Early dynamics of T helper cell cytokines and T regulatory cells in response to treatment of active Mycobacterium tuberculosis infection

Abstract

Biomarkers that can identify tuberculosis (TB) disease and serve as markers for efficient therapy are requested. We have studied T cell cytokine production [interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-α] and degranulation (CD107a) as well as subsets of CD4(+) T regulatory cells (Tregs ) after in-vitro Mycobacterium tuberculosis (Mtb) antigen stimulation [early secretory antigenic target (ESAT)-6, culture filtrate protein (CFP)-10, antigen 85 (Ag85)] in 32 patients with active tuberculosis (TB) disease throughout 24 weeks of effective TB treatment. A significant decline in the fraction of Mtb-specific total IFN-γ and single IFN-γ-producing T cells was already observed after 2 weeks of treatment, whereas the pool of single IL-2(+) cells increased over time for both CD4(+) and CD8(+) T cells. The Treg subsets CD25(high) CD127(low) , CD25(high) CD147(++) and CD25(high) CD127(low) CD161(+) expanded significantly after Mtb antigen stimulation in vitro at all time-points, whereas the CD25(high) CD127(low) CD39(+) Tregs remained unchanged. The fraction of CD25(high) CD127(low) Tregs increased after 8 weeks of treatment. Thus, we revealed an opposing shift of Tregs and intracellular cytokine production during treatment. This may indicate that functional signatures of the CD4(+) and CD8(+) T cells can serve as immunological correlates of early curative host responses. Whether such signatures can be used as biomarkers in monitoring and follow-up of TB treatment needs to be explored further.

Keywords: T cells; Tregs; cytokines; treatment; tuberculosis.

Fig 1

Total Mycobacterium tuberculosis (Mtb) antigen-specific interferon (IFN)-γ⁺, interleukin (IL)-2⁺ or tumour necrosis factor (TNF)-α⁺ T cell responses at different time-points of tuberculosis (TB) treatment: baseline (n = 20), week 2 (n = 11), week 8 (n = 18), week 24 (n = 20). (a) Early secretory antigenic target (ESAT)-6/culture filtrate protein (CFP)-10 (E6C10)-stimulated CD4⁺ T cells. (b) Antigen 85 (Ag85)-stimulated CD4⁺ T cells. (c) E6C10-stimulated CD8⁺ T cells. (d) Ag85-stimulated CD8⁺ T cells. P-values calculated by Wilcoxon's matched-pairs test. Plots are shown with median, interquartile range (IQR) and minimum/maximum values.

Fig 2

Mycobacterium tuberculosis (Mtb) antigen-specific cytokine T cell subsets at different time-points of tuberculosis (TB) treatment: baseline (n = 20), week 2 (n = 11), week 8 (n = 18), week 24 (n = 20). Boolean gating strategy was used to create cytokine combinations of single-producing, duo-producing and polyfunctional T cells. (a) Early secretory antigenic target (ESAT)-6/culture filtrate protein (CFP)-10 (E6C10)-stimulated CD4⁺ T cells. (b) Antigen 85 (Ag85)-stimulated CD4⁺ T cells. (c) E6C10 stimulated CD8⁺ T cells. (d) Ag85-stimulated CD8⁺ T cells. P-values calculated by Wilcoxon's matched-pairs test. Plots are shown with median, interquartile range (IQR) and minimum/maximum values.

Fig 3

Piecharts representing percentages of the various cytokine combinations to the overall pool of cytokine producing CD4⁺ and CD8⁺ T cells at different time-points during treatment: week 0 (n = 20), week 2 (n = 11), week 8 (n = 18) and week 24 (n = 20). (a) Early secretory antigenic target (ESAT)-6/culture filtrate protein (CFP)-10 (E6C10)-stimulated CD4⁺ T cells. (b) Antigen 85 (Ag85)-stimulated CD4⁺ T cells. (c) E6C10-stimulated CD8⁺ T cells. (d) Ag85-stimulated CD8⁺ T cells.

Fig 4

The fraction of regulatory T cell (T_reg) subsets in tuberculosis (TB) patients before (n = 12) and after 8 (n = 11) and 24 weeks (n = 11) of TB treatment in unstimulated (white bars) and early secretory antigenic target (ESAT)-6/culture filtrate protein (CFP)-10 (E6C10)-stimulated peripheral blood mononuclear cells (PBMC) (hatched bars). (a) CD4⁺CD25^hiCD127^low, (b) CD4⁺CD25^hiCD127^lowCD147⁺⁺, (c) CD4⁺CD25^hiCD127^lowCD39⁺ and (d) CD4⁺CD25^hiCD127^lowCD161⁺. *Significant changes (P < 0·005) between unstimulated and E6C10-stimulated samples at corresponding time-points. P-values calculated by Wilcoxon's matched-pairs test. Plots are shown with median, interquartile range (IQR) and minimum/maximum values.

Fig 5

Fraction of regulatory T cells (T_regs) (CD4⁺CD25^hiCD127^low) in unstimulated and early secretory antigenic target (ESAT)-6/culture filtrate protein (CFP)-10 (E6C10)-stimulated peripheral blood mononuclear cells (PBMC) during treatment at baseline (week 0), week 8 and week 24 in (a) high (white bars, n = 6) versus low (hatched bars, n = 6) symptom score and (b) pulmonary tuberculosis (PTB) (white bars, n = 6) versus extrapulmonary tuberculosis (EPTB) (dotted bars, n = 6). Plots are shown with median, interquartile range (IQR) and minimum/maximum values. P-values were calculated by Mann–Whitney U-test.