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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

Analysis of cytokine production by CD4+ cells at the single-cell level. (a) ROC curve (plotting sensitivity versus 1 − specificity) to discriminate infected (active TB and LTBI) from uninfected patients. The area under curve (AUC) was 0.9722. (b) Analysis of activated M. tuberculosis-specific CD4+T cells producing any of the 3 cytokines (IFN-γ, IL-2, or TNF-α), using a cut-off to score responses as either positive or negative. The subjects were considered as positive (black) whether the frequency of CD4+ T cells was >0.45% and negative (white) when the frequency was <0.45%. Horizontal bars represent the median values and horizontal dashed line indicates the cut-off of 0.45%. ((c), (d), and (e)) Frequency of “total IFN-γ+,” “total IL-2+,” and “total TNF-α+” Mtb-specific CD4+T cells in active TB patients (n = 18), in LTBI patients (n = 10), and in healthy controls (n = 10) is shown. Horizontal bars represent the median values. Statistical analysis was performed using Kruskal-Wallis ANOVA with Dunn's posttest comparison and significant differences are indicated by asterisks (**P < 0.01).
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fig2: Analysis of cytokine production by CD4+ cells at the single-cell level. (a) ROC curve (plotting sensitivity versus 1 − specificity) to discriminate infected (active TB and LTBI) from uninfected patients. The area under curve (AUC) was 0.9722. (b) Analysis of activated M. tuberculosis-specific CD4+T cells producing any of the 3 cytokines (IFN-γ, IL-2, or TNF-α), using a cut-off to score responses as either positive or negative. The subjects were considered as positive (black) whether the frequency of CD4+ T cells was >0.45% and negative (white) when the frequency was <0.45%. Horizontal bars represent the median values and horizontal dashed line indicates the cut-off of 0.45%. ((c), (d), and (e)) Frequency of “total IFN-γ+,” “total IL-2+,” and “total TNF-α+” Mtb-specific CD4+T cells in active TB patients (n = 18), in LTBI patients (n = 10), and in healthy controls (n = 10) is shown. Horizontal bars represent the median values. Statistical analysis was performed using Kruskal-Wallis ANOVA with Dunn's posttest comparison and significant differences are indicated by asterisks (**P < 0.01).

Mentions: The expression of CD4+ T cells producing any of the 3 cytokines (IFN-γ or IL-2 or TNF-α) was assessed in patients with active TB, LTBI, and healthy controls, after simulation with Mtb-specific antigens. A significantly higher frequency of these CD4+ T cells was found in active TB patients (median 1.197%, range 0.219%–3.59%) and in LTBI patients (1.666%, 0.234–5.762%) if compared to healthy controls (0.246%, 0–0.423%; P < 0.0001 by Kruskal-Wallis test); on the other hand, no significant differences were found between the 2 infected group subjects. Following this observation, we performed a ROC analysis (Figure 2(a)) and a cut-off >0.45% for activated CD4+ T cells was found as the value allowing the best combination of sensitivity (94.44%, 95% CI: 72.2–99.8%) and specificity (100%, 95% CI: 69.15–100%; AUC 0.9722; 95% CI: 0.9141–1.030%, P < 0.0001) to differentiate Mtb-infected patients (active TB and LTBI) from healthy controls. Using this cut-off, we scored as positive 17/18 (95%) of active TB patients, 9/10 (90%) of LTBI patients, and none of 10 healthy controls (Figure 2(b)). Thus, the analysis of Mtb-specific CD4+ T cells allowed the discrimination between Mtb-infected and uninfected patients.


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)

Analysis of cytokine production by CD4+ cells at the single-cell level. (a) ROC curve (plotting sensitivity versus 1 − specificity) to discriminate infected (active TB and LTBI) from uninfected patients. The area under curve (AUC) was 0.9722. (b) Analysis of activated M. tuberculosis-specific CD4+T cells producing any of the 3 cytokines (IFN-γ, IL-2, or TNF-α), using a cut-off to score responses as either positive or negative. The subjects were considered as positive (black) whether the frequency of CD4+ T cells was >0.45% and negative (white) when the frequency was <0.45%. Horizontal bars represent the median values and horizontal dashed line indicates the cut-off of 0.45%. ((c), (d), and (e)) Frequency of “total IFN-γ+,” “total IL-2+,” and “total TNF-α+” Mtb-specific CD4+T cells in active TB patients (n = 18), in LTBI patients (n = 10), and in healthy controls (n = 10) is shown. Horizontal bars represent the median values. Statistical analysis was performed using Kruskal-Wallis ANOVA with Dunn's posttest comparison and significant differences are indicated by asterisks (**P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Analysis of cytokine production by CD4+ cells at the single-cell level. (a) ROC curve (plotting sensitivity versus 1 − specificity) to discriminate infected (active TB and LTBI) from uninfected patients. The area under curve (AUC) was 0.9722. (b) Analysis of activated M. tuberculosis-specific CD4+T cells producing any of the 3 cytokines (IFN-γ, IL-2, or TNF-α), using a cut-off to score responses as either positive or negative. The subjects were considered as positive (black) whether the frequency of CD4+ T cells was >0.45% and negative (white) when the frequency was <0.45%. Horizontal bars represent the median values and horizontal dashed line indicates the cut-off of 0.45%. ((c), (d), and (e)) Frequency of “total IFN-γ+,” “total IL-2+,” and “total TNF-α+” Mtb-specific CD4+T cells in active TB patients (n = 18), in LTBI patients (n = 10), and in healthy controls (n = 10) is shown. Horizontal bars represent the median values. Statistical analysis was performed using Kruskal-Wallis ANOVA with Dunn's posttest comparison and significant differences are indicated by asterisks (**P < 0.01).
Mentions: The expression of CD4+ T cells producing any of the 3 cytokines (IFN-γ or IL-2 or TNF-α) was assessed in patients with active TB, LTBI, and healthy controls, after simulation with Mtb-specific antigens. A significantly higher frequency of these CD4+ T cells was found in active TB patients (median 1.197%, range 0.219%–3.59%) and in LTBI patients (1.666%, 0.234–5.762%) if compared to healthy controls (0.246%, 0–0.423%; P < 0.0001 by Kruskal-Wallis test); on the other hand, no significant differences were found between the 2 infected group subjects. Following this observation, we performed a ROC analysis (Figure 2(a)) and a cut-off >0.45% for activated CD4+ T cells was found as the value allowing the best combination of sensitivity (94.44%, 95% CI: 72.2–99.8%) and specificity (100%, 95% CI: 69.15–100%; AUC 0.9722; 95% CI: 0.9141–1.030%, P < 0.0001) to differentiate Mtb-infected patients (active TB and LTBI) from healthy controls. Using this cut-off, we scored as positive 17/18 (95%) of active TB patients, 9/10 (90%) of LTBI patients, and none of 10 healthy controls (Figure 2(b)). Thus, the analysis of Mtb-specific CD4+ T cells allowed the discrimination between Mtb-infected and uninfected patients.

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