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Competition of peptide-MHC class I tetrameric complexes with anti-CD3 provides evidence for specificity of peptide binding to the TCR complex.
Cytometry 2000; 41(4):321-8C

Abstract

BACKGROUND

Major histocompatibility complex (MHC)-peptide tetrameric complexes (tetramers) are valuable tools for detecting and characterizing peptide-specific T cells. Because the frequency of these cells is generally very low, it may be difficult to discriminate between nonspecific and specific tetramer binding.

METHODS

A four-color flow cytometric assay that simultaneously measures tetramer, CD3, CD8, and CD14 was used to investigate the sensitivity and specificity of MHC class I tetramer staining. This was accomplished by using the influenza virus matrix protein peptide, GILGFVFTL (FLU), as a model recall antigen and the human immunodeficiency virus (HIV) reverse transcriptase peptide, ILKEPVHGV (HIV), as a model novel antigen. Peripheral blood mononuclear cells (PBMC) from 31 HLA-A2.1(+) and 10 HLA-A2.1(-) healthy individuals 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 were 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 the specificity of tetramer binding to the T-cell receptor (TCR) complex. The competition assay was validated by evaluating several anti-CD3 antibodies and showing that in PBMC from HLA-A2(-) subjects, spurious tetramer-positive events (1/20,000) failed to compete with CD3 binding. For the "recall" FLU tetramer, the degree of competition was proportional to the frequency, suggesting a selection of high avidity cells. Although CD3 competition was also highly correlated with the intensity of tetramer staining, competition allowed the identification of false positive cases with relatively high tetramer staining intensity.

CONCLUSION

The data indicate that competition of CD3 binding allows confirmation of the specificity of tetramer binding to the TCR, extending the usefulness of tetramers in the frequency analysis of peptide-specific T lymphocytes.

Authors+Show Affiliations

University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11084618

Citation

Hoffmann, T K., et al. "Competition of peptide-MHC Class I Tetrameric Complexes With anti-CD3 Provides Evidence for Specificity of Peptide Binding to the TCR Complex." Cytometry, vol. 41, no. 4, 2000, pp. 321-8.
Hoffmann TK, Donnenberg VS, Friebe-Hoffmann U, et al. Competition of peptide-MHC class I tetrameric complexes with anti-CD3 provides evidence for specificity of peptide binding to the TCR complex. Cytometry. 2000;41(4):321-8.
Hoffmann, T. K., Donnenberg, V. S., Friebe-Hoffmann, U., Meyer, E. M., Rinaldo, C. R., DeLeo, A. B., ... Donnenberg, A. D. (2000). Competition of peptide-MHC class I tetrameric complexes with anti-CD3 provides evidence for specificity of peptide binding to the TCR complex. Cytometry, 41(4), pp. 321-8.
Hoffmann TK, et al. Competition of peptide-MHC Class I Tetrameric Complexes With anti-CD3 Provides Evidence for Specificity of Peptide Binding to the TCR Complex. Cytometry. 2000 Dec 1;41(4):321-8. PubMed PMID: 11084618.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Competition of peptide-MHC class I tetrameric complexes with anti-CD3 provides evidence for specificity of peptide binding to the TCR complex. AU - Hoffmann,T K, AU - Donnenberg,V S, AU - Friebe-Hoffmann,U, AU - Meyer,E M, AU - Rinaldo,C R, AU - DeLeo,A B, AU - Whiteside,T L, AU - Donnenberg,A D, PY - 2000/11/21/pubmed PY - 2001/2/28/medline PY - 2000/11/21/entrez SP - 321 EP - 8 JF - Cytometry JO - Cytometry VL - 41 IS - 4 N2 - BACKGROUND: Major histocompatibility complex (MHC)-peptide tetrameric complexes (tetramers) are valuable tools for detecting and characterizing peptide-specific T cells. Because the frequency of these cells is generally very low, it may be difficult to discriminate between nonspecific and specific tetramer binding. METHODS: A four-color flow cytometric assay that simultaneously measures tetramer, CD3, CD8, and CD14 was used to investigate the sensitivity and specificity of MHC class I tetramer staining. This was accomplished by using the influenza virus matrix protein peptide, GILGFVFTL (FLU), as a model recall antigen and the human immunodeficiency virus (HIV) reverse transcriptase peptide, ILKEPVHGV (HIV), as a model novel antigen. Peripheral blood mononuclear cells (PBMC) from 31 HLA-A2.1(+) and 10 HLA-A2.1(-) healthy individuals 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 were 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 the specificity of tetramer binding to the T-cell receptor (TCR) complex. The competition assay was validated by evaluating several anti-CD3 antibodies and showing that in PBMC from HLA-A2(-) subjects, spurious tetramer-positive events (1/20,000) failed to compete with CD3 binding. For the "recall" FLU tetramer, the degree of competition was proportional to the frequency, suggesting a selection of high avidity cells. Although CD3 competition was also highly correlated with the intensity of tetramer staining, competition allowed the identification of false positive cases with relatively high tetramer staining intensity. CONCLUSION: The data indicate that competition of CD3 binding allows confirmation of the specificity of tetramer binding to the TCR, extending the usefulness of tetramers in the frequency analysis of peptide-specific T lymphocytes. SN - 0196-4763 UR - https://www.unboundmedicine.com/medline/citation/11084618/Competition_of_peptide_MHC_class_I_tetrameric_complexes_with_anti_CD3_provides_evidence_for_specificity_of_peptide_binding_to_the_TCR_complex_ L2 - http://www.iedb.org/pmId/11084618 DB - PRIME DP - Unbound Medicine ER -