Competition of peptide-MHC class I tetrameric complexes with anti-CD3 provides evidence for specificity of peptide binding to the TCR complex

Background: Major histocompatibility complex (MHC)peptide tetrameric comple xes (tetramers) are valuable tools for detecting and characterizing peptide -specific T cells. Because the frequency of these cells is generally very l ow, it may be difficult to discriminate between nonspecific and specific te tramer binding. Methods: A four-color flow cytometric assay that simultaneously measures te tramer, CD3, CD8, and CD14 was used to investigate the sensitivity and spec ificity of MHC class I tetramer staining. This was accomplished by using th e influenza virus matrix protein peptide, GILGFVFTL (FLU), as a model recal l antigen and the human immunodeficiency virus (HIV) reverse transcriptase peptide, ILKEPVHGV (HIV), as a model novel antigen. Peripheral blood mononu clear cells (PBMC) from 31 HLA-A2.1(+) and 10 HLA-A2.1(-) healthy individua ls were stained with the tetramers. Results: The lower limit of detection was established at approximately 1/8, 000. In HLA-A2(+) PMBC, frequencies of tetramer-positive CD8(+) T cells wer e log normally distributed and were high for FLU (1/910) but low for HIV (1 /6,067). A novel competition assay, in which tetramer binding was shown to diminish subsequent staining with anti-CD3 antibody, was used to confirm th e specificity of tetramer binding to the T-cell receptor (TCR) complex. The competition assay was validated by evaluating several anti-CDS antibodies and showing that in PBMC from HLA-A2(-) subjects, spurious tetramer-positiv e events (1/20,000) failed to compete with CD3 binding. For the "recall" FL U tetramer, the degree of competition was proportional to the frequency, su ggesting a selection of high avidity cells. Although CD3 competition was al so highly correlated with the intensity of tetramer staining, competition a llowed the identification of false positive cases with relatively high tetr amer staining intensity. Conclusion: The data indicate that competition of CD3 binding allows confir mation of the specificity of tetramer binding to the TCR, extending the use fulness of tetramers in the frequency analysis of peptide-specific T lympho cytes. Cytometry 41:321-328, 2000. (C) 2000 Wiley-Liss, Inc.