Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Summary

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

Abstract

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8⁺ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8⁺ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8⁺ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8⁺ T cells.

Introduction

Qa-1 belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules in mice. Its human homolog is HLA-E. Previous evidence has demonstrated that Qa-1 molecules have important biological functions. Firstly, Qa-1 molecules play an important role in surveying cells for structural and functional integrity. In this regard, Qa-1 molecules have evolved several strategies to monitor the normal function of a cell. One such strategy enables Qa-1 molecules to form complexes with a processed leader peptide (epitope), i.e. the Qa-1 determinant modifier (Qdm) that is processed from classical MHC-Ia molecules in the endoplasmic reticulum

Protocol

All experiments were done in compliance with an Institutional Animal Care and Use Protocol approved by Animal Care and Use Committee at the University of Texas at El Paso and Loma Linda University.

1. Generation of an OLP Library Covering the Whole Length of a Protein

1. Design an OLP library in which all peptides are 15-mer in length, and adjacent peptides overlap by 11 amino acids.
   NOTE: In the MOG OLP study, sequence of the MOG precursor [Mus musculus] was retrieved from NCBI protein database by following this link: https://www.ncbi.nlm.nih.gov/protein/NP_034944. The MOG precursor (247 amino acids), which contained both s....

Results

Design of an OLP library covering the whole length of a protein

Beginning at the N-terminus of a protein, each peptide is 15 amino acids (15-mer). Hence, the first peptide spans position 1 to position 15. The N-terminus of the second peptide overlaps with the C-terminus of the first peptide by 11 amino acid. Hence, the second peptide spans the position 5 to position 19. Design the rest of the peptides to the end of C-terminus of th.......

Discussion

Here, we have described a protocol for analyzing Qa-1 epitopes in a protein. In relation to this protocol, several other strategies were also reported previously. First, allogeneic CD8⁺ T cell lines and clones were used for the identification of the Qdm¹. Second, a putative Qa-1-binding motif from the analysis of Qdm was used for the identification of the HSP60p216-224 and a TCRBV8.1 epitope^9,18. Third, individual overlapping pe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
The protein to be analyzed	N/A	N/A	Sequence of the protein can be obtained from NCBI
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat#: D2650 SIGMA	DMSO should be sterile and cell culture tested.
Kb-/-Db-/- mice	The Lackson Laboratory	Stock#: 019995	We used Taconic H2-KbH2-Db doube knockout mice (Cat#: 4215-F and 4215-M) which however are not available anymore.
AIM V Serum Free Medium	ThermoFisher Scientific	Cat#: 12055091
2-mercaptoethanol	ThermoFisher Scientific	Cat#: 21985023
Sodium pyruvate	ThermoFisher Scientific	Cat#: 11360070
Nonessential Amino Acids	ThermoFisher Scientific	Cat#: 11140076
Dynabeads CD8 Positive Isolation Kit	ThermoFisher Scientific	Cat#: 11333D
Bio-Gel P-100	Bio-Rad	Cat#: 150-4171
Phoshate Balanced Solution (PBS)	ThermoFisher Scientific	Cat#: 20012027
Trasfer pipette	Globe Scientific	Mfg#: 137238
Murine M-CSF	PeproTech	Cat#: 315-02
48-well tissue culture plates	USA Scientific	Cat#: CC7682-7548
Corning Costar TC-treated Multiple well Plates, 96-well, V-shaped bottom	Sigma-Aldrich	Cat#: Z372129 Sigma
1ml deep 06-well PP plate, sterile	USA Scientific	Item#: 1896-1110
Recombinant murine IL-2	PeproTech	Cat#: 212-12
Recombinant murine IL-7	PeproTech	Cat#L: 217-17
Capture anti-IFN-γ antibody	BD Biosciences	Cat#: 551881
ELISPOT plate	Sigma-Aldrich	Cat#: S2EM004M99
C1R	ATCC	Cat#: ATCC CRL-1993
C1R.Qa-1b			Custom made (GenBank access#: NM_010398.3)
Qa-1 lentiviral vector	GeneCopoeia	Product#: Mm02955
Detection anti-IFN-γ antibody	BD Biosciences	Cat#: 551881
Tween20	Sigma-Aldrich	Cat#: P9416
Streptavidin-HRP	BD Biosciences	Cat#: BD557630
AEC substrate	BD Biosciences	Cat#: 551951
ImmunoSpot Analyzer	ImmunoSpot		Any immunoSpot analyer should work for this purpose.