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 methodolo- gies have been established for the analysis of humoral re- sponses, 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 recog- nize 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-spe- cific T cells and how they have impacted our understanding of cellular immune responses. In the past, several methods were used to quantitate antigen- specific T-cell responses. Traditionally, these assays were func- tional 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 func- tions, 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 under- estimated the true number of antigen-specific CTL and also lacked further detailed information about the characteristics of antigen-specific CD8 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- ful in analyzing the antigen-specific T-cell response in vivo.