Multicolor flow cytometric analyses of CD4+ T cell responses to Mycobacterium tuberculosis-related latent antigens
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Updated: May 11, 2026
Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection
Published on: September 18, 2020
Yoshiro Yamashita1, Yoshihiko Hoshino, Mayuko Oka
1Department of Clinical Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
Insights
Detecting latent tuberculosis (TB) requires sensitive tests. New CD4(+) T cell cytokine responses, including IL-17, show promise for diagnosing TB infection, improving upon interferon-gamma release assays (IGRAs).
Area of Science:
- Immunology
- Infectious Diseases
- Microbiology
Background:
- Interferon-gamma release assays (IGRAs) offer specificity for tuberculosis (TB) detection but early, sensitive diagnosis of latent TB reactivation remains a challenge.
- Multicolor flow cytometry analyzing CD4(+) T cell cytokine responses (IFN-γ/IL-2/TNF-α) to TB antigens has shown potential but diagnostic relevance needs further investigation.
Purpose of the Study:
- To investigate additional CD4(+) T cell cytokine responses (IL-10/IL-13/IL-17) to latent mycobacterial antigens and standard antigens.
- To assess the diagnostic utility of these cytokine responses in distinguishing TB cases from healthy controls.
Main Methods:
- Analysis of IL-10, IL-13, and IL-17 responses to various mycobacterial antigens (α-crystallin, HBHA, MDP-1, PPD, ESAT-6/CFP-10) in 12 IGRA(+) TB cases and 8 healthy controls.
- Utilized multicolor flow cytometry to quantify CD4(+) T cell cytokine production.
Main Results:
- No significant difference in IFN-γ response was observed between TB cases and controls due to high individual variation.
- Significant increases in IL-2 response to HBHA were found in recovery stage TB cases.
- Elevated numbers of M. tuberculosis-specific polyfunctional CD4(+) T cells and increased IL-17 responses to HBHA and MDP-1 were observed in on-treatment and recovery stage TB cases.
Conclusions:
- A combination of T cell cytokine parameters, including IL-2 and IL-17 responses, may enhance the accurate diagnosis of latent TB infection.
- These findings suggest novel biomarkers for TB diagnosis beyond traditional IFN-γ responses.
Abstract:
Although IFN-γ release assays (IGRAs) provide increased specificity over tuberculin skin tests, the early and sensitive detection of reactivation of latently infected Mycobacterium tuberculosis is required to control tuberculosis (TB). Recently, a multicolor flow cytometry has been developed to study CD4(+) T cell cytokine responses (IFN-γ/IL-2/TNF-α) to purified protein derivatives (PPD) and M. tuberculosis-specific antigens (ESAT-6/CFP-10) and provided useful information regarding anti-TB immunity. However, the diagnostic relevancy remains uncertain. Here, we analyzed three additional CD4(+) T cell cytokine responses (IL-10/IL-13/IL-17) to latent mycobacterial antigens (α-crystallin, methylated heparin-binding hemagglutinin [HBHA], and mycobacterial DNA-binding protein 1 [MDP-1]) as well as PPD and ESAT-6/CFP-10 in 12 IGRA(+) TB cases and 8 healthy controls. No significant difference in IFN-γ response was observed between TB cases and controls, which was likely due to the high variation among the individuals. However, we found a significant increase over healthy controls in (i) the IL-2 response to HBHA in recovery stage TB cases, (ii) the number of M. tuberculosis-specific polyfunctional CD4(+) T cells in on-treatment and recovery stage cases, and (iii) the IL-17 response to HBHA and MDP-1 in on-treatment and recovery stage cases. These results suggest that a combination of these T cell cytokine parameters could aid in accurate diagnosis of latent TB infection.
