Development and Use of Multimeric Major Histocompatibility Complex Molecules

Enumeration and analysis of antigen-specific T cells play a central role in cellular immunology.While many methodologies have been established for the analysis of humoral responses, until recently the direct analysis of antigen-specific T-cell responses was extremely difficult.Difficulty in analyzing T cells was partially due to the fact that T lymphocytes recognize cell surface peptide-major histocompatibility complex (MHC) complexes and also due to the intrinsic low affinity of T-cell receptors (TCR) for cognate ligand.In this review, we discuss some new methodologies for visualizing antigen-specific T cells and how they have impacted our understanding of cellular immune responses.In the past, several methods were used to quantitate antigenspecific T-cell responses.Traditionally, these assays were functional assays, dependent on the proliferation or lytic activity of antigen-specific T cells in vitro.To improve the sensitivity of these assays, it was necessary to expand the population of antigen-specific T cells in vitro before measuring effector functions, such as cytolysis, proliferative responses, or cytokine release.Serial limiting-dilution assays (LDA) were the old "gold standard" for quantitative analysis of antigen-specific cytotoxic T lymphocytes (CTL) (19).This assay depended on the ability of antigen-specific T cells to survive, function, and proliferate in vitro upon stimulation.Therefore, the number of antigen-specific CTL determined by LDA significantly underestimated the true number of antigen-specific CTL and also lacked further detailed information about the characteristics of antigen-specific CD8 ϩ T cells in vivo.Recently, new assays based on cytokine induction and/or release have been developed.These assays do not require in vitro proliferation of antigen-specific T cells but do depend on peptide-induced production of cytokines.Cytokine production can be detected in an ELISPOT assay (34), intracellularly (29, 31), or captured on the cell surface (8) and analyzed by flow cytometry.Analysis for cytokine-producing cells has influenced our estimate of antigen-specific T-cell precursor frequency.While these methods have been useful in visualizing cells on the basis of effector cytokine release, there is also a skewing that occurs depending on which cytokine is being assayed that is not present if one can visualize T cells solely based on TCR specificity.In addition, due to technical aspects of the assays, one cannot keep cells alive for additional studies after ELI-SPOT assay or intracellular cytokine staining.Anticlonotypic TCR-specific antibodies have also been use-