Cell-Mediated Immune Responses to in vivo-Expressed and Stage-Specific Mycobacterium tuberculosis Antigens in Latent and Active Tuberculosis Across Different Age Groups

Abstract

A quarter of the global human population is estimated to be latently infected by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). TB remains the global leading cause of death by a single pathogen and ranks among the top-10 causes of overall global mortality. Current immunodiagnostic tests cannot discriminate between latent, active and past TB, nor predict progression of latent infection to active disease. The only registered TB vaccine, Bacillus Calmette-Guérin (BCG), does not adequately prevent pulmonary TB in adolescents and adults, thus permitting continued TB-transmission. Several Mtb proteins, mostly discovered through IFN-γ centered approaches, have been proposed as targets for new TB-diagnostic tests or -vaccines. Recently, however, we identified novel Mtb antigens capable of eliciting multiple cytokines, including antigens that did not induce IFN-γ but several other cytokines. These antigens had been selected based on high Mtb gene-expression in the lung in vivo, and have been termed in vivo expressed (IVE-TB) antigens. Here, we extend and validate our previous findings in an independent Southern European cohort, consisting of adults and adolescents with either LTBI or TB. Our results confirm that responses to IVE-TB antigens, and also DosR-regulon and Rpf stage-specific Mtb antigens are marked by multiple cytokines, including strong responses, such as for TNF-α, in the absence of detectable IFN-γ production. Except for TNF-α, the magnitude of those responses were significantly higher in LTBI subjects. Additional unbiased analyses of high dimensional flow-cytometry data revealed that TNF-α+ cells responding to Mtb antigens comprised 17 highly heterogeneous cell types. Among these 17 TNF-α+ cells clusters identified, those with CD8+TEMRA or CD8+CD4+ phenotypes, defined by the expression of multiple intracellular markers, were the most prominent in adult LTBI, while CD14+ TNF-α+ myeloid-like clusters were mostly abundant in adolescent LTBI. Our findings, although limited to a small cohort, stress the importance of assessing broader immune responses than IFN-γ alone in Mtb antigen discovery as well as the importance of screening individuals of different age groups. In addition, our results provide proof of concept showing how unbiased multidimensional multiparametric cell subset analysis can identify unanticipated blood cell subsets that could play a role in the immune response against Mtb.

Keywords: IVE-TB antigens; LTBI; Mycobacterium tuberculosis (Mtb); TB; cell responses; cytokines.

Figure 1

IVE-TB Mtb antigens, Mtb DosR-regulon, and Rpf antigens activate multiple-cytokine-producing blood cells of Mtb exposed subjects. The levels of eight cytokines were measured in diluted whole blood supernatants from a cohort of Mtb exposed individuals (n = 35) after 6 days stimulation with either IVE-TB, DosR, Rpf (10 μg/ml), control antigens ESAT6/CFP10 (E/C) (10 μg/ml), PPD (5 μg/ml), or the positive control, mitogen PHA (2 μg/ml). The statistical significance of the differences between cytokine levels in stimulated and unstimulated samples was evaluated by Mann-Whitney U-test with FDR multiple test correction for IL-13, IL-22, IL-17A, IFN-γ, IP-10, IL-10, GM-CSF, IL-32, and TNF-α. (A) Results are shown only for TNF-α, GM-CSF, IP-10, IFN-γ, and IL-17A for which significant differences were found among single Mtb antigens and unstimulated samples. Each dot represents a donor. Bars indicate medians. Purple bars indicate significantly increased responses compared to the unstimulated samples with a p < 0.05 (Mann-Whitney U-test with FDR multiple test correction). (B) The polar histogram displays the different p-values found among single Mtb antigens (or control) and unstimulated samples. Stimuli are ordered clockwise as follows: controls and Mtb antigens grouped according to how many cytokines were induced. These groups of antigens are separated by interruptions of the histogram. In red are indicated Mtb antigens that induced cytokines other than IFN-γ.

Figure 2

LTBI individuals' and TB patients' blood cells secrete different amounts of cytokines in response to IVE-TB and stage specific Mtb antigens. The levels of nine cytokines were measured in diluted whole blood supernatants after 6 days stimulation with either IVE-TB, DosR, Rpf antigens (10 μg/ml), ESAT6/CFP10 (E/C) (10 μg/ml), PPD (5 μg/ml), or PHA (2 μg/ml). The cytokine responses were compared between LTBI donors (n = 17) and TB patients (n = 18). This comparison is displayed for five cytokines, TNF-α, GM-CSF, IFN-γ, IP-10, and IL-17A, which were found to be significantly increased in stimulated samples compared to unstimulated ones (Figure 1). Within each donor, prior to further analysis, the pg/ml values detected in response to every antigen were normalized to the background-values, i.e., divided by the medium, and then log₂ transformed. Results are reported only for Mtb antigens that induced increased cytokine levels compared to the background-values. (A) Cytokine differences were assessed by Mann–Whitney U-test (significant for p-values < 0.05) and by median log₂ fold-changes [log₂FC(LTBI/TB) > 1 or <-1). P-values and median log₂ fold-changes are reported on the y and x axes, respectively. Thresholds for p-values and median log₂ fold-changes are indicated by dotted lines intersecting the axes (x:−1 and 1; y: 1.30103). Closed circles define when the log₂FC(LTBI/TB) is >1 (red) or <−1 (blue), such that closed red circles define antigens inducing higher responses in LTBI donors than TB patients, while blue circles depict antigens inducing higher responses in TB patients than LTBI subjects. Open circles define when the log₂FC(LTBI/TB) is >−1 and <1 (gray). (B) Differences between LTBI and TB patients were analyzed separately among adults (TB n = 8; LTBI n = 12) and adolescents (TB n = 9; LTBI n = 6). X and y-axes indicate the median log₂ fold-changes in adolescents and adults, respectively. The color-coded dots indicate p-values: ≥0.05 in adults and adolescents (gray), <0.05 in adults (orange), <0.05 in adolescents (blue), and <0.05 in adults and adolescents (green). The gray area around the regression line (dotted line) represents the range in which the true regression line lies at a 95% level of confidence. Both in (A,B), the Rv numbers are coupled to antigens recognized differently by LTBI and TB according to both p-values and median log₂ fold-changes.

Figure 3

Quantitative and qualitative differences are present in specific TNF-α+ cell subsets between LTBI and TB patients. Intracellular cytokine staining was performed on whole blood (ICS-WB) of a limited number of Mtb exposed individuals recruited in this study (n = 11). Samples were left unstimulated or stimulated for 12 h with a pool of three Mtb antigens (Rv1131, Rv2461, and Rv3616c) or PPD, in the presence of (T cell) co-stimulants (anti-CD28 and anti-CD49d). After staining cryopreserved fixed blood cells with an extensive 14-color FACS panel, TNF-α+ cells were quantified in each sample. (A) The gating strategy is shown for one sample (TB02) to illustrate the selection of single debris-free TNF-α+ cells in unstimulated (AIM-V i.e., medium) and stimulated (antigen pool or PPD) sample. In the left upper table, the number of TNF-α+ cells is indicated for each donor. (B) The difference in the absolute number of TNF-α+ cells between LTBI and TB was defined by median log₂ fold change [log₂FC(LTBI/TB)] and calculated separately in adolescents and adults. Blue or red bars represent log₂FC(LTBI/TB) >0.5 or <-0.5, respectively. (C) For each of the 11 donors, TNF-α+ cells responding to the antigen pool and PPD were used as input in Cytosplore. Input cells were randomly downsampled to 153 events leading to a total of 3,366 TNF-α+ cells to explore via hierarchical stochastic neighbor embedding (HSNE). In the upper left HSNE plot, blue, and red dots indicate cells isolated from LTBI or TB patients (color tonality distinguishes adolscents from adults). In the other 11 HSNE plots the colored dots indicate the expression range of 11 singular cellular markers on the cells analyzed (high expression, red; medium expression, yellow; low expression, blue). (D) By applying a Gaussian mean-shift clustering to the FACS data, 17 distinct TNF-α+ cell clusters (displayed in the HSNE plot, upper panel) were defined by a unique combination of expressed markers as shown by the heatmap (middle panel). Differences in the size of colored squares indicate the contribution of each marker in defining the specific cell subset, while colors indicate the relative expression of each cellular marker compared to all the other markers (red indicates high; blue indicates low). Lower heatmap (lower panel) shows the proportion of cells composing each cluster per age group of donors (LTBI and TB) and stimuli (PPD and antigen pool). The numbers in the last row refer to the cell clusters.