Research paper
Expression mapping using a retroviral vector for CD8+ T cell epitopes: Definition of a Mycobacterium tuberculosis peptide presented by H2-Dd

https://doi.org/10.1016/j.jim.2004.12.015Get rights and content

Abstract

Identification of CD8+ T cell epitopes is important because detection of specific CD8+ T cells after infection or immunization requires prior knowledge of epitope specificity. Furthermore, identification of CD8+ T cell epitopes permits the development of specific preventive and therapeutic approaches to both infections and tumors. Thus far, CD8+ T cell epitopes have been identified either using an overlapping peptide library covering an entire protein, or using algorithms designed to identify likely peptides that bind to major histocompatibility complex (MHC) class I molecules. The synthesis of overlapping peptides can be prohibitively expensive, and the algorithm programs used to predict CD8+ T cell epitopes are not always accurate. Here we describe a retroviral expression system that specifically allows longer polypeptides and shorter peptides to be expressed in the cytoplasm, and thereby to be processed onto class I MHC molecules. T cells from mice that were immunized with a DNA vaccine encoding MPT-51 were probed against MHC-compatible cell lines retrovirally transduced with overlapping gene fragments encoding 120–140 amino acids of the MPT-51 molecule. After further testing of shorter peptide sequences, we identified a CD8+ T cell epitope using cell lines expressing a relatively small number of algorithm-predicted candidate epitopes. We found that one of the requirements for cell surface display of the 20-mer peptide was the need for cotranslational ubiquitination. The restriction molecule was identified as Dd following transduction with MHC class I genes followed by transduction with the oligonucleotide encoding the epitope. The retroviral expression system described here is cost-effective, particularly if the target molecule is large, and could be adapted to identifying T cell epitopes recognized in infectious disease and against tumor cell antigens.

Introduction

CD8+ cytotoxic T lymphocytes (CTL) play a pivotal role in protective immunity against infection with intracellar pathogens, such as certain bacteria and viruses (Uchijima et al., 1998, Flynn and Chan, 2001, Gulzar and Copeland, 2004). During an infection, many proteins derived from the pathogen are secreted or produced in the host cells. These proteins are processed by the MHC class I pathway. The endogenous MHC class I pathway targets cytosolic proteins, either self-derived or pathogen-derived, that are processed to peptides by the proteasome. The resulting peptides are actively transported into the endoplasmic reticulum by transporter associated with antigen processing (TAP) molecules, and are finally presented to CTL in the context of MHC class I molecules (Ortmann et al., 1994, Heemels and Ploegh, 1995). Following CTL recognition of pathogen-derived MHC/peptides complexes on the cell surface, the infected cells are killed and infection eliminated by the secretion of effector molecules, such as perforin, granzyme, and interferon-γ (Barry and Bleackley, 2002).

Although intracellular pathogens possess a large number of peptides which have the potential to bind to MHC class I molecules, the CD8+ T cell response is more limited, with only a minority of the potential binding motif-containing peptides from a given foreign protein being actually immunogenic (Sercarz et al., 1993, Barber and Parham, 1994). Various factors have been implicated in this limitation (epitope selection): (1) antigen processing by the proteasome (Eggers et al., 1995, Niedermann et al., 1996); (2) TAP-dependent peptide transport (Lauvau et al., 1999); (3) affinity of the peptide for the MHC (Chen et al., 1994, Sette et al., 1994); (4) the transport of MHC/peptide complexes to the cell surface (Levitsky et al., 1997); and (5) the response of the T-cell repertoire (Connolly, 1994, Cao et al., 1996).

To better understand CD8+ T cell responses during infection, it is important to identify epitopes that are recognized by CD8+ T cells. Identification of the epitopes, which typically consist of 8–10-mer peptides, is essential to detect specific CD8+ T cells by the tetramer assay (Altman et al., 1996), intracellular cytokine staining, or the enzyme-linked immunosorbent spot assay (ELISPOT). In addition, identification of these epitopes permits the development of specific preventive and therapeutic approaches in both infectious disease and cancer (Nagata et al., 2004, Chen et al., 2004, Faure et al., 2004, Romero et al., 2004).

Overlapping peptide libraries have been widely employed for the identification of T cell epitopes. The libraries, consisting of 15–20-mer peptides overlapping by at least nine residues, span the whole protein of interest (Van der Zee et al., 1989). Once a T cell epitope has been mapped in a 15–20-mer peptide, the minimal T cell epitope can be delineated by preparing a set of peptides, which have been truncated from the N- or C-terminal end of the known active peptide. This is a very powerful method for mapping T cell epitope(s), but it requires numerous rounds of peptide synthesis, which can be prohibitively expensive for a large protein.

Recently, a number of programs that utilize algorithms that have been useful in predicting T cell epitopes for peptide mapping studies have been developed (Parker et al., 1994, Rammensee et al., 1999, Reche et al., 2002). These programs list and rank the possible candidates of T cell epitopes within a given protein sequence. However, it has been shown that these program methods are not infallible, and in some cases are no better than the use of the simple anchor residue motifs (Andersen et al., 2000). In general the immunogenicity of individual peptides predicted by such programs must be confirmed empirically, using any number of T cell assays, although some programs have incorporated various factors involved in the epitope processing (Peters et al., 2003). Currently, the combination of an overlapping peptide library with an epitope prediction program seems to be the most comprehensive and effective method for identification of CD8+ T cell epitopes within a protein (Suzuki et al., 2004).

In this report, we describe a novel cost-effective retroviral expression method for the identification of CD8+ T cell epitopes within antigens of interest. As a model antigen, we used MPT51, a major secreted protein of Mycobacterium tuberculosis (Wilson et al., 2004), since we recently demonstrated that MPT51 can induce T-cell-mediated immune responses and protective immunity upon challenge with M. tuberculosis (Miki et al., 2004). The retroviral expression system makes it possible to express large DNA fragments in cells so that the location of CD8+ T cell epitopes can be rapidly identified without preparing numerous synthesized peptides. Minimal CD8+ T cell epitopes can be identified from the candidate DNA fragments using a computer-assisted algorithm. This method is particularly useful for mapping epitopes from large target molecules where the cost of synthesizing peptides is particularly high.

Section snippets

Mice

BALB/c mice (Japan SLC, Hamamatsu, Japan) were kept under specific pathogen-free conditions and fed autoclaved food and water ad libitum at the Institute for Experimental Animals, Hamamatsu University School of Medicine. Two-month-old female mice were used in all experiments. All animal experiments were performed according to the Guidelines for Animal Experimentation, Hamamatsu University School of Medicine.

Cell lines

P815 mastocytoma cell line (H2d) and BW5147 T lymphoma cell line (H2k) were cultured in

P815 cells retrovirally transduced with MPT51 can stimulate MPT51 immune splenocytes from BALB/c mice

We examined whether MPT51-transduced P815 cells were capable of stimulating splenocytes from BALB/c mice immunized with pCI-MPT51. The immune splenocytes were stimulated with MPT51 gene-transduced or nontransduced P815 for 24 h, and the IFN-γ concentrations in the culture supernatants were determined by ELISA. As shown in Fig. 1, robust IFN-γ production was observed after stimulation with P815 cells that had been transduced with MPT51. However, no significant IFN-γ production was observed in

Discussion

In this study, we have used a molecular approach for the identification of CD8+ T cell epitopes in the MPT51 protein of M. tuberculosis, a novel secreted antigen that was previously shown to be protective in vaccinated mice (Miki et al., 2004). Employing the retroviral expression system, we successfully identified one CD8+ T cell epitope, MPT51 24–32 (GGPHAVYLL), in BALB/c mice.

Progressing from longer expressed fragments (120–140 aa in length) of MPT51 to shorter peptides, we were able to

Acknowledgments

We thank the NIH Tetramer Facility for providing the H2-Dd–peptide tetramer complex, Dr. N. Ohara (Nagasaki University Graduate School of Biomedical Science) for providing plasmid pMB49, Mr. K. Shibata for technical assistance with flow cytometry (Equipment Center, Hamamatsu University School of Medicine), and Dr. John Spencer (Colorado State University) for critical reading of this manuscript.

This work was supported by grants-in-aid for scientific research and centers of excellence from the

References (43)

  • L.D. Barber et al.

    The essence of epitopes

    J. Exp. Med.

    (1994)
  • M. Barry et al.

    Cytotoxic T lymphocytes: all roads lead to death

    Nat. Rev., Immunol.

    (2002)
  • W. Cao et al.

    The weak CD8+ CTL response to an influenza hemagglutinin epitope reflects limited T cell availability

    J. Immunol.

    (1996)
  • W. Chen et al.

    Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues

    J. Exp. Med.

    (1994)
  • Y.F. Chen et al.

    Cytotoxic-T-lymphocyte human papillomavirus type 16 E5 peptide with CpG-oligodeoxynucleotide can eliminate tumor growth in C57BL/6 mice

    J. Virol.

    (2004)
  • J.M. Connolly

    The peptide p2Ca is immunodominant in allorecognition of Ld by beta chain variable region V β8+ but not V β8− strains

    Proc. Natl. Acad. Sci. U. S. A.

    (1994)
  • M. Eggers et al.

    The cleavage preference of the proteasome governs the yield of antigenic peptides

    J. Exp. Med.

    (1995)
  • O. Faure et al.

    Inducible Hsp70 as target of anticancer immunotherapy: identification of HLA-A*0201-restricted epitopes

    Int. J. Cancer

    (2004)
  • J.L. Flynn et al.

    Immunology of tuberculosis

    Annu. Rev. Immunol.

    (2001)
  • F. Grignani et al.

    High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein

    Cancer Res.

    (1998)
  • N. Gulzar et al.

    CD8+ T-cells: function and response to HIV infection

    Curr. HIV Res.

    (2004)
  • Cited by (13)

    • Profiling T cell interaction and activation through microfluidics-assisted serial encounter with APCs

      2021, Sensors and Actuators, B: Chemical
      Citation Excerpt :

      BW5147 (H2k) lymphoma cell line (JCRB9002) was obtained from JCRB Cell Bank (Japan). BW5147 cells were retrovirally transduced with H-2Kb as previously described [62] We used this H-2Kb expressing BW5147 cells as antigen presenting cells (APCs) throughout this study. APCs were cultured in RPMI 1640 medium supplemented with 10 % Fetal bovine serum (FBS) and 1 % Penicillin.

    • Identification of HLA-DR4-restricted T-cell epitope on MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis using MPT51 overlapping peptides screening and DNA vaccination

      2010, Vaccine
      Citation Excerpt :

      Using DNA vaccine encoding MPT51, we found that MPT51 can induce specific cellular immune responses and the protective immunity against challenge with M. tuberculosis in murine infection model [15]. We identified murine T-cell epitopes using C57BL/6 and BALB/c mouse strains [16,17] and an HLA-A*0201-restricted CD8+ T-cell epitope in MPT51 molecule [18]. Here, we identified an HLA-DRB1*0401-restricted CD4+ T-cell epitope on MPT51 with a strategy using HLA-DRB1*0401-transgenic (Tg) mice, gene gun immunization with expression plasmid DNA encoding MPT51, overlapping synthetic peptides spanning the entire mature MPT51 amino acid (aa) sequence, and the computer-assisted major histocompatibility complex (MHC) binding peptide prediction algorithms.

    • Chemokine receptor-mediated delivery of mycobacterial MPT51 protein efficiently induces antigen-specific T-cell responses

      2008, Vaccine
      Citation Excerpt :

      An MPT51 24–32 peptide/H2-Dd tetramer complex was kindly supplied by the NIH Tetramer Facility. MHC/peptide tetramer assay was performed as described previously [8]. In brief, 3 days after the last immunization, spleen cells were prepared and stained with PE-conjugated MPT51 24–32 peptide/H2-Dd tetramer complexes and fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (BD PharMingen) monoclonal antibody for 30 min at 4 °C.

    View all citing articles on Scopus
    View full text