In the Russian Federation, tuberculosis (TB) is on the decline [1]. However, despite the decreasing morbidity and mortality rates, the disease is still a serious threat, especially considering the spread of HIV infection and drug-resistant strains of Mycobacterium tuberculosis (Mtb). Other factors that contribute to TB spread are new immunity disrupting factors, such as commonization of transplantation, spread of autoimmune diseases and allergies, population ageing, insufficient physical activity. There are reasons to believe that they will play an increasingly important role. In this connection, host-oriented therapy aimed to optimize host immunity during TB disease and new TB vaccines able to prevent TB disease show promise. However their development requires understanding the mechanisms of antituberculous defense and knowing immunological correlates of protection. The latter is especially crucial for preclinical and clinical studies of new TB vaccines, as assessment of their effectiveness is challenging and largely based on the evaluation of vaccine immunogenicity. Unfortunately, exact mechanisms of TB protection are not fully clear, and TB protection correlates remain unidentified. Antigen-specific Th1 lymphocytes have long been considered as the main correlate of TB protection. However, recent data have not confirmed this concept. This article discusses debatable issues concerning the role for Th1 lymphocytes in antituberculosis immunity and their potential usage as TB correlate of protection.

Dependence of protective antituberculous immunity on Th1 lymphocytes response

 Since the immunology of TB became a subject of research, protective antituberculous immunity has been attributed to CD4⁺ Th1 lymphocytes that activate macrophages for mycobacteria killing [2–7]. There are a large number of experiments and clinical studies supporting this concept. Indeed, CD4 T cell deficiency, either due to HIV infection or induced experimentally, increases TB risk in people and makes the disease severe in laboratory animals [8–12]. In mice that have IFNγ, TNFα, IL12, iNOS or other genes involved in IFNγ-dependent response knocked out, infection with Mtb leads to severe conditions and rapid death [13–19]. Children with mutations in genes of IL12/IFNγ axis (i.e., IFNGR1, IFNGR2, IL12B, IL12RB1, STAT1, IRF8, ISG15, NEMO, CYBB) are more susceptible to mycobacterial infections, including TB, and the diseases developed thereof typically take severe forms [20–29]. Cytokine anti-TNF therapy is another factor known to heighten the risk of TB development [30, 31]. In mice, antimycobacterial activity of macrophages depends on the production of active oxygen and nitrogen activated by type 1 cytokines IFNγ and TNFα [32–37].

The data mentioned laid the foundation of the concept stating that Th1 lymphocytes are the main activators of macrophages and mediators of TB protection. However, in fact, the data summarized above indicate that deficiency in Th1 response leads to TB development, but this does not mean that TB always results from Th1 response deficiency. Moreover, a series of experimental studies and clinical observations of the recent years have challenged the existence of association between TB development and Th1/IFNγ deficiency.

Lack of correlation between the levels of Th1 responses and TB protection: experimental findings

BCG-vaccinated mice infected with Mtb have shown no correlation between the level of BCG-induced protection and the level of IFNγ synthesized by CD4⁺ lymphocytes [38, 39]. Several studies have reported that CD4⁺ lymphocytes, derived from IFNγ^-/- mice and differentiated in Th1-polarizing conditions, are capable of controlling the multiplication of Mtb in vitro [40] and in vivo when transferred adoptively [41, 42]. Thus, the lack of IFNγ does not prevent sufficiently effective control over Mtb multiplication in mice.

In contrast to the control of Mtb multiplication, protection against pathological reactions in the lung tissue did require IFNγ. Nandi & Behar [42] have adoptively transferred CD4⁺ IFNγ^-/- lymphocytes to RAG^-/- Mtb-infected mice. IFNγ^-/- lymphocytes protected recipient mice against Mtb multiplication as effectively as lymphocytes derived from wild-type mice, however unlike the latter, IFNγ^-/- lymphocytes did not protect mice from pathological reactions in their lungs and death. The authors linked protective activity of IFNγ to its ability to decrease the induction of "pathological" Th17 population and neutrophilic infiltration, i.e., inflammation control. At the same time, Barber and coauthors have recently shown that excessively high production of IFNγ can do damage and lead to death of Mtb-infected mice [43, 44].

Thus, recent studies have demonstrated that Th1/IFNγ response can be more complex than plain activation of the macrophages' antimycobacterial properties, and that the state of protection is largely determined by the organism's ability to control inflammatory responses to the infection. Moreover, no correlation between the level of vaccine-induced Th1/IFNγ response and protection against experimental tuberculosis infection was found.

Th1/IFNγ responses to mycobacteria in humans: contradictory data

Despite the afore-mentioned fact that Mtb multiplication in mice can be controlled in the absence of T-cell derived IFNγ, the mainstay concept considers IFNγ as the main part of the pathway “T cells – IFNγ – iNOS – active forms of nitrogen – macrophage activation – suppression of Mtb growth". However this pathway does not seem to describe the processes ongoing in human macrophages: several studies reported that in human macrophages IFNγ did not stimulate active nitrogen production and did not cause significant suppression of Mtb multiplication [33, 45, 46]. Interestingly, a recent study by Meyer and coauthors found no significant impact of IFNγ pathway gene variants on tuberculosis susceptibility in a West African population (analysis included 20 genes in samples obtained from 23 TB patients and 46 healthy donors, and exon gene analysis of IFNGR1 in 1999 samples from TB patients and 2589 control samples) [47].

One of the most common approaches to analyze the contribution of various immune responses to TB protection in human beings implies comparing the responses in TB patients and TB contacts who did not develop disease. The results of such comparative studies are ambiguous. Some of them have reported smaller numbers of Mtb-specific Th1 lymphocytes and weaker IFNγ production in TB patients, which is taken as an argument proving that these types of responses contribute to TB protection [48–52]. However, in other studies the amount of cells producing IFNγ and the levels of IFNγ and TNFα production in TB patients were higher than those seen in people with latent tuberculosis infection (LTBI) and healthy donors [53–55]. In our studies, the levels of antigen-stimulated IFNγ production were higher in TB patients compared to TB contacts and individuals with LTBI; moreover, we have registered higher IFNγ production in patients with active TB compared to patients with residual post-tuberculous lung tissue alterations [56]. We have also shown that the group of patients with recently diagnosed TB had greater percentages of IFNγ and TNFα producing CD4⁺ lymphocytes than people with LTBI, TB contacts and healthy donors [57]. In contrast to patients with recently diagnosed TB, patients with chronic TB do exhibit signs of Th1 inhibition, but apparently this is a secondary process [58].

Another approach that allows investigating mechanisms of immune protection implies comparison of immunological parameters in TB patients with diverse TB severity. The approach is based on a thorough assessment of the severity of diverse TB manifestations in each patient included in the study. TB manifestations considered in our study included clinical TB forms (tuberculoma, infiltrative TB, focal TB, cavernous and fibrous-cavernous TB, disseminated TB); TB extent (evaluated based on the number of lung segments and lobes affected by the pathology); the degree of lung tissue destruction (i.e., number and size of foci of destruction); bacterial excretion (presence, level of); clinical severity of the disease (assessed by temperature and other clinical signs of intoxication). Correlation and cluster analyzes did not reveal significant associations between these TB manifestations and the levels of Th1 responses (i.e, the percentages and absolute numbers of CD4 lymphocytes producing IFNγ, TNFα, IL2, their various combinations, the level of antigen-induced IFNγ production in the Quantiferon-TB gold in-tube test) [56, 57]. Thus, it can be deduced that in most cases the intensity of Th1 response does not affect the post-infection development or non-development of the disease, and neither does it influence the course of TB disease. It seems that, provided there are no significant defects (like HIV-related deficiency of CD4 lymphocytes or mutations in IL12 / IFNγ chain genes), the host organism is capable of mounting a Th1 response proportionate to the threat, and the quantitative characteristics of such response (which differ from person to person), do not have a significant effect on the outcome of the infection.

This conclusion is in line with the results of studies researching the relationship between the vaccine-induced Th1 response and protection against TB disease. For example, Kagina and coauthors evaluated BCG-specific CD4, CD8 lymphocytes and γδ T-cells producing IFNγ, TNFα, IL2 and IL17 in children who received the BCG vaccine at birth [59]. Two year follow-up allowed identifying a group of children for whom the protection was ineffective, i.e. TB developed in them, and a group of children whose protection was effective, i.e. the disease did not develop in them in spite of their contacts with TB patients. The percentages and the cytokine profile of Mtb-specific T lymphocytes in these groups did not differ significantly, so the authors deduced that the IFNγ-producing lymphocytes induced by BCG vaccination cannot signal of the vaccine effectiveness [59].

CONCLUSIONS

The data available suggest that the levels of Th1/IFNγ responses reflect the activity of TB infection rather than a degree of protection. This in turn means that Th1 response is not a reliable correlate of protection, nor does it allow evaluating (even preliminary) the potential effectiveness of new candidate vaccines. Unfortunately, the current practice is often the contrary: Th1 response is taken as the main (and often the only) indicator of the immunogenicity and the potential efficacy of new candidate TB vaccines. The search for new markers of protection goes on. Some studies have already shown the dependence of vaccine-induced protection on Th17 lymphocytes [60–63]; several clinical research suggested population of the so-called nonclassical Th1 lymphocytes as a new protection correlate [64–66]. Validation of these data and the search for other reliable protection markers are important for further development and testing of TB vaccines.