Multiplex mapping of CD4 T cell epitopes using class II tetramers

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Abstract

With the advent of class II tetramer technology, a tetramer-guided epitope mapping (TGEM) technique was developed for the identification of CD4+ T cell epitopes. This allowed the direct identification of epitopes recognized by the responding T cells, which were restricted to the single MHC allele of interest. However, as each individual carries multiple class II alleles, it would be advantageous to design an approach to identify CD4+ epitopes presented by different class II alleles at the same time. In the present study, a multiplex TGEM approach was developed to identify antigenic epitopes presented by multiple HLA class II alleles simultaneously. In this new approach, CD4+ T cells were stained with multiple sets of MHC class II tetramers—each labeled with a unique fluorescent label. Using this multiplex approach, novel epitopes from influenza antigens hemagglutinin and matrix protein presented by multiple class II alleles were identified in a single experimental setting.

Introduction

Understanding T cell epitope recognition is important as a basic immunological question and for practical applications such as vaccine development and immune modulation in the contexts of allergy and autoimmunity. It is well known that the selection of epitopes from an antigenic protein is dependent on antigen processing, peptide binding to the MHC, and interaction of the MHC/peptide complex with the spectrum of available TCR. In this way, the myriad of possible peptide patterns is narrowed to a much smaller number of dominant epitopes. A variety of approaches have been developed for identification of T cell epitopes within a given antigenic protein. These approaches are summarized in Table 1.

Each of these methods is based either on the ability of antigenic peptides to bind MHC or the ability of antigenic peptides to elicit a measurable functional response. While each of these approaches has facilitated epitope identification, some limitations remain. The first three approaches–CFSE assays, ELISpot (enzyme-linked immunosorbent spot assay), and Intracellular cytokine staining–are sensitive and capable of identifying epitopes without any prior knowledge of the HLA genotypes of the subject [1], [2], [3], [4]. However, subsequent determination of the HLA restriction for each specific epitope is difficult and time consuming. Computer-assisted prediction of MHC peptide binding is a useful approach to identify possible epitopes [5], [6]. However, the accuracy of these predictions must be verified empirically. In addition, peptide binding to the MHC does not assure the presence of T cells specific for the MHC/peptide complex. Similarly, the elution of peptides from antigen presenting cells and subsequent analysis by chromatography and mass spectrometry can yield important information about naturally processed epitopes from a given antigen [7]. But again, the resulting epitopes must be interrogated by a secondary method to measure the ability of the epitope to elicit a T cell response. The use of combinatorial peptide libraries can generate substantial information about the reactivity to various peptide patterns [8]. However, there is a disconnection that must be bridged between the reactive mimotopes and their biological counterparts. In many cases, determination of the “natural” epitope that corresponds to the synthetic mimotope is difficult or impossible [9]. HLA transgenic mice have been used to map various T cell epitopes [10], but the time and expenses associated with maintaining a mouse colony and generating transgenic mice with the desired HLA genotypes have limited the widespread use of this approach.

With the advent of HLA class II tetramers, it is now possible to detect antigen-specific CD4+ T cells by flow cytometry. Our laboratory has developed a method for producing functional tetramers without using a linked peptide [11]. This allows the production of numerous peptide/MHC combinations by loading the MHC with exogenous peptide. More importantly, the MHC can be loaded with mixtures (or pools) of peptide, facilitating the simultaneous screening of multiple peptides. Based on these innovations, the tetramer guided epitope mapping (TGEM) method was developed to identify class II restricted T cell epitopes [12], [13]. With this approach, T cell epitopes restricted by one class II allele of interest were identified in a single experimental setting within 3 weeks. Following this and other successes [12], [13], [14], [15], we reasoned that as each individual carries multiple alleles for HLA-DR, DQ and DP, a large amount of epitope information could be gleaned from each individual particularly if multiple alleles could be tested using a single sample. The goal of this present study, then, was to extend TGEM to allow the mapping of antigenic epitopes for multiple class II alleles simultaneously. Our results demonstrated that multiplex TGEM can be used to identify T cell epitopes for as many as four class II alleles simultaneously—by using a distinct fluorescent marker to label tetramers for each allele. These experiments demonstrated that TGEM provides a high throughput approach for T cell epitope identification.

Section snippets

Blood samples

All donors were healthy subjects recently immunized with the trivalent type A and B influenza virus “Fluzone” vaccine (Aventis, Bridgewater, NJ). PBMC were isolated from heparinized venous blood by Ficoll gradient centrifugation. HLA class II typing of donors was performed by reverse dot blot hybridization as described [16].

Chromophore-labeled antibodies and streptavidin

FITC-labeled anti-human CD4 antibody, PerCP-Cy5.5-labeled streptavidin, and PerCP-labeled streptavidin were purchased from BD Biosciences (San Jose, CA). PE-labeled

Defining of a set of chromophores for multiplex detection of T cells with tetramers

To date, MHC class II tetramers have been applied almost exclusively using PE as the fluorescent label. For this study, it was necessary to define a set of chromophores suitable for simultaneous use in tetramer staining on a 4-color flow cytometry instrument. To this end, we assembled tetramers using DR0103/HA306–318 monomer and streptavidin conjugated with a panel of readily available chromophores: FITC, PE, PerCP, PerCP-Cy5.5 and APC. PBMC from a healthy donor carrying the HLA-DRB1*0103

Discussions

In our previous work, we developed a tetramer-guided epitope mapping (TGEM) method to identify the class II restricted epitopes of a single antigen for individual class II alleles. The basis for this TGEM approach is the ability of tetramers to reveal precise class II restricted T cell epitopes directly, using a two-step staining procedure [12], [13]. In this way, T cell epitopes could be identified in a single experimental setting within 3 weeks. As each individual possesses multiple alleles

Summary

In summary, we have demonstrated that multiplex TGEM provides a high throughput approach to identify T cell epitopes restricted by multiple class II alleles. These findings significantly expand the utility of the technique, allowing epitope mapping with as many as four distinct sets of tetramers at one time. The TGEM approach provides some advantages over other approaches—most notably the rapid and robust nature of the assay and the precise determination of the class II restriction element for

Acknowledgments

We thank Matt Warren for the secretarial assistance. This work was supported in part by NIH contract HHSN266200400028C and the Immune Tolerance Network.

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