High IFN-γ Release and Impaired Capacity of Multi-Cytokine Secretion in IGRA Supernatants Are Associated with Active Tuberculosis

Abstract

Interferon gamma (IFN-γ) release assays (IGRAs) detect Mycobacterium tuberculosis (Mtb) infection regardless of the active (ATB) or latent (LTBI) forms of tuberculosis (TB). In this study, Mtb-specific T cell response against region of deletion 1 (RD1) antigens were explored by a microbead multiplex assay performed in T-SPOT TB assay (T-SPOT) supernatants from 35 patients with ATB and 115 patients with LTBI. T-SPOT is positive when over 7 IFN-γ secreting cells (SC)/250 000 peripheral blood mononuclear cells (PBMC) are enumerated. However, over 100 IFN-γ SC /250 000 PBMC were more frequently observed in the ATB group compared to the LTBI group. By contrast, lower cytokine concentrations and lower cytokine productions relative to IFN-γ secretion were observed for IL 4, IL-12, TNF-α, GM-CSF, Eotaxin and IFN-α when compared to LTBI. Thus, high IFN-γ release and low cytokine secretions in relation with IFN-γ production appeared as signatures of ATB, corroborating that multicytokine Mtb-specific response against RD1 antigens reflects host capacity to contain TB reactivation. In this way, testing cytokine profile in IGRA supernatants would be helpful to improve ATB screening strategy including immunologic tests.

Fig 1. Cytokines response in T-SPOT supernatants.

A- The expression of IFN-γ in ESAT-6 stimulated T cells was quantified by the T-SPOT assay. Results are expressed by numbers of secreted cells (SC) per 250 000 PBMC for 36 patients with ATB (red circles) and 115 patients with LTBI (green squares). The median is shown in each group. The p value is calculated by the Mann-Whitney U test. ** corresponds to p≤0.01. B- The ROC curve displays sensitivity vs specificity for IFN-γ in differentiating the ATB group from the LTBI group. The AUC and a threshold at 100 SC/250000 PBMC are indicated in the graph. C-E-G-I-K-M- IL-4, IL-12, TNF-α, GM-CSF, Eotaxin and IFN-α indexes (cytokine/IFN-γ *100), ATB are represented by red circles and LTBI by green squares. The median indexes are shown in each group of each panel. p values are calculated by the Mann-Whitney U test. ** corresponds to p≤0.01, *** to p≤0.001 and **** to p≤0.0001. D-F-H-J-L-N- Biparametric graphs between the numbers of IFN-γ immunospots in response to ESAT-6 stimulation and the level of cytokines (pg/ml) in the corresponding supernatants. ATB are represented by red circles and LTBI by green squares. Defined risk groups are circled.

Fig 2. Identification of cytokine profiles associated with ATB.

A. Unsupervised hierarchical clustering was performed according to the similarities in cytokine expression profiles which were visualised using the indicated color scale. Cytokine concentrations were indicated using a color scale that ranges from green (low) through black to red (high). The dendrogram above the heat map illustrated degrees of relatedness between the expression profiles evident within the various patients. Subjects with ATB (n = 35; black boxes on the strip above the heat-map), with high IFN-γ and low IL-12, TNF-α, GM-CSF, Eotaxin and IFN-γ secretions tended to cluster together (cluster I on the dendogram), while subjects with LTBI (n = 125; grey boxes) with IFN-γ and high IL-12, TNF-α, GM-CSF, Eotaxin and IFN-γ secretions also tended to cluster together (cluster II). The dendrogram on the right-hand side of the heat map indicates relationships between the expression profiles of the analysed cytokines across all the patients assessed in each study. B. Partial least squares analysis model of all 7 cytokines classified individuals with 61% overall accuracy for classification and 55% accuracy for cross-validation (red circles, ATB; green squares, LTBI). Latent Variable 1 (LV1), linear combination of the variables included in the model, LV2, orthogonal to the first combination. 95% of each population was circled. C. Latent variable 1 best separated individuals who are ATB from those LTBI. Cytokine loadings indicate multivariate cytokines associated with ATB.