Identification of immunological biomarkers which may differentiate latent tuberculosis from exposure to environmental nontuberculous mycobacteria in children

Abstract

A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic.

FIG 1

Collection of archived samples and recruitment of study subjects. In a previous study cohort, 13 of 98 children at 3 years post-BCG vaccination showed positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10. In this study, 24 sets of archived culture supernatant samples from the children at 3 years post-BCG vaccination were collected for a 19-plex bead assay. For the new follow-up study at 8 years post-BCG vaccination, 55 subjects, including 40 children at 8 years post-BCG vaccination and 15 TB patients, were recruited; the 40 healthy children included 11 previous IFN-γ responders to ESAT-6/CFP-10 and 29 nonresponders from the previous study. Fifteen TB patients were recruited as positive controls for IFN-γ response to M. tuberculosis ESAT-6/CFP-10 and included those at diagnosis or on treatment for <3 months. The blood samples obtained from the 55 participants were used for the IFN-γ ELISA, the IFN-γ ELISpot assay, and the 42-plex bead assay.

FIG 2

IFN-γ responses to M. tuberculosis (M. tb) ESAT-6/CFP-10, M. tuberculosis PPD, and PHA in 40 children at 8 years post-BCG vaccination and 15 TB patients. (A) TB patients showed significantly greater IFN-γ production in response to M. tuberculosis ESAT-6 (P < 0.0001) and M. tuberculosis PPD (P = 0.013) than children at 8 years post-BCG vaccination, while all of the children and TB patients had positive IFN-γ responses to PHA-M (P = 0.82) in the IFN-γ ELISA. The median levels of IFN-γ are indicated in red, and the cutoff value for positivity (>62.5 pg/ml) is marked in blue. Values of >4,000 pg/ml were considered to be 4,000 pg/ml. IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 were measured from 11 previous responders (B) and 29 previous nonresponders (C) at 3 years post-BCG vaccination. The subjects who showed positive IFN-γ responses (>62.5 pg/ml) at a follow-up time point of 8 years post-BCG vaccination are marked in red. Two of 11 previous responders had a marked increase in IFN-γ in response to M. tuberculosis ESAT-6/CFP-10. In the previous nonresponder group, IFN-γ was increased to 101 pg/ml in one subject.

FIG 3

Cytokine/chemokine responses in archived samples from children at 3 years post-BCG vaccination. The levels of cytokines and chemokines in response to M. tuberculosis ESAT-6/CFP-10 measured by multiplex bead assays were compared between positive IFN-γ responders (R) (black circles) and nonresponders (NR) (white circles); 6 of 19 different cytokines and chemokines were highly produced in IFN-γ responders, compared with nonresponders, with a >5-fold difference in median responses. The significance of difference of immune responses (P values) between IFN-γ responders and nonresponders is marked on each graph. The median level of each cytokine is indicated in red.

FIG 4

Cytokine/chemokine responses to M. tuberculosis ESAT-6/CFP-10 at 3 and 8 years post-BCG vaccination. Among the 11 subjects who had positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 at 3 years post-BCG vaccination, only two subjects showed positive IFN-γ responses to M. tuberculosis ESAT-6/CFP-10 at 8 years postvaccination (marked in red; subjects 103738 and 103278). Compared with 9 IFN-γ nonresponders at 8 years post-BCG vaccination, 2 IFN-γ responders (marked in red) showed greater increases of cytokine and chemokine responses, particularly in the production of IL-17 and Th2 cytokines such as IL-5, IL-9, and IL-13.

FIG 5

Comparison of cytokine and chemokine signatures between the subject who responded to M. tuberculosis ESAT-6 and those who responded to ESAT-6 homologues of M. avium and M. kansasii. (A) Subject 103738 had a higher number of spot-forming cells (SFCs) in response to M. tuberculosis ESAT-6 peptides than that in response to M. avium or M. kansasii ESAT-6 peptides, while subject 104043 showed a much higher number of SFCs in response to M. kansasii ESAT-6 than that in response to M. tuberculosis ESAT-6. Another subject, 104041, showed higher numbers of SFCs in response to M. avium_57-97 than those in response to M. tuberculosis ESAT-6 and a strong positive response to M. kansasii ESAT-6 peptide. (B) An analysis of 42 cytokine and chemokine signatures to M. tuberculosis ESAT-6/CFP-10 showed that IL-17 and Th2 cytokines, such as IL-5, IL-9, and IL-13, were produced in greater quantities in the IFN-γ responder to M. tuberculosis ESAT-6 (subject 103738) than in the IFN-γ responders to M. avium and M. kansasii.

FIG 6

The cytokines induced by M. tuberculosis (M. tb) ESAT-6/CFP-10 in 3 categorized groups. The diagram shows how the cytokine production following M. tuberculosis ESAT-6/CFP-10 stimulation can improve the diagnosis of latent TB infection. Among the cytokines which were tested in children at 3 and 8 years post-BCG vaccination, only 4 cytokines (IL-17, IL-5, IL-9, and IL-13) were able to distinguish the responders to M. tuberculosis ESAT-6 (subjects 103278 and 103738) from those to ESAT-6 homologues of M. avium and M. kansasii (subjects 104041 and 104043).