A Key Role of CD8+ T Cells in Controlling of Tuberculosis Infection

The main role in the control of tuberculosis infection is played by macrophages and Th1 and CD8+ T cells. The study aimed to identify the most diagnostically significant CD8+ T cell subsets in tuberculosis patients. Methods: Peripheral blood samples from patients with clinical, radiological, and bacteriologically confirmed pulmonary tuberculosis (TB, n = 32) and healthy subjects (HC, n = 31) were collected and analyzed using 10-color flow cytometry. Results: The frequency of the EM4 CD3+CD8+ cells was reduced in the peripheral blood of patients with pulmonary tuberculosis, while the relative and absolute number of EM1 CD3+CD8+ cells increased compared to the control group. CD57 expression was reduced in patients with pulmonary tuberculosis on EM1, EM2, and pE1 CD3+CD8+ cells, whereas the EM3 cells had a high level of CD57 expression. The relative and absolute number of Tc2 (CCR6–CXCR3–) cells in peripheral blood in patients with pulmonary tuberculosis was increased, while the frequency of Tc1 (CCR6–CXCR3+) was decreased, compared to healthy donors. Conclusions: Patients with pulmonary tuberculosis have an abnormal CD3+CD8+ cell profile and demonstrate their impaired maturation and functional activity.


Introduction
Tuberculosis (TB) remains a deadly infectious disease that causes a large number of deaths worldwide [1].At the UN Assembly in 2021, the World Health Organization's report on the global fight against TB declared that the COVID-19 pandemic had severely undermined the gains made against the disease worldwide, with TB mortality rates rising for the first time in more than a decade [2].
According to the WHO, after the COVID-19 pandemic, there was an increase in mortality due to the disease from 1.4 million TB deaths in 2019 to 1.5 million in 2021.At the same time, there has been an 18% decrease in the number of reported new tuberculosis (TB) cases from 7.1 million in 2019 to 5.8 million in 2020 [2].At the same time, analyses have shown that global TB notification rates are expected in 40% of children and 21% of adults [3,4], highlighting the need to focus maximum attention on the issues of early detection.
Before the COVID-19 tuberculosis pandemic, a quarter of the world's population was estimated by the WHO to be infected with M. tuberculosis.At the same time, 4% of those infected with M. tuberculosis with latent TB infection may develop active TB under the influence of various factors [5][6][7].
It is known that the presence of concomitant pathology, immunosuppression, HIV infection, malnutrition, and diabetes mellitus are the main risk factors for the development of tuberculosis infection [2,8,9].The emergence of COVID-19 has led to adjustments being made, as it may be one of the factors influencing the development of active tuberculosis due to changes in immune status against the background of infection with the SARS-CoV-2 virus [10].Therefore, obtaining new data on changes in immunologic parameters in tuberculosis patients will be of high importance in determining the prognosis of the course of the disease in the future.
Despite the existence of a large number of studies on the immune status of tuberculosis patients, there are certain gaps in understanding the interaction of M. tuberculosis with a host organism.Currently, it remains unclear what immunological processes lead to the activation, progression, and generalization of tuberculosis infection [11,12].
It is known that the immune response in tuberculosis is cell-mediated [13,14].More than 65% of the total number of lymphocytes are T cells, which are the most important human immune effector cells.T cells mainly include helper or inducible T cells (CD3+CD8-CD4+), cytotoxic T cells (CD3+CD8+CD4-), and NKT cells [15,16].Moreover, it is the protective cellular immunity against tuberculosis that is mainly mediated by CD4+ Tlymphocytes but supported by CD8+ T-lymphocytes [17].However, the role of CD8+ T-lymphocytes in the development and course of tuberculosis infection has not been clearly defined to date, which is the purpose of our study.The study aims to identify the most diagnostically significant CD8+T-lymphocyte subpopulations in tuberculosis patients.

Patients
A prospective study from 2017 to 2021, analyzing the results of an immunological study of the peripheral blood of patients with pulmonary tuberculosis (n = 32, main group), aged 18 to 65 years in comparison with a group of healthy subjects (n = 31, control group), was conducted.Inclusion criteria: presence of clinical, radiological, and bacteriologically confirmed pulmonary tuberculosis.Exclusion criteria: Presence of immunosuppression, HIV infection, cancer, autoimmune pathology, pregnancy, breastfeeding, alcoholism, drug addiction, and chronic pathology in the exacerbation stage.The control group included healthy individuals not in contact with tuberculosis patients, without chronic pathology, with no changes in chest radiography, and with negative results on immunodiagnostics the immunologic test (Diaskintest ® , Generium, Russia).We also want to mention that acute COVID-19 and COVID-19 convalescents were excluded from the TB and healthy control groups.
The characteristics of the tuberculosis patients are presented in Table 1.The diagnosis of pulmonary tuberculosis was verified through the detection of M. tuberculosis in sputum and/or MBT DNA according to molecular-genetic and bacteriological methods, with the presence of typical changes according to radiation examination.

Sample Collection
Peripheral blood samples from the patients were collected before treatment initiation.Five milliliters of peripheral blood was collected from each TB patient and healthy subject using K3EDTA anticoagulant tubes.Collected peripheral blood samples were processed immediately.CD8+ T cell subset immunophenotyping was performed within several hours (less than 6 h at 20-22 • C) after blood collection.

Immunophenotyping of Peripheral Blood CD8+ T Cell Subset Maturation Stages and CD57 Expression
For the immunophenotyping of CD8+ T cell subset maturation stages, 200 µL of whole peripheral blood samples was stained with mouse anti-human monoclonal antibodies that recognized cell-surface human CD57, CD62L, CD28, CD27, CD4, CD8, CD3, CD45RA, and CD45.The detailed characteristics of antibodies are given in Supplementary Table S1.All antibodies were manufactured by Beckman Coulter Inc. (Indianapolis, IN, USA), and were used according to the manufacturer's recommendations.Next, all samples were incubated at room temperature in the dark for 15 min, followed by red blood cell lysis for 15 min in the dark using 2 mL of VersaLyse Lysing Solution (Beckman Coulter, Inc., USA) supplied with 50 µL of IOTest 3 Fixative Solution (Beckman Coulter, Inc., USA).Finally, 200 µL of Flow-Count Fluorospheres (Beckman Coulter, Indianapolis, IN, USA) was added, and sample acquisition was performed using a 3/10 Navios flow cytometer (Beckman Coulter, Indianapolis, IN, USA).At least 20,000 CD8+ T cells were analyzed in each sample.The gating strategy for the CD8+ T cell subset maturation stages is shown in Supplementary Figure S1 and has been described in detail previously [18].

Immunophenotyping of Peripheral Blood 'Polarized' CD8+ T Cell Subsets
For the immunophenotyping of 'polarized' CD8+ T cell subsets, 200 µL of whole peripheral blood samples was stained with the following mouse anti-human monoclonal antibodies, which interacted with human CD45RA (Beckman Coulter, Indianapolis, IN, USA), human CD62L (Beckman Coulter, Indianapolis, IN, USA), human CXCR5 (CD185, BioLegend, Inc., San Diego, CA, USA), human CCR6 (BioLegend, Inc., San Diego, CA, USA), human CXCR3 (CD183, BioLegend, Inc., San Diego, CA, USA), human CD3 (Beckman Coulter, Indianapolis, IN, USA), human CD8 (BioLegend, Inc., San Diego, CA, USA), and human CCR4 (CD194, BioLegend, Inc., San Diego, CA USA).The detailed characteristics of antibodies are given in Supplementary Table S2.All antibodies were used according to the manufacturer's recommendations.Blood samples were stained with antibodies at room temperature for 15 min in the dark.Then, red blood cells were lysed by adding 2 mL of Versa Lyse Lysing Solution (Beckman Coulter, Inc., Indianapolis, IN, USA) with 25 µL of IOTest 3 Fixative Solution (Beckman Coulter, Inc., Indianapolis, IN, USA) in the dark at room temperature for 15 min.Next, all samples were washed (330× g for 8 min) twice with sterile PBS supplemented with 2% of fetal calf serum (FCS) (Sigma-Aldrich Co., Saint Louis, MO, USA), resuspended in 500 µL of fresh PBS with 2% neutral formalin (Sigma-Aldrich Co., Saint Louis, MO, USA), and subjected to a flow cytometry analysis.At least 20,000 CD3+CD8+ cells were collected from each sample.The gating strategy for the major 'polarized' CD8+ T cell subsets is shown in Supplementary Figure S2 and has been described in detail previously [18].

Statistical Analysis
The flow cytometry data were analyzed using Kaluza software v2.3 (Beckman Coulter, Indianapolis, IN, USA).Statistical analysis was performed with Statistica 7.0 (StatSoft, Tulsa, OK, USA) and GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) software packages.Normality was checked using Pearson's chi-squared test.Flow cytometry data were presented as a percentage of cells, and the absolute number of CD8+ T cell subsets was presented as the number of cells per 1 µL of whole peripheral blood.All data were presented as the median and interquartile range, Me (Q25; Q75).The differences between patients with pulmonary tuberculosis and the healthy controls were analyzed using a nonparametric Mann-Whitney U-test with a p < 0.05 value.

Main Peripheral Blood T Cell Subsets in Patients with Pulmonary Tuberculosis
Surface expression of CD3 was used to define the total T cell subset in patients with pulmonary tuberculosis (Figure 1), and we found no significant differences in the relative and absolute numbers of CD3+ T cells between the TB and HC groups (79.23% (74.31; 84.95) vs. 76.72%(70.60; 84.08) and 1415 cells/1 µL (1170; 1813) vs. 1481 cells/1 µL (1044; 1811) with p = 0.191 and p = 0.591, respectively).Notably, we also did not find changes in CD3+CD4+ and CD3+CD8+ frequencies between groups (Figure 1).

Alterations in CD57 Expression on CD8+ T Cell Subsets from Patients with Pulmonary Tuberculosis
Next, we analyzed CD57 expression using CD8+ T cells.Previously, it was demonstrated that CD57 is a marker of highly differentiated mature CD8+ T cells that are needed to control intracellular pathogens, including CMV and other viruses in humans [22,23].Furthermore, CD57 cell-surface expression was closely linked with intracellular granzyme A, granzyme B, and perforin accumulation [24].We found that several CD8+ T cell subsets from patients with tuberculosis, including EM1, EM2, and pE1 cells showed decreased levels of CD57+ cells, while EM3 cells were enriched with CD57+ cells compared to healthy controls (Table 2).Our data indicated alterations in the effector capacities of CD8+ T cells during M. tuberculosis infection.Interestingly, we found no significant differences in the absolute numbers of CD57-positive cells in diverse CD8+ T cell subsets (Supplementary Table S3).

Imbalance in Tc1, Tc2, and Tc17 Cells in Peripheral Blood CD8+ T Cell Maturation Sub in Patients with Pulmonary Tuberculosis
Finally, we analyzed the frequencies of inflammatory CD8+ T cell subsets in d ent maturation stages of CD8+ T cells.Thus, we first analyzed the frequency of Tc1, Tc17, and Tc17.1 cells within the 'naïve' CD8+ T cells that were generated in the thy and were not activated by their corresponding antigens.We found that, in TB pati the related numbers on Tc1 and Tc17.1 were decreased vs. the control group (69 (65.14; 75.68) vs. 74.96%(68.55; 81.60) and 1.19% (0.75; 2.05) vs. 2.05% (1.40; 3.28) wit 0.017 and p = 0.006, respectively, Figure 6A,D), while the frequency of Tc2 was sig cantly increased (25.04% (18.74; 32.49)

Discussion
The main role in the local immunological response against M. tuberculosis is pla by macrophages and Th1 [27].These cells are the main participants in the delayedhypersensitivity reaction and contribute to limiting the spread of mycobacterial infec [28].However, Th2, when producing IL-4, suppresses the Th1 response and worsen course of the infection, contributing to its spread [27].Reports have shown an increa Th2 cells in patients with cavitary tuberculosis [29].Cytokines released by these CD cells induce alternative macrophage activation and collagen deposition at the site o flammation [27].This process is observed in common forms of tuberculosis.There in patients with pulmonary tuberculosis, the balance of Th1/Th2 is very important, determines the further course of this infection.

Discussion
The main role in the local immunological response against M. tuberculosis is played by macrophages and Th1 [27].These cells are the main participants in the delayed-type hypersensitivity reaction and contribute to limiting the spread of mycobacterial infection [28].However, Th2, when producing IL-4, suppresses the Th1 response and worsens the course of the infection, contributing to its spread [27].Reports have shown an increase in Th2 cells in patients with cavitary tuberculosis [29].Cytokines released by these CD4+ T cells induce alternative macrophage activation and collagen deposition at the site of inflammation [27].This process is observed in common forms of tuberculosis.Therefore, in patients with pulmonary tuberculosis, the balance of Th1/Th2 is very important, as it determines the further course of this infection.
M. tuberculosis uses multiple immune evasion strategies and can also effectively modulate adaptive immune responses via the inhibition of different T cell functional activities [30].It was found that M. tuberculosis was able suppress T cells by stimulating mesenchymal stem cells, recruiting them to the site of infection; meanwhile, mesenchymal stem cells produced different mediators that down-regulated effector T cell functions [31].Furthermore, M. tuberculosis-specific CD4+CD25+FoxP3+ T cells isolated from the blood and pleural fluid were capable of suppressing IFN-g production in TB patients that potently restricted protective immune responses during tuberculosis by suppressing the effector T cell re-sponses against the bacterium in the site of infection [32].Finally, M. tuberculosis antigens could chronically stimulate antigen-specific CD4+ T cells, making ESAT-6-specific T cells functionally exhausted due to chronic antigenic stimulation [33].
We found no differences in the CD3+CD8+ frequencies between patients with pulmonary tuberculosis and healthy controls.Similarly, Ocaña-Guzmán et al. found that the frequency of CD8+ T cell distribution was similar in HC and TB groups [34].On the contrary, Chávez-Galán et al. found that TB patients had a high frequency of CD8+ cells in peripheral blood [35].Absolute counts of CD8+ T cells decreased significantly with an increase in the number of pulmonary lobes involved, and the peripheral blood CD8+ T cells' concentration had the most impact on the presence or absence of cavities [36].Moreover, Chen et al. noticed that patients with active pulmonary tuberculosis had fewer CD8+ T cells compared to the healthy control and patients with a latent M. tuberculosis infection [37].Furthermore, in patients with active pulmonary tuberculosis, CD8+ T cell counts decreased and were negatively associated with the extent of the lesions detected in the chest using computed tomography; however, they increased significantly after 4 weeks of antituberculosis treatment [38].
We described an abnormal CD8+ T cell profile in patients with pulmonary tuberculosis, which resulted in imbalanced distribution of CD8+ maturation and 'polarized' subsets.We found that EM and TEMRA CD8+ T cell subsets were enriched with immature EM1 and pE1 cells, respectively, while effector CD8+ cells were decreased.Previously, Ocaña-Guzmán et al. observed a decrease in the levels of the 'naïve' CD8+ T cell subset and an increase in the EM cells in groups of patients infected with M. tuberculosis [34].But, the expression of granzyme A, granzyme B, and perforin in patients with active pulmonary tuberculosis was higher than those in the healthy controls and patients with latent M. tuberculosis infection, and it decreased after curing [37].
Interestingly, CD57 was previously proposed as a cell molecule of end-stage, senescent CD8+ T cells in HIV patients exhibiting highly cytotoxic potential, while exhausted CD8+ T cells tended to have a low expression of CD57 [39].Furthermore, exhausted CD57+ CD8+ T cells had a low proliferative capacity [40].Thus, the decreased levels of CD57 in immature EM1, EM4, and pE1 (expressing high levels of CD27 and CD28) cells could be linked with their ineffective function activities, including altered clonal expansion in response to antigen stimulation or even the slow homeostatic turnover of memory CD8+ T cells and the altered formation of effector CD8+ T cells in general.Recently, Chen et al. found that CD8+ T cells from patients with tuberculosis exhibited a high expression of LAG-3 that inhibited the proliferation and maturation of these cells, and elevated LAG-3 expression correlated with the functional defects of CD8+ T cells and TB severity [41].Moreover, CD8+CD28− T cells in the peripheral blood of patients with pulmonary tuberculosis were significantly higher than those in the healthy control group, and the increase in atypical CD8+CD28− T cells was related to the progression of M. tuberculosis infection [42].Finally, the CD8+ T cells of patients with tuberculosis presented a decrease in the frequency of CD8+ cells expressing activating receptor NKG2D, but the frequencies of the CD8+ T cells that co-expressed the NKG2A inhibitor receptor were similar to the control group [35].Thus, all these data point to an imbalance in the effector and regulatory mechanisms of CD8+ T cells, which compromised cell function and could promote pathogen immune escape during M. tuberculosis infection.
We noted that, in peripheral blood samples from patients with M. tuberculosis infection, markedly decreased levels of CXCR3+ Tc1 cells are known to produce high levels of IFNγ and TNFα, as well as granzyme and perforin, which contribute to the killing of yeastinfected host cells [43].Similarly, CXCR3+CD8+ T cells were depleted from the circulation in TB, and this CD8+ T cell subset slightly and progressively recovered by the end of the anti-tuberculosis therapy [34].Furthermore, the levels of CXCR3 ligands-chemokine MIG, IP-10, and I-TAC-in plasma samples from patients with active pulmonary tuberculosis were discovered to be higher than those in patients with latent M. tuberculosis infection and cured pulmonary tuberculosis patients [37].Moreover, the pleural fluid concentration of MIG, IP-10, and I-TAC from patients with active pulmonary tuberculosis increased significantly compared to their plasma levels.Thus, CXCR3 and its ligands could play an important part in the regulation of Tc1 CD8+ T cell migration to the site of infection.Immunohistochemically stained lung biopsies show a large number of CD8+ T cells in tuberculosis granulomas [27].
It was shown that the effector subsets of CD8+ T cells can migrate to sites of inflammation, perform cytolytic functions, and produce effector cytokines [44].Moreover, effector perforin-and granulysin-expressing CD8+ T cells were detected in tuberculous granulomas in patients with pulmonary tuberculosis [45].Thus, CD8+ T cells expressed effector molecules and infiltrated pulmonary tissue.Furthermore, effector CD8+ T cells upregulated CXCR3 expression on their cell membranes following infection with a variety of intracellular pathogens [46].Hickman et al. reported that CXCR3 could affect not only CD8+ T cell migration into sites of infection but also migration within inflamed tissues, as CD8+ T cells locate pathogen-infected targets for killing [47].Therefore, a decrease in the peripheral blood of Tc1 and the most mature effector subset within TEMRA cells may indicate their migration to the site of the tuberculosis infection.
However, these cytotoxic cells have a reduced expression of perforin, granzyme, and granulizing, which indicates the functional disorders of these cells during tuberculosis infection.CD8+ T cells have a peripheral localization in granulomas, while CD4+ T cells occupy their center [27,45].M. tuberculosis is located in the center of the granuloma.Therefore, IFN-γ-producing CD4+ T cells are the most involved in the anti-tuberculosis response, and CD8+ T cells are not in close contact with M. tuberculosis-infected cells, which impairs pathogen clearance by these cytotoxic lymphocytes.
We also noticed increased levels of Tc2 cells in peripheral blood samples from patients with tuberculosis.It was shown that Tc2 cells have reduced cytotoxic activity compared to Tc1 cells [48].However, Tc2 cells have a similar cytokine profile to Th2 cells that can suppress Th1 and Tc1 effector functions [49].Therefore, an increase in the peripheral blood Tc2 cells in patients with tuberculosis can be closely linked with the low effectiveness of the type 1 immune response.Furthermore, type 2 immune response dominance may lead to disease progression, since enhanced Th2 polarization was one of the consequences of the progressive TB disease [50].Interestingly, significantly augmented levels of the IL-17+CD8+ T cells were detected in patients with TB infection compared to the healthy group, suggesting a potentially deleterious role of Tc17 CD8+ T cells during active pulmonary tuberculosis [51].Previously, it was shown that circulating CD8+ T cells from TB patients had increased the levels of the activation marker HLA-DR as well as chemokine receptors (CCR2, CCR3, and CXCR4) [51].
It is necessary to study further changes in the level of CD8+ T cells in tuberculosis patients after COVID-19 infection, as well as in the post-COVID-19 syndrome.As the results of past studies have shown, COVID-19 patients have certain immunogenetic features that determine the severity of COVID-19 infection and the consequences of post-COVID-19 infection [52].Also, in patients with tuberculosis after COVID-19, there were disorders of immune response with changes in the level of naive T cells, and their ratio with B-cells changed in patients who underwent 53].
Our research had some limitations.We did not assess the functional capacity of CD8+ T cell subsets, and we also did not analyze serum cytokines levels.Moreover, we did not conduct immunohistochemical studies of the affected lung tissues.

Conclusions
Thus, the obtained results and literary data suggest that CD8+ T cells play an important role in controlling M. tuberculosis infection.We are sure that the determination of the subpopulation of CD8+ T cells can further help in predicting the course of tuberculosis.

Figure 1 .
Figure 1.Comparison of relative and absolute numbers of main circulating T cell subsets in tients with pulmonary tuberculosis.Scatter plots (A-F) showing the percentages (the percentage T cell subset within the total lymphocyte subset, LY) and absolute numbers (number of cells pe μL of whole peripheral blood) of T cells (CD3+), T-helpers (Th, CD3+CD4+), and CD8+ T ce (Tcyt, CD3+CD8+), respectively.Black circles denote patients with pulmonary tuberculosis (TB, 32); white circles-the healthy control (HC, n = 31).

Figure 1 .
Figure 1.Comparison of relative and absolute numbers of main circulating T cell subsets in patients with pulmonary tuberculosis.Scatter plots (A-F) showing the percentages (the percentage of T cell subset within the total lymphocyte subset, LY) and absolute numbers (number of cells per 1 µL of whole peripheral blood) of T cells (CD3+), T-helpers (Th, CD3+CD4+), and CD8+ T cells (Tcyt, CD3+CD8+), respectively.Black circles denote patients with pulmonary tuberculosis (TB, n = 32); white circles-the healthy control (HC, n = 31).

Figure 1 .
Figure 1.Comparison of relative and absolute numbers of main circulating T cell subsets in p tients with pulmonary tuberculosis.Scatter plots (A-F) showing the percentages (the percentage T cell subset within the total lymphocyte subset, LY) and absolute numbers (number of cells pe μL of whole peripheral blood) of T cells (CD3+), T-helpers (Th, CD3+CD4+), and CD8+ T ce (Tcyt, CD3+CD8+), respectively.Black circles denote patients with pulmonary tuberculosis (TB, n 32); white circles-the healthy control (HC, n = 31).

Figure 4 .
Figure 4. Alterations in the relative and absolute number of TEMRA CD8+ T cell subsets with different patterns of CD27 and CD28 expression in patients with pulmonary tuberculosis.Scatter plots

Table 1 .
Demographic, clinical, and bacteriological characteristics of Tbc patients.

Table 2 .
CD57 expression on diverse CD8+ T cell subsets in patients with pulmonary tuberculosis.
19.19.92%(13.87;26.78)withp = 0.006, Figure We also noticed elevated levels of Tc2 cells in TB patients within central memory CD