We report that, after newborn BCG vaccination, the magnitude and profile of cytokine expression of BCG-specific CD4 and CD8 T cells did not correlate with protection against childhood TB. Importantly, there were no differences in polyfunctional BCG-specific CD4 T cells, which coexpress IFN-γ, TNF-α, and IL-2. We also confirm production of IFN-γ by γδ T cells when whole blood is stimulated with BCG; however, expression of this cytokine by this subset was not associated with protection against childhood TB.
Multiple experimental studies have shown that the Th1 cytokines, IFN-γ and TNF-α, are required for immunity against M.tb
infection and disease (10
). This is supported by findings from experimental TB vaccine studies that evaluated biomarkers of protection (34
). For example, in a heterologous prime-boost strategy with BCG followed by adenovirus-expressing Ad85A, Forbes and colleagues (23
) reported a correlation between magnitude of polyfunctional Ad85A-specific Th1 responses in the lungs after M.tb
challenge and protection against disease. Transfer of early secretory antigenic target (ESAT)-6–specific Th1 memory cells to recipient mice before M.tb
challenge enhanced protection, suggesting the importance of the quantity of antigen-specific T cells at the disease site (37
). In contrast, multiple other experimental studies have shown that IFN-γ production at the disease site does not correlate with protection against TB; rather, expression of the cytokine may be a marker of the magnitude of the inflammatory response (38
). For example, Mittrucker and colleagues (38
) reported no correlation of BCG-induced T cell responses and protection in a mouse TB challenge model. Furthermore, in a clinical study, Sutherland and colleagues (41
) reported that patients with TB disease had a higher polyfunctional CD4+
T cell response after overnight stimulation of whole blood with ESAT-6 and purified protein derivative compared with healthy individuals with a positive tuberculin skin test.
In our study, we did not measure IFN-γ at a disease site—this is not possible in healthy 10-week-old infants—but in peripheral blood. We found no association between the frequency of BCG-specific Th1 cells and protection against TB. This finding is of particular importance, because this peripheral blood outcome is used to assess vaccine-induced immunity in most clinical trials of new TB vaccines. The latter studies often focus on the quality of the CD4 T cell response, with the hypothesis that polyfunctionality (i.e., combined expression of IFN-γ, TNF-α, and IL-2 by individual cells) is a marker of protective immunity. The interest to evaluate mycobacteria-specific polyfunctional T cells is based on observations from experimental mouse models of protection against other intracellular organisms, such as Leishmania major
), and, to a limited extent, from animal studies of novel TB vaccination (23
). We showed no correlation between polyfunctional BCG-specific CD4 T cell responses in peripheral blood and protection against TB. In an experimental mouse model with L. major
infection, Darrah and colleagues (32
) reported that MFI of cytokine expression could be used as an additional measure of quality of the T cell response, as polyfunctional cells had the highest MFI of cytokine expression. In this study, we assessed the MFI of BCG-specific T cell cytokine expression, and showed no association with protection against TB.
We evaluated IL-17 expression in CD4 T cells based on evidence that this cytokine plays a protective role against TB (25
). This is the first study to demonstrate induction of BCG-specific IL-17 cells in infants after BCG vaccination at birth; however, frequencies of BCG-specific Th17 cells did not correlate with protection against TB.
We investigated CD8 T cell responses as possible correlates of protection, based on the important role of this subset suggested by recent experimental and clinical studies (13
). For example, Chen and colleagues (13
) reported that depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a decrease in induced immunity upon subsequent challenge of the animals with M.tb
. Furthermore, Bruns and colleagues (14
) showed that patients undergoing anti-TNF therapy had decreased antimicrobial activity against M.tb
due to diminished numbers of antigen-specific effector memory CD8 T cells, with an associated increased incidence of TB disease. We observed no differences when comparing specific CD8 T cell responses between protected and unprotected infants.
We assessed γδ T cell responses based on a report that only the γδ T cells expanded from PBMCs of purified protein derivative–positive donors incubated with BCG, and not γδ T cells expanded by phosphoantigen, were able to inhibit growth of M.tb
in autologous macrophages (42
). However, we found no association between the frequency of BCG-induced γδ T cells and protection against TB in our study.
Our results strongly suggest that aspects of BCG-specific CD4 and CD8 T cell immunity, or γδ T cell immunity, measured in this whole blood assay at 10 weeks of age, may not correlate with protection against TB. We cannot exclude the possibility that these outcomes, measured at another time point after newborn BCG vaccination, or with different antigens or assay systems, might correlate with protection. An infant biomarker study of this size (n
= 5,662) has not been reported to date; the magnitude of the project required that we limit our blood collection to one practical time point, before M.tb
infection, and at an age before significant exposure to environmental mycobacteria. This was also the reason for selecting a single viable bacterial antigen that can be processed for recognition by a wide range of lymphocytes. The results generated by using BCG as an antigen are likely to be specific, as we have recently demonstrated that responses with BCG in a whole blood assay are detectable at 10 weeks of age only in infants who have been vaccinated at birth, and not in unvaccinated infants (43
). Regardless, individual mycobacterial antigens might yield different results. Furthermore, the T cell response to mycobacteria is complex (44
), and involves cytotoxic activity (46
), for example, in addition to cytokine production. These additional aspects of T cell immunity might correlate with outcome, whereas routine vaccine take measurements focus on cytokine production, using a short-term whole blood assay. Similarly, innate host responses may also be important. We propose that biomarkers of protection against TB may only be unraveled when multiple host factors are examined together in a system biology approach. Ongoing, complementary studies will address whether biomarkers of protection against TB may be identified through other approaches. These include measuring soluble levels of cytokines, chemokines, and growth factors after 7-hour incubation of whole blood with BCG, the cytotoxic, proliferative, and cytokine-expressing capacity of T cells after incubation of PBMCs with BCG for 3–6 days, and gene expression profiles in PBMCs incubated with BCG for 12 hours.
Overall, our results strongly suggest caution when interpreting T cell immune markers commonly evaluated in new TB vaccine clinical trials. More importantly, our results indicate that protective immunity against M.tb may be very complex, and suggest a need to look beyond the classical Th1 immunity when assessing the efficacy of novel TB vaccines in clinical trials.