image_1_High Frequencies of Caspase-3 Expressing Mycobacterium tuberculosis-Specific CD4+ T Cells Are Associated With Active Tuberculosis.tiff

<p>Antigen-specific CD4⁺ T cell responses to Mycobacterium tuberculosis (Mtb) infection are important for host defense against tuberculosis (TB). However, Mtb-specific IFN-γ-producing T cells do not distinguish active tuberculosis (ATB) patients from individuals with asymptomatic latent Mtb infection (LTBI). We reasoned that the immune phenotype of Mtb-specific IFN-γ⁺CD4⁺ T cells could provide an indirect gauge of Mtb antigen load within individuals. We sought to identify immune markers in Mtb-specific IFN-γ⁺CD4⁺ T cells and hypothesized that expression of caspase-3 Mtb-specific CD4⁺ T cells would be associated with ATB. Using polychromatic flow cytometry, we evaluated the expression of caspase-3 in Mtb-specific CD4⁺ T cells from LTBI and ATB as well as from ATB patients undergoing anti-TB treatment. We found significantly higher frequencies of Mtb-specific caspase-3⁺IFN-γ⁺CD4⁺ T cells in ATB compared to LTBI. Caspase-3⁺IFN-γ⁺CD4⁺ T cells were also more activated compared to their caspase-3-negative counterparts. Furthermore, the frequencies of caspase-3⁺IFN-γ⁺CD4⁺ T cells decreased in response to anti-TB treatment. Our studies suggest that the frequencies of caspase-3-expressing antigen-specific CD4⁺ T cells may reflect mycobacterial burden in vivo and may be useful for distinguishing Mtb infection status along with other host biomarkers.</p

image_2_High Frequencies of Caspase-3 Expressing Mycobacterium tuberculosis-Specific CD4+ T Cells Are Associated With Active Tuberculosis.tiff

<p>Antigen-specific CD4⁺ T cell responses to Mycobacterium tuberculosis (Mtb) infection are important for host defense against tuberculosis (TB). However, Mtb-specific IFN-γ-producing T cells do not distinguish active tuberculosis (ATB) patients from individuals with asymptomatic latent Mtb infection (LTBI). We reasoned that the immune phenotype of Mtb-specific IFN-γ⁺CD4⁺ T cells could provide an indirect gauge of Mtb antigen load within individuals. We sought to identify immune markers in Mtb-specific IFN-γ⁺CD4⁺ T cells and hypothesized that expression of caspase-3 Mtb-specific CD4⁺ T cells would be associated with ATB. Using polychromatic flow cytometry, we evaluated the expression of caspase-3 in Mtb-specific CD4⁺ T cells from LTBI and ATB as well as from ATB patients undergoing anti-TB treatment. We found significantly higher frequencies of Mtb-specific caspase-3⁺IFN-γ⁺CD4⁺ T cells in ATB compared to LTBI. Caspase-3⁺IFN-γ⁺CD4⁺ T cells were also more activated compared to their caspase-3-negative counterparts. Furthermore, the frequencies of caspase-3⁺IFN-γ⁺CD4⁺ T cells decreased in response to anti-TB treatment. Our studies suggest that the frequencies of caspase-3-expressing antigen-specific CD4⁺ T cells may reflect mycobacterial burden in vivo and may be useful for distinguishing Mtb infection status along with other host biomarkers.</p

Higher frequencies of PD-1 expression on antigen-specific memory CD4⁺ T cells in LTBI relative to BCG individuals.

<p>(A) A representative plot of CD4⁺ T cells expressing PD-1 upon CW stimulation, displaying the percentage of antigen-specific IFN-γ producing cells. The summary of percentage (B) and MFI (C) of PD-1 expression on antigen-specific memory CD4⁺ T cells in LTBI (n = 18) and BCG (n = 18) groups. Each data point corresponds to a single donor. Polyfunctional cytokine production by memory PD-1⁺ (D) and PD-1⁻ (E) T cells from LTBI. Data are represented as the mean percentage of responding PD-1⁺ or PD-1⁻ T cells that are triple producers (3+), double producers (2+) or single producers (1+) of IFN-γ, TNF-α, and IL-2 and summarized by the pie charts. Each slice of the pie represents the fractions of the total response that consists of PD-1⁺ or PD-1⁻ cells positive for a given function.</p

Cytokines production by effector memory CD4⁺ T cells from LTBI and BCG individuals.

<p>(A) Functional profile of IFN-γ, IL-2 and TNF-α CD4⁺ T cells are shown in representative LTBI and BCG individuals. (B) Polyfunctional cytokine production by memory CD4⁺ T cells from LTBI and BCG. Data are represented as the mean percentage of responding CD4⁺ T cells that are triple producers (3+), double producers (2+) or single producers (1+) of IFN-γ, TNF-α, and IL-2 and summarized by the pie charts. Each slice of the pie represents the fractions of the total response that consists of CD4⁺ T cells positive for a given function.</p

Identification of LTBI individuals by IFN-γ ELISPOT assays.

<p>PBMC isolated from 40 healthy BCG-vaccinated individuals were stimulated with Mtb cell wall antigens (CW) or peptide pools derived from Mtb-specific proteins ESAT-6 and CFP-10 (10 mg/ml). Data are represented as spot forming cells (SFC) per 2×10⁵ PBMC. Each data point corresponds to a single donor.</p

Statistical modeling to evaluate the predictive value of CD27 and PD-1 in healthy individuals.

<p>The frequencies of PD-1⁺ IFN-γ⁺ and CD27⁺ IFN-γ⁺ T cells for the following pairs (A) LTBI versus BCG, (B) LTBI versus TB-treated and (C) BCG versus TB-treated, are graphically represented. Receiver Operating Characteristic (ROC) curve analysis and logistic regression models were used to evaluate the predictive value of CD27 and PD-1 individually and in combination. The area under the curve (AUC) indicates the predictive value for each marker.</p

CD27⁺ and CD27⁻ antigen-specific memory CD4⁺ T cell subsets are polyfunctional.

<p>(A) Distribution of CD27⁺ and CD27⁻ memory CD4⁺ T cells from LTBI (n = 12) and BCG (n = 9) that produce IFN-γ, TNF-α or IL-2 upon stimulation with CW antigen. The antigen-specific T cells are CD28⁺, CCR7⁻ and CD45RA⁻. Each data point corresponds to a single donor. (B) Polyfunctional cytokine responses from CD27⁺ and CD27⁻ subsets in LTBI. Data are represented as the mean percentage of responding CD4⁺ T cells that are triple producers (3+), double producers (2+) or single producers (1+) of IFN-γ, TNF-α, and IL-2 and summarized by the pie charts. Each slice of the pie represents the fractions of the total response that consists of CD4⁺ T cells positive for a given function.</p

Neutrophil extracellular trap (NET) levels in human plasma are associated with active TB

<div><p>Neutrophils are increasingly associated with tuberculosis (TB) disease. Neutrophil extracellular traps (NETs), which are released by neutrophils as a host antimicrobial defense mechanism, are also associated with tissue damage. However, a link between NET levels and TB disease has not been studied. Here we investigate plasma NETs levels in patients with active pulmonary tuberculosis using an ELISA assay that is suitable for high-throughput processing. We show that plasma NETs levels at baseline correlated with disease severity and decreased with antibiotic therapy. Our study demonstrates the biologic plausibility of measuring NETs in plasma samples from patients with TB.</p></div

Neutrophil extracellular traps (NETs) plasma levels.

<p>Panel A: comparison of NET plasma levels among subjects with latent tuberculosis infection, healthy controls, and baseline samples of patients with active tuberculosis. Panel B: NET plasma levels during treatment for active tuberculosis and comparison with subjects with latent tuberculosis infection. Abbreviations: NET, Neutrophil Extracellular Traps; MPO, Myeloperoxidase; HNE, Human Neutrophil Elastase; LTBI, Latent Tuberculosis Infection; ATB, Active Tuberculosis; HC, Healthy Control. * P ≤ 0.05; ** P > 0.05; *** P > 0.01.</p