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Multifunctional Analysis of CD4+ T-Cell Response as Immune-Based Model for Tuberculosis Detection.

Lichtner M, Mascia C, Sauzullo I, Mengoni F, Vita S, Marocco R, Belvisi V, Russo G, Vullo V, Mastroianni CM - J Immunol Res (2015)

Bottom Line: Among TB-infected subjects, the frequencies of multifunctional CD4(+) T cells, simultaneously producing all 3 cytokines, are lower in active TB than LTBI subjects (P = 0.003).Thus, assigning to triple-positive CD4(+) T cells a cut-off <0.182%, TB-infected individuals could be classified as active TB subjects (<0.182%) or LTBI subjects (>0.182%).The magnitude of CD8(+) T-cell responses showed no differences between active TB and LTBI.

View Article: PubMed Central - PubMed

Affiliation: Department of Public Health and Infectious Diseases, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, Italy ; Infectious Diseases Unit, Sapienza University, Corso della Repubblica 79, 04100 Latina, Italy.

ABSTRACT
Mono- and multifunctional specific CD4(+) and CD8(+) T-cell responses were evaluated to improve the immune-based detection of active tuberculosis (TB) and latent infection (LTBI). We applied flow cytometry to investigate cytokines profile (IFN-γ, TNF-α, and IL-2) of T cells after stimulation with TB antigens in 28 TB-infected subjects (18 active TB and 10 LTBI) and 10 uninfected controls. Cytokines production by CD4(+) T cells at single-cell levels was higher in TB-infected subjects than uninfected controls (P < 0.0001). Assigning to activated CD4(+) T cells, producing any of the three cytokines, a cut-off >0.45%, it was possible to differentiate TB-infected (>0.45%) by uninfected subjects (<0.45%). Among TB-infected subjects, the frequencies of multifunctional CD4(+) T cells, simultaneously producing all 3 cytokines, are lower in active TB than LTBI subjects (P = 0.003). Thus, assigning to triple-positive CD4(+) T cells a cut-off <0.182%, TB-infected individuals could be classified as active TB subjects (<0.182%) or LTBI subjects (>0.182%). The magnitude of CD8(+) T-cell responses showed no differences between active TB and LTBI. Multifunctional CD4(+) T-cell responses could have the potential to identify at single time point subjects without TB infection and patients having active or latent TB.

No MeSH data available.


Related in: MedlinePlus

Representative flow cytometry “combination gates” analysis of CD4+ T cells of LTBI, active TB, and control subject under stimulation of TB antigens. Whole blood was analysed using a gating strategy to exclude debris and to identify CD4+ and CD8+ T cells on CD45+ lymphocytes. The subsequent analysis was on CD4+ gate to describe IFN-γ, IL-2, and TNF-α producing T cells. The percentages of the seven different population cells were showed at bottom and were defined in CD4+ cell gate on the basis of total IFN-γ, IL-2, and TNF-α producing cells.
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fig1: Representative flow cytometry “combination gates” analysis of CD4+ T cells of LTBI, active TB, and control subject under stimulation of TB antigens. Whole blood was analysed using a gating strategy to exclude debris and to identify CD4+ and CD8+ T cells on CD45+ lymphocytes. The subsequent analysis was on CD4+ gate to describe IFN-γ, IL-2, and TNF-α producing T cells. The percentages of the seven different population cells were showed at bottom and were defined in CD4+ cell gate on the basis of total IFN-γ, IL-2, and TNF-α producing cells.

Mentions: In brief, after 18 hrs of incubation, the cell surface staining was performed with the following markers, anti-CD45-VioBlue, anti-CD4 PE-Vio770, and anti-CD8 PerCP (Miltenyi Biotec, Germany), and the red cells were lysed with 1 mL FACS lysing solution (BD Bioscience). Cells were then permeabilized with 0.5 mL FACS permeabilizing solution (BD Bioscience) and intracellularly stained with anti-IFN-γ FITC, anti-TNF-α APC, and anti-IL-2 PE (Miltenyi Biotec). Cells were fixed in 1% paraformaldehyde and analysed within 1 hr using a MACSQuant Analyzer flow cytometer (Miltenyi Biotec) after calibration and automatic compensation. We acquired at least 100,000 cells in the lymphocyte gate. FlowJo Software version 7.6.5 was used to perform a “combination gates” analysis. Seven different population cells were detected in CD4+ and in CD8+ cell gate on the basis of IFN-γ, IL-2, and TNF-α produced by CD4+ and CD8+ T cells (Figure 1). Background cytokine production in negative control (saline buffer) was subtracted from each stimulated condition. Intra-assay coefficient of variation and interassay coefficient variation were estimated and were <5% and <10%, respectively.


Multifunctional Analysis of CD4+ T-Cell Response as Immune-Based Model for Tuberculosis Detection.

Lichtner M, Mascia C, Sauzullo I, Mengoni F, Vita S, Marocco R, Belvisi V, Russo G, Vullo V, Mastroianni CM - J Immunol Res (2015)

Representative flow cytometry “combination gates” analysis of CD4+ T cells of LTBI, active TB, and control subject under stimulation of TB antigens. Whole blood was analysed using a gating strategy to exclude debris and to identify CD4+ and CD8+ T cells on CD45+ lymphocytes. The subsequent analysis was on CD4+ gate to describe IFN-γ, IL-2, and TNF-α producing T cells. The percentages of the seven different population cells were showed at bottom and were defined in CD4+ cell gate on the basis of total IFN-γ, IL-2, and TNF-α producing cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4539126&req=5

fig1: Representative flow cytometry “combination gates” analysis of CD4+ T cells of LTBI, active TB, and control subject under stimulation of TB antigens. Whole blood was analysed using a gating strategy to exclude debris and to identify CD4+ and CD8+ T cells on CD45+ lymphocytes. The subsequent analysis was on CD4+ gate to describe IFN-γ, IL-2, and TNF-α producing T cells. The percentages of the seven different population cells were showed at bottom and were defined in CD4+ cell gate on the basis of total IFN-γ, IL-2, and TNF-α producing cells.
Mentions: In brief, after 18 hrs of incubation, the cell surface staining was performed with the following markers, anti-CD45-VioBlue, anti-CD4 PE-Vio770, and anti-CD8 PerCP (Miltenyi Biotec, Germany), and the red cells were lysed with 1 mL FACS lysing solution (BD Bioscience). Cells were then permeabilized with 0.5 mL FACS permeabilizing solution (BD Bioscience) and intracellularly stained with anti-IFN-γ FITC, anti-TNF-α APC, and anti-IL-2 PE (Miltenyi Biotec). Cells were fixed in 1% paraformaldehyde and analysed within 1 hr using a MACSQuant Analyzer flow cytometer (Miltenyi Biotec) after calibration and automatic compensation. We acquired at least 100,000 cells in the lymphocyte gate. FlowJo Software version 7.6.5 was used to perform a “combination gates” analysis. Seven different population cells were detected in CD4+ and in CD8+ cell gate on the basis of IFN-γ, IL-2, and TNF-α produced by CD4+ and CD8+ T cells (Figure 1). Background cytokine production in negative control (saline buffer) was subtracted from each stimulated condition. Intra-assay coefficient of variation and interassay coefficient variation were estimated and were <5% and <10%, respectively.

Bottom Line: Among TB-infected subjects, the frequencies of multifunctional CD4(+) T cells, simultaneously producing all 3 cytokines, are lower in active TB than LTBI subjects (P = 0.003).Thus, assigning to triple-positive CD4(+) T cells a cut-off <0.182%, TB-infected individuals could be classified as active TB subjects (<0.182%) or LTBI subjects (>0.182%).The magnitude of CD8(+) T-cell responses showed no differences between active TB and LTBI.

View Article: PubMed Central - PubMed

Affiliation: Department of Public Health and Infectious Diseases, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, Italy ; Infectious Diseases Unit, Sapienza University, Corso della Repubblica 79, 04100 Latina, Italy.

ABSTRACT
Mono- and multifunctional specific CD4(+) and CD8(+) T-cell responses were evaluated to improve the immune-based detection of active tuberculosis (TB) and latent infection (LTBI). We applied flow cytometry to investigate cytokines profile (IFN-γ, TNF-α, and IL-2) of T cells after stimulation with TB antigens in 28 TB-infected subjects (18 active TB and 10 LTBI) and 10 uninfected controls. Cytokines production by CD4(+) T cells at single-cell levels was higher in TB-infected subjects than uninfected controls (P < 0.0001). Assigning to activated CD4(+) T cells, producing any of the three cytokines, a cut-off >0.45%, it was possible to differentiate TB-infected (>0.45%) by uninfected subjects (<0.45%). Among TB-infected subjects, the frequencies of multifunctional CD4(+) T cells, simultaneously producing all 3 cytokines, are lower in active TB than LTBI subjects (P = 0.003). Thus, assigning to triple-positive CD4(+) T cells a cut-off <0.182%, TB-infected individuals could be classified as active TB subjects (<0.182%) or LTBI subjects (>0.182%). The magnitude of CD8(+) T-cell responses showed no differences between active TB and LTBI. Multifunctional CD4(+) T-cell responses could have the potential to identify at single time point subjects without TB infection and patients having active or latent TB.

No MeSH data available.


Related in: MedlinePlus