Expanded polyfunctional T cell response to mycobacterial antigens in TB disease and contraction post-treatment

Abstract

Background: T cells producing multiple factors have been shown to be required for protection from disease progression in HIV but we have recently shown this not to be the case in TB. Subjects with active disease had a greater proportion of polyfunctional cells responding to ESAT-6/CFP-10 stimulation than their infected but non-diseased household contacts (HHC). We therefore wanted to assess this profile in subjects who had successfully completed standard TB chemotherapy.

Methods: We performed a cross-sectional study using PBMC from TB cases (pre- and post-treatment) and HHC. Samples were stimulated overnight with TB antigens (ESAT-6/CFP-10 and PPD) and their CD4+ and CD8+ T cells were assessed for production of CD107a, IFN-gamma, IL-2 and TNF-alpha and the complexity of the responses was determined using SPICE and PESTLE software.

Results and conclusions: We found that an increase in complexity (i.e., production of more than 1 factor simultaneously) of the T cell profile was associated with TB disease and that this was significantly reduced following TB treatment. This implies that T cells are able to respond adequately to TB antigens with active disease (at least initially) but the ability of this response to protect the host from disease progression is hampered, presumably due to immune evasion strategies by the bacteria. These findings have implications for the development of new diagnostics and vaccine strategies.

Figure 1. Representative flow cytometry profiles demonstrating the gating strategy used throughout the study.

A) PBMC were stimulated overnight and cells were analysed by flow cytometry for expression of CD107a, TNF-α, IL-2 and IFN-γ. Following lymphocyte gating, CD4+ and CD8+ T cells were analysed separately for combinations of all 4 factors. B) Representative dot plots illustrating cytokine production from CD4+ and CD8+ T cells following PHA, PPD, EC and no stimulation.

Figure 2. Increased CD107a, IFN-γ and TNF-α expression in TB cases pre-treatment and reduction post-treatment.

Bar indicates median of TB cases pre-treatment (n = 12, filled circles), TB cases post-treatment (n = 10, open circles) and 17 HHC (triangles). Data were analysed using a Kruskal-Wallis ANOVA followed by Dunn's post-test comparison and p-values indicated.

Figure 3. The functional profile of CD4+ and CD8+ T cells is increased in TB cases but reduced post-treatment.

PBMC were stimulated overnight using ESAT-6/CFP-10 and CD4+ and CD8+ cells were analysed by flow cytometry for different combinations of IFN-γ, IL-2, TNF-α and CD107a. Responses are grouped and colour coded according to the number of factors: White  = 1 function, black  =  any 2 factors, blue  =  any 3 factors and red  =  all 4 factors. The pie charts summarise the results shown in the dot plots indicating the combination of cytokines as a percent of total responding cells for CD4+ (A) and CD8+ (B). Significant differences are indicated and were determined using a Kruskal-Wallis ANOVA followed by Dunn's post-test comparison. Pie chart segments were analysed using a permutation test and p-values indicated.