Th1, Th17, and Th1Th17 Lymphocytes during Tuberculosis: Th1 Lymphocytes Predominate and Appear as Low-Differentiated CXCR3+CCR6+ Cells in the Blood and Highly Differentiated CXCR3+/-CCR6- Cells in the Lungs.

Th1 lymphocytes are considered the main mediators of protection against tuberculosis (TB); however, their phenotypic characteristics and relationship with Th17 and Th1Th17 populations during TB are poorly understood. We have analyzed Th1, Th17, and Th1Th17 lymphocytes in the blood and pulmonary lesions of TB patients. The populations were identified based on the production of IFN-γ and/or IL-17 and the coexpression of CXCR3 (X3) and CCR6 (R6). In the blood, IL-17+ and IFN-γ+IL-17+ lymphocytes were barely detectable (median, <0.01% of CD4+ lymphocytes), whereas IFN-γ+ lymphocytes predominated (median, 0.45%). Most IFN-γ+ lymphocytes (52%) were X3+R6+, suggesting their "nonclassical" (ex-Th17) nature. In the lungs, IL-17+ and IFN-γ+IL-17+ lymphocytes were more frequent (0.3%, p < 0.005), yet IFN-γ+ cells predominated (11%). Phenotypically, lung CD4+ cells were X3+/loR6- The degree of differentiation of blood effector CD4+ lymphocytes (evaluated based on CD62L/CD27/CD28 coexpression) increased as follows: X3+R6+ < X3+R6- < X3-R6-, with X3-R6- cells being largely terminally differentiated CD62L-CD27-CD28- cells. Lung CD4+ lymphocytes were highly differentiated, recalling blood X3+/-R6- populations. Following in vitro stimulation with anti-CD3/anti-CD28 Abs, X3+R6+CD4+ lymphocytes converted into X3+R6- and X3-R6- cells. The results demonstrate that, during active TB, Th1 lymphocytes predominate in blood and lungs, document differences in X3/R6 expression by blood and lung CD4+ cells, and link the pattern of X3/R6 expression with the degree of cell differentiation. These findings add to the understanding of immune mechanisms operating during TB and are relevant for the development of better strategies to control it.

HLA-DR-Positive NK Cells Expand in Response to Mycobacterium Tuberculosis Antigens and Mediate Mycobacteria-Induced T Cell Activation.

NK cells play an important role in the control of tuberculosis infection: they are not only able to kill the infected cells, but also control the activity of macrophages and development of the adaptive immune response. Still, there is little information on the role of specific NK cell subsets in this network. In this study, we focused on the mycobacteria-driven responses of the NK cells expressing HLA-DR - a type of MHC class II. We have revealed that this subset is increased in the peripheral blood of patients with primary diagnosed tuberculosis, and expands in response to in vitro stimulation with ultrasonically destroyed Mycobacterium tuberculosis cells (sonicate). The expanded HLA-DR+ NK cells had less differentiated phenotype, higher proliferative activity and increased expression of NKp30 and NKp46 receptors. HLA-DR+CD56dim NK cells showed higher IFNγ production and degranulation level than the respective HLA-DR- NK cells in response to both 24 h and 7 day stimulation with sonicate, while HLA-DR+CD56bright NK cells mostly demonstarted similar high responsiveness to the same stimulating conditions as their HLA-DR-CD56bright counterparts. After preliminary incubation with destroyed mycobacteria, cytokine-activated HLA-DR-expressing NK cells were able to mediate mycobacteria-induced and HLA-DR-dependent cytokine production in autologous CD4+ T cells. Thus, functionally active HLA-DR+ cells seem to be one of the NK cell subsets providing an important link to the adaptive immunity.

Severe Tuberculosis in Humans Correlates Best with Neutrophil Abundance and Lymphocyte Deficiency and Does Not Correlate with Antigen-Specific CD4 T-Cell Response.

It is generally thought that Mycobacterium tuberculosis (Mtb)-specific CD4+ Th1 cells producing IFN-γ are essential for protection against tuberculosis (TB). In some studies, protection has recently been associated with polyfunctional subpopulation of Mtb-specific Th1 cells, i.e., with cells able to simultaneously secrete several type 1 cytokines. However, the role for Mtb-specific Th1 cells and their polyfunctional subpopulations during established TB disease is not fully defined. Pulmonary TB is characterized by a great variability of disease manifestations. To address the role for Mtb-specific Th1 responses during TB, we investigated how Th1 and other immune cells correlated with particular TB manifestations, such as the degree of pulmonary destruction, TB extent, the level of bacteria excretion, clinical disease severity, clinical TB forms, and "Timika X-ray score," an integrative parameter of pulmonary TB pathology. In comparison with healthy Mtb-exposed controls, TB patients (TBP) did not exhibit deficiency in Mtb-specific cytokine-producing CD4+ cells circulating in the blood and differed by a polyfunctional profile of these cells, which was biased toward the accumulation of bifunctional TNF-α+IFN-γ+IL-2- lymphocytes. Importantly, however, severity of different TB manifestations was not associated with Mtb-specific cytokine-producing cells or their polyfunctional profile. In contrast, several TB manifestations were strongly correlated with leukocyte numbers, the percent or the absolute number of lymphocytes, segmented or band neutrophils. In multiple alternative statistical analyses, band neutrophils appeared as the strongest positive correlate of pulmonary destruction, bacteria excretion, and "Timika X-ray score." In contrast, clinical TB severity was primarily and inversely correlated with the number of lymphocytes in the blood. The results suggest that: (i) different TB manifestations may be driven by distinct mechanisms; (ii) quantitative parameters and polyfunctional profile of circulating Mtb-specific CD4+ cells play a minor role in determining TB severity; and (iii) general shifts in production/removal of granulocytic and lymphocytic lineages represent an important factor of TB pathogenesis. Mechanisms leading to these shifts and their specific role during TB are yet to be determined but are likely to involve changes in human hematopoietic system.

Antigen-Specific IFN-γ Responses Correlate with the Activity of M. tuberculosis Infection but Are Not Associated with the Severity of Tuberculosis Disease.

IFN-γ is a key cytokine in antituberculosis (TB) defense. However, how the levels of its secretion affect M. tuberculosis (Mtb) infection is not clear. We have analyzed associations between IFN-γ responses measured in QuantiFERON®-TB Gold In-tube (QFT) assay, TB disease severity, and Mtb infection activity. TB severity was evaluated based on the results of radiological, microbiological, and clinical examinations. Antigen-driven IFN-γ secretion did not correlate with TB severity. Mitogen-induced IFN-γ secretion correlated inversely with the form of pulmonary pathology and the area of affected pulmonary tissue; the levels of spontaneous IFN-γ secretion correlated with patients' age (r = 0.395, p = 0.001). Mtb infection activity was evaluated based on radiological data of lung tissue infiltration, destruction, dissemination or calcification, and condensation. The rate of positive QFT results and the levels of antigen-driven IFN-γ secretion increased in a row: patients with residual TB lesions < patients with low TB activity < patients with high TB activity. Thus, antigen-driven IFN-γ secretion and QFT results did not associate with TB severity but associated with the infection activity. The results suggest that quantitative parameters of IFN-γ secretion play a minor role in determining the course of TB disease but mirror the activity of the infectious process.

A side-by-side comparison of T cell reactivity to fifty-nine Mycobacterium tuberculosis antigens in diverse populations from five continents.

We compared T cell recognition of 59 prevalently recognized Mycobacterium tuberculosis (MTB) antigens in individuals latently infected with MTB (LTBI), and uninfected individuals with previous BCG vaccination, from nine locations and populations with different HLA distribution, MTB exposure rates, and standards of TB care. This comparison revealed similar response magnitudes in diverse LTBI and BCG-vaccinated cohorts and significant correlation between responses in LTBIs from the USA and other locations. Many antigens were uniformly recognized, suggesting suitability for inclusion in vaccines targeting diverse populations. Several antigens were similarly immunodominant in LTBI and BCG cohorts, suggesting applicability for vaccines aimed at boosting BCG responses. The panel of MTB antigens will be valuable for characterizing MTB-specific CD4 T cell responses irrespective of ethnicity, infecting MTB strains and BCG vaccination status. Our results illustrate how a comparative analysis can provide insight into the relative immunogenicity of existing and novel vaccine candidates in LTBIs.