptors (CARs) 19 , MCRs expressed in TCR − T cell lines assemble with the native CD3 complex and can use the whole signaling machinery of the TCR (Fig.…”

“…Current methods to identify T cell epitopes [2][3][4][5][6][7][8][9] follow two major strategies. The first is based on detecting pMHC-TCR interactions by staining T cells with recombinant major histocompatibility complex-(MHC)-tetramers or by selecting phage particles, yeast or insect cells displaying peptide-MHC complexes with recombinant TCRs [4][5][6]10 . These methods are severely constrained by the binding affinities of the recombinant reagents to their ligands, which can differ substantially (for example, specific staining of OVA 323-339reactive OT-II T cells requires 400 times more tetramer than the specific staining of lymphocytic choriomeningitis virus (LCMV) gp 66-77 reactive SMARTA T cells 11 ).…”

Phage display—A powerful technique for immunotherapy

“…Human immunization allows to obtain phage display libraries from the peripheral blood or spleen lymphocytes and provides phage-displayed antibodies to red cell antigens D and B, 47 platelet antigen HPA-Ia, 48 glycoprotein IIb/IIIa, 49 native T-cell receptor TCR-Vα 50 and specific major histocompatibility complex (MHC)/peptide construct. 51 Furthermore, humanized immune repertoires are obtained using preparation of RNA from spleen material taken from hyperimmunised animals such as chickens, 52 rabbits, 53 sheep, 54 cows, nonhuman primates. 55 Moreover for this purpose the human antibody-producing XenoMouse strain, 56 has been engineered.…”

Diversity‐oriented approaches for interrogating T‐cell receptor repertoire, ligand recognition, and function

“…Three systems have been utilized to create pMHC libraries for selections, based upon baculovirus, yeast, and phage display ( Table ). Although the large library sizes obtainable through phage display make it a tempting system to use for pMHC libraries, there are relatively few examples of robust and functional pMHC displayed on phage . There is one example of novel peptides being isolated from a phage display library, but this library only expresses peptide and relies upon the addition of exogenous MHC .…”