Confirmation of Mycobacterium tuberculosis infection by flow cytometry after ex vivo incubation of peripheral blood T cells with an ESAT-6-derived peptide pool

Lydia Tesfa, Franz Wilhelm Koch, Wulf Pankow, Hans Dieter Volk, Florian Kern

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Background: The presence of a T-cell response to the early secretory antigenic target-6 (ESAT-6) indicates previous infection with or exposure to Mycobacterium tuberculosis. Measuring this response is useful for identifying individuals infected with M. tuberculosis. It was also reported that the frequencies of ESAT-6-specific T cells correlate with disease state. Established procedures measure secreted T-cell cytokines following whole blood stimulation with recombinant ESAT-6 protein or use Elispot as a read-out. Methods: A single ESAT-6-spanning pool of overlapping peptides (15 amino acids length with 11 overlaps) was used for overnight stimulation of peripheral blood mononuclear cells (PBMCs) from 15 patients infected with Mycobacterium tuberculosis and 11 healthy controls. T-cell responses were rated positive if interferon-γ (IFN-231γ)-producing T cells were identified above background level, using 4-color cytokine flow cytometry. Results: Thirteen of 15 (87%) patients, but none of the healthy controls, had a positive CD4 T-cell response to the ESAT-6 spanning peptide pool. The frequencies of IFN-γ-producing cells varied between 1 and 167 per 10,000 CD4 T cells. The test performed as well as the tests described in the literature. Conclusions: Cytokine flow cytometry following PBMC stimulation with an ESAT-6 spanning peptide pool is a useful laboratory test for ESAT-6-specific T cells combining precise counting and multi-parameter phenotyping.

Original languageEnglish (US)
Pages (from-to)47-53
Number of pages7
JournalCytometry Part B - Clinical Cytometry
Volume60
Issue number1
StatePublished - Jul 2004
Externally publishedYes

Fingerprint

Mycobacterium Infections
Mycobacterium tuberculosis
Blood Cells
Flow Cytometry
T-Lymphocytes
Peptides
Cytokines
Interferons
Color
Amino Acids
Infection

Keywords

  • ESAT-6
  • Flow cytometry
  • Protein spanning peptide pools, peptides
  • Tuberculosis

ASJC Scopus subject areas

  • Hematology
  • Cell Biology
  • Pathology and Forensic Medicine
  • Biophysics
  • Endocrinology

Cite this

Confirmation of Mycobacterium tuberculosis infection by flow cytometry after ex vivo incubation of peripheral blood T cells with an ESAT-6-derived peptide pool. / Tesfa, Lydia; Koch, Franz Wilhelm; Pankow, Wulf; Volk, Hans Dieter; Kern, Florian.

In: Cytometry Part B - Clinical Cytometry, Vol. 60, No. 1, 07.2004, p. 47-53.

Research output: Contribution to journalArticle

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AB - Background: The presence of a T-cell response to the early secretory antigenic target-6 (ESAT-6) indicates previous infection with or exposure to Mycobacterium tuberculosis. Measuring this response is useful for identifying individuals infected with M. tuberculosis. It was also reported that the frequencies of ESAT-6-specific T cells correlate with disease state. Established procedures measure secreted T-cell cytokines following whole blood stimulation with recombinant ESAT-6 protein or use Elispot as a read-out. Methods: A single ESAT-6-spanning pool of overlapping peptides (15 amino acids length with 11 overlaps) was used for overnight stimulation of peripheral blood mononuclear cells (PBMCs) from 15 patients infected with Mycobacterium tuberculosis and 11 healthy controls. T-cell responses were rated positive if interferon-γ (IFN-231γ)-producing T cells were identified above background level, using 4-color cytokine flow cytometry. Results: Thirteen of 15 (87%) patients, but none of the healthy controls, had a positive CD4 T-cell response to the ESAT-6 spanning peptide pool. The frequencies of IFN-γ-producing cells varied between 1 and 167 per 10,000 CD4 T cells. The test performed as well as the tests described in the literature. Conclusions: Cytokine flow cytometry following PBMC stimulation with an ESAT-6 spanning peptide pool is a useful laboratory test for ESAT-6-specific T cells combining precise counting and multi-parameter phenotyping.

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