Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Abstract

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines.

Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-γ production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus.

Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-α and IFN-γ) in T cells from non-human primates (NHPs) after BCG vaccination.

Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Figure 1

Visualization of differences in IFN-γ production in response to M.tb peptide pools using heatmaps in individuals from Belarus. Darker colors (red) represent higher and lighter colors (white) lower INF-γ production. Top panel: Healthy individuals (n = 15); Middle panel: Individuals recovered from TB (15 =); Bottom panel: TB+ patients (n = 15) from Belarus. The negative controls and constitutive IFN-γ production is subtracted.

Figure 2

Visualization of differences in IFN-γ production in response to M.tb peptide pools in PBMCs from healthy individuals from Belarus as compared to Sweden. Darker colors (red) represent higher and lighter colors (white) lower INFγ production. Top panel: Healthy individuals from Belarus (n = 15) and Bottom panel: from Sweden (n = 15).

Figure 3

Visualization of differences in IFN-γ production in response to M.tb proteins. Blood was obtained from individuals from Belarus and incubated with different M.tb proteins followed by detection of IFN-γ production. Darker colors (red) represent higher and lighter colors (white) lower INF-γ production. Top panel: Healthy individuals (n = 15); Middle panel: Individuals recovered from TB (15 =). Bottom panel: TB + patients (n = 15) from Belarus.

Figure 4

Visualization of differences in IFN-γ production in response to proteins. Comparison between healthy individuals from Belarus versus Sweden. Darker colors (red) represent higher and lighter colors (white) lower INF-γ production. Top panel: Healthy individuals from Belarus (n = 15) and bottom panel: from Sweden (n = 15).

Figure 5

Strong polyfunctional CD4+ and CD8+ T cell responses directed against Rv0477c M.tb target peptides. PMBCs were obtained after BCG vaccination and tested by ICS for IFN-γ, TNF-α and IL-2 production in CD4+ and CD8+ T cells. First, cells were gated on site and forward scatter, followed by gating on CD3+ events and CD4+ versus CD8+ T-cells. Responding T-cells were first tested for IFN-γ production, followed by IL-2 versus TNF-α production in the IFN- γ + and IFN-γ - T cell populations. Controls included stimulation with medium or with PMA/ionomycin (data not shown).