Flow Cytometric Analysis of Antigen Specific T Cells Using PeptideMHC Tetramers

Recently, it has become possible to directly visualize antigen-specific T cells by using soluble multimeric MHC-peptide complexes. In 1997, Altman and Davis (31) demonstrated that flourescently labeled, tetrameric peptide-MHC complexes could indeed bind stably, specifically, avidly to antigen-specific T cells. Another methodology for multimer generation involves using an antibody to link two peptide-MHC complexes, forming peptide-MHC dimers, often termed MHC-Ig complexes (32,33). In theory, only T cells with receptors specific for the peptide used in the complex will be recognized by these approaches. Using standard flow cytometric analysis, one can gate on the T cells and look for expression of the antigen-specific TCR. Analysis of peripheral blood T cells specific for potent immunogens such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV) demonstrated that between 0.2 and 6% of circulating CD8+ cells were specific for peptides representing these antigens. The quantitation of antigen-specific CD8+ T cells by flow cytometry using peptide-MHC tetramers has been shown to correlate well with traditional in vitro cytotoxicity assays (34). In addition, the intensity of staining of CD8+ T cells with peptide-MHC tetramers appears to correlate well with T-cell avidity for the antigen (34). A number of studies have shown the utility of flow cytometric analysis using peptide-MHC tetramers to quantitate CD8+ T cells specific for tumor antigens or control antigens often used in immunotherapy protocols (15,35,36). Dunbar and colleagues (37) used peptide-MHC tetramers to isolate antigen-specific T cells from peripheral blood or lymph nodes by cell sorting. Following cloning of these selected T cells they were shown to respond to specific antigen by cytokine production. However, it is not known whether simple binding of T cells to peptide-MHC tetramer accurately predicts functional activity.

Although peptide-MHC tetramers are powerful tools, they have certain limitations. In particular, they can only be used to detect known immune responses, as the peptide of interest must be loaded into the peptide-MHC tetramer and thus must already be known and synthesized. Immune responses to unknown antigens thus cannot be detected. Additionally, only class I MHC tetramers have been routinely available for widespread use; construction of class II tetramers has required considerable technical challenges to be overcome but these reagents have been described and are soon to be commercially available.

The exquisite sensitivity of peptide-MHC tetramers for quantitating antigen-specific T cells has raised the question of whether CD8(+) cells that bind to peptide-MHC tetramers are naïve or antigen-experienced ("memory" T cells). Pittet and colleagues (18) observed that 10 of 13 melanoma patients and 6 of 10 healthy individuals had high frequencies (greater than or equal to 1/2500 CD8+ T cells) of Melan-A-specific cells in the peripheral blood. All of these Melan-A-specific cells from the healthy individuals and seven of the patients displayed a naïve CD45RA(hi)/RO(-) phenotype. In three of the patients, "memory" CD45RA(lo)/RO(+) Melan-A-specific cells were observed. In contrast, influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype. One patient was observed to have an evolution of the Melan-A-specific cell phenotype over time. This study suggests that in addition to simply detecting peptide-MHC positive cells, it may be important to assess if antigen-specific T cells are naïve or memory T cells in order to determine if the detected antigen-specific T cells have been stimulated by the immunization strategy.

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