Monitoring CD4+ T cell responses against viral and tumor antigens using T cells as novel target APC

https://doi.org/10.1016/S0022-1759(03)00209-6Get rights and content

Abstract

CD4+ T cells play an important role in the induction and maintenance of an effective antiviral and antitumor immune response. However, standardized monitoring of antigen-specific CD4+ T cells has not been established at the single-cell level. We now present a sensitive, specific, and simple methodology in which purified memory CD4+ T cells are expanded from PBMC in a single cycle of antigen-driven stimulation and quantitatively assayed by interferon-γ ELISPOT. Issues of nonspecific background in assays were resolved with the use of innovative target cells, autologous PHA-expanded CD4+ T cells (T-APC). Remarkably, T-APC could not only present peptide epitopes from model antigens NY-ESO-1 and influenza nucleoprotein, but could also process full-length antigen endogenously expressed from recombinant fowlpox vector. This approach makes it possible to monitor CD4+ T cells in large series of patients, regardless of HLA haplotype, against the full peptide repertoire of a given antigen.

Introduction

It is now clear that the immune system can influence the initiation and development of cancer (Shankaran et al., 2001). It is widely believed that CD8+ cytotoxic T lymphocytes (CTL) represent the most potent antitumor effector cells. Accordingly, most clinical cancer vaccination studies have thus far focused on activating tumor-specific CD8+ T cells Jager et al., 2000a, Banchereau et al., 2001. However, several studies have indicated that CD4+ T helper cells in vivo have the capacity to enhance CTL activity (Keene and Forman, 1982) and, most importantly, help maintain the immune response for sustained periods of time (Matloubian et al., 1994). Therefore, it seems likely that optimal antitumor activity can be achieved only if both CD4+ and CD8+ tumor-specific T cells are induced (Greten and Jaffee, 1999).

MHC class II restricted epitopes of several tumor antigens, including the cancer-testis antigen NY-ESO-1, have recently been described Jager et al., 2000b, Zeng et al., 2000, Zarour et al., 2002. In addition, initial vaccination studies aiming at specifically eliciting a CD4+ response against tumor antigens have been undertaken in humans (Schuler-Thurner et al., 2002). Although the final goal in clinical studies of cancer vaccines is tumor regression or a reduction in tumor recurrence, the first step that needs to be taken is to demonstrate that the vaccination results in an increase in immunity against the chosen tumor antigen. “Traditional” methods like the measurement of proliferative capacity (Dhodapkar et al., 1999) or determination of cytokine concentration in the supernatant of bulk cultures Zeng et al., 2000, Hural et al., 2002 are still being widely used to detect responses of CD4+ T cells to antigens. While these methods may be suitable to simply distinguish between responders and nonresponders, they fail to quantitate increases or decreases in CD4+-mediated immunity at a single-cell level following vaccination.

More accurate and quantitative assays for the monitoring of CD4+ responses are warranted. Recently, MHC class II tetramers have been shown to provide a useful tool for the detection and quantification of human antigen-specific CD4+ T cells (Novak et al., 1999). However, MHC class II-peptide complexes are not easy to construct and are therefore not widely available yet. In addition, this technique does not allow the monitoring of responses against the whole sequence of a given antigen in patients with various MHC class II alleles. Thus, a standardized method to reliably monitor tumor-specific CD4+ responses to single epitopes as well as the full length of a tumor antigen has thus far not been presented.

Section snippets

Peptides and viral vectors

The synthetic influenza matrix protein peptide 58–66 (GILGFVFTL), influenza A nucleoprotein (NP) peptide 206–229 (FWRGENGRKTRIAYERMCNILKGK), NY-ESO-1 peptides 157–165 (SLLMWITQC), 80–109 (ARGPESRLLEFYLAMPFATPMEAELARRSL), and 121–138 (VLLKEFTVSGNILTIRLT), 145–174 (DHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQP), and the MAGE-3 peptide 243–258 (KKLLTQHFVQENYLEY) were obtained from Bio-Synthesis (Lewisville, TX), with a purity of >90%. The fowlpox virus recombinant for full-length influenza A nucleoprotein or

A mixture of monocytes and B cells can be used to expand antigen-specific CD4+ T cells in a single cycle of antigen-driven presensitization

In certain infectious diseases, the frequency of peripheral Ag-specific T cells is high enough to perform ex vivo enumerative assays Dunbar et al., 1998, Pathan et al., 2001. However, in the case of most tumor antigens, the unambiguous ex vivo detection of antigen-specific CD4+ and CD8+ T cells in the peripheral blood has proven difficult. Therefore, a method for amplifying specific CD8+ T cell responses has been established in our laboratory a few years ago (Gnjatic et al., 2000). In this

Acknowledgments

D.A. was supported by a grant from the Deutsche Krebshilfe, Mildred Scheel-Stiftung and by the Cancer Research Institute.

References (28)

  • S. Gnjatic et al.

    Strategy for monitoring T cell responses to NY-ESO-1 in patients with any HLA class I allele

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

    (2000)
  • T.F. Greten et al.

    Cancer vaccines

    J. Clin. Oncol.

    (1999)
  • J.A. Hural et al.

    Identification of naturally processed CD4 T cell epitopes from the prostate-specific antigen kallikrein 4 using peptide-based in vitro stimulation

    J. Immunol.

    (2002)
  • E. Jager et al.

    Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers

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

    (2000)
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