The aim of this study was to assess the relationship between the frequency and functional signature of antigen-specific T cells secreting IFN-γ and IL-2 and antigen load in tuberculosis. Our statistical analysis revealed a significant decline in the frequency of ESAT-6/CFP-10-specific T cells secreting IFN-γ over 28 months and a significant increase in the number of ESAT-6/CFP-10-specific T cells secreting IL-2 during 6 months of treatment. These contrasting dynamics for the two cytokines led to a progressive convergence of the frequencies of IFN-γ and IL-2-secreting cells over 28 months, during and after treatment. From simultaneous analysis of IFN-γ and IL-2 secretion at the single cell level in a subset of our cohort using the ultra-sensitive cytokine secretion assay, we found this was due to a change in the functional signature of M. tuberculosis-specific T cells over time. There was a shift in the IFN-γ and IL-2 cytokine profile from a co-dominance of IFN-γ-only and IFN-γ/IL-2-secreting T cells in active tuberculosis to a dominance of IFN-γ/IL-2-secreting T cells and the appearance of IL-2 only secreting T cells during and after treatment. Thus, IFN-γ measured with the ex vivo ELISpot is secreted by two functional subsets of CD4+ T cells with different relative proportions in untreated active tuberculosis compared with during and after treatment.
The decline in ESAT-6 and CFP-10-specific IFN-γ T cell responses ex vivo is consistent with the decline observed during treatment of active tuberculosis in other studies (15
). However, in our study, which is the longest longitudinal study of antigen-specific T cells in tuberculosis to-date, the decline in ESAT-6 and CFP-10-specific IFN-γ-secreting T cells was only statistically significant over 28 months whereas in previous studies the decline in ESAT-6 or CFP-10-specific IFN-γ-secreting T cells was significant over 6 or 12 months using different statistical analyses (; (16
)). An increase in IFN-γ secretion from cultured PBMC during anti-tuberculosis therapy has been observed in other studies (34
), where IFN-γ was measured after several days in vitro culture. These results reflect IFN-γ derived from in vitro antigen-stimulated proliferating memory T cells. The increased IFN-γ production after treatment with these assays likely reflect the fact that antigen-specific lymphoproliferation is inhibited by the non-specific immunosuppresion associated with active, untreated tuberculosis (16
). This read-out is different to the direct ex vivo enumeration of IFN-γ-secreting T cells (18 hours), as performed in this study (28
Harari and colleagues showed that functional T cell heterogeneity is associated with changes in HIV antigen load (8
) and we have now demonstrated that functional T cell heterogeneity is also associated with changes in M. tuberculosis
antigen load. In active disease IFN-γ is secreted from two functional subsets of IFN-γ only and IFN-γ/IL-2 dual secreting T cells whereas after treatment IFN-γ is predominantly secreted from one subset of IFN-γ/IL-2 dual secreting T cells. These observations would not have been possible from the IFN-γ and IL-2 ELISpot assays alone. It is possible that IFN-γ/IL-2 functional profiles might correlate with specific clinical parameters such as disease severity or anatomical site of disease. However, the small numbers of patients within each clinical subgroup precluded statistically meaningful comparisons which would require a larger study population. Although our data are from cases of active tuberculosis not paired with follow up samples, the demographic and clinical characteristics of the untreated patients and the patients tested during and after treatment were similar.
The IFN-γ and IL-2 profile after treatment is similar to a model of antigen clearance, past influenza infection (13
). This suggests that after anti-tuberculosis treatment viable bacilli and antigen may be cleared. This is consistent with the clinical observation that less than 10% of tuberculosis patients relapse within the first year after completing anti-tuberculosis treatment (42
) and none of the patients we studied relapsed during 28 months of follow up. Interestingly in a different model of antigen clearance where infection with live organisms is not involved, past tetanus toxoid vaccination, IL-2-only secreting T cells persisted years after vaccination with minimal levels of IFN-γ/IL-2 dual-secreting T cells. Whilst detailed phenotyping of M. tuberculosis
-specific IFN-γ and IL-2-secreting T cells was beyond the scope of this study, previous studies have identified a relationship between the function and phenotype of memory CD4+ T cells, and proposed that the IL-2-only-secreting cells that are typical TCM
that persist after antigen clearance while the IFN-γ/IL-2 and IFN-γ-only secreting T cells are typical of TEM
IFN-γ is an important mediator of macrophage activation and resistance to M. tuberculosis
infection and is therefore crucial in the effector response to this intracellular pathogen (44
). The paradoxical functions of IL-2 could explain why IL-2 is secreted from cells also secreting IFN-γ both in the early and in the later stages of infection and why IL-2 is secreted when antigen load has declined or been cleared by treatment (47
). Dual IFN-γ/IL-2-secreting cells can support their own expansion as IL-2 is a potent T cell growth factor. The presence of these cells in active tuberculosis when antigen load is high may therefore suggest their involvement in the initiation phase of the immune response through the expansion of effector cells which may further differentiate into IFN-γ-only secreting cells. The relative increase in the proportion of CD4+ T cells secreting both cytokines during and after treatment may reflect the maintenance of a stable effector response. Secretion of IL-2 when antigen load is reduced or cleared may reflect its function in the termination of T cell responses. This proposed signalling function augments the growth and survival of regulatory T cells which control inflammatory responses (48
). Regulatory T cells express high levels of CD25, the IL-2 receptor α-chain, and have recently been described in active tuberculosis where they suppressed IFN-γ-secreting ESAT-6 and CFP-10-specific T cells ex vivo (49
As a quantitative measure of T cell function, the IFN-γ ELISpot assay for diagnosis of M. tuberculosis
) holds promise as a tool for tracking antigen load and monitoring disease activity (16
). But T cell function defined solely by quantification of IFN-γ secretion may prove an insufficient biomarker of antigen load and clinical disease status and other measures of T cell function will probably be required in addition (14
). Through simultaneous measurement of IFN-γ and IL-2 secretion we noted a shift in the cytokine profile of M. tuberculosis
-specific T cells associated with the treatment-induced change from high to low antigen load. Our data thus suggest that IFN-γ and IL-2 functional signatures are associated with antigen load and clinical disease status.
We propose this dynamic functional signature could be used as an immunological marker of mycobacterial load. Existing clinical, radiological and microbiological parameters to monitor the response to treatment in active tuberculosis have several limitations. The IFN-γ and IL-2 functional signature could be used to evaluate new therapies for active tuberculosis entering clinical trials where new biomarkers are urgently needed. In latent tuberculosis infection, for which there are no clinical, radiological or microbiological parameters for assessing response to therapy, the IFN-γ and IL-2 functional signature could be used to monitor the impact of conventional and novel preventive treatments, as well as new vaccines. The IFN-γ and IL-2 functional signature could also be used to monitor individuals infected with M. tuberculosis at high risk of progression to active tuberculosis, e.g. patients with HIV co-infection or on anti-TNF therapy, to guide the early initiation of treatment as antigen load increases prior to clinical reactivation. Whether such changes in the M. tuberculosis antigen-specific T cell functional signature predict specific clinical outcomes is the subject of our ongoing prospective studies.