Mucosal-Associated Invariant T Cells in Tuberculosis Pleurisy

Mucosal-Associated Invariant T (MAIT) cells, which is a prevalent and unique innate T-cell population that expresses an evolutionarily conserved invariant T cell receptor TCRVα7.2, are present at high frequencies at mucosal tissue sites and have an intrinsic capacity to respond to microbial infections. However, the local immune responses of MAIT cells at the site of M.tb infection is unclear. We compared the PFMCs from TB (n = 57) with the PBMCs from TB (n = 57) and HD (n = 50), and characterized those T-cell phenotypes and functions. Our direct ex vivo analysis demonstrated that the frequencies of MAIT cells in PFMCs were much higher than those in PBMCs from TBP patients (P<0.001), however, lower than those in PBMCs from HD (P<0.01). Those infiltrating MAIT cells expressed high levels of tissue-tropism chemokine receptors (CXCR3hiCXCR4hiCXCR6hiCCR6hiCXCR5hiCCR5hi) and displayed an effector memory phenotype (CD45RO+CCR7-CD62L-), which indicated preferential accumulating these cells into infected lung lesions. Further, the majority of MAIT cells in PFMCs expressed CD69, a marker for tissue resident memory T cells, which suggested that specialization of these T cells into unique tissue-resident subsets given the host enhanced regional immunity. In addition, MAIT cells from PFMCs produced IFN-γ and TNF-α, and exhibited cytotoxic activity molecules CD107a/b, suggesting that poly functional M.tb-reactive MAIT cells played an significant role against M.tb infection in the local lesions. This study addressed that the M.tb-reactive MAIT cells exerted unique innate functions in immune responses to M.tb at local infection sites.


Introduction
Invariant Natural Killer T (iNKT) cells and Mucosal-Associated Invariant T (MAIT) cells represent peculiar T-lymphocyte subpopulations with innate-like properties that differ from conventional T cells.CD1d-restricted iNKT cells and MR1-restricted MAIT cells are defined by invariant or semiinvariant repertoires [1][2][3].These invariant lymphocyte subsets follow specific onto genic pathways, home to particular tissues and have larger clonal sizes than do conventional lymphocytes [4,5] MAIT cell is an abundant population of innate-like T-cell subset in humans, which is characterized by expression of an evolutionarily conserved invariant T Cell Receptor (TCR) carrying the canonical Vα7.2-Jα33 TCR rearrangement and activated by an antigen bound to the major Histocompatibility Complex (MHC) class I-like molecule MR1 [6][7][8].
In humans, contrary to NKT cells, MAIT cells display a naive phenotype in the thymus as well as in cord blood where they are in low numbers.However, after birth, MAIT cells have a memory phenotype early in life and are abundant in human blood (1-8% of T cells versus 0.01-1% for natural killer T cells), which suggests that MAIT cell populations expand after birth and acquire their memory phenotype in the presence of commensal flora [9,10].It is shown that the development of MAIT cells are dependent upon the expression of MR1 on bone marrow-derived cells, and are selected by MR1 in the thymus on a non-B non-T hematopoietic cells [11].However, they acquire a memory phenotype and expand in the Volume 6; Issue 02 J Vaccines Immunol, an open access journal ISSN: 2575-789X periphery in a process dependent both upon B cells and bacterial flora [12].In support of that hypothesis, MAIT cells are not detectable in germ-free mice and/or B-cell-deficient patients and mice, but can be induced to expand after microbial colonization, which is a complex relationship between microbes and MAIT cells [13].MAIT cells are abundant within the CD8αα + , an intermediate level of CD8αβ and scarce in the CD4 + T cell subset, which account for up to ～15% of the CD8 + T-cell population in the blood of healthy individuals [14].Meanwhile, MAIT cells gradually obtain some other functional surface makers in the process of development.The specific anti-Va7.2antibody allows the human MAIT cell population to be characterized in detail.This marker, together with the high expression of CD161 identifies MAIT cells in peripheral blood and other tissues [15].CD161, is a c-type lectin family member, which is expressed on a significant proportion of tissue-infiltrating T cells, such as the majority of NKT cells, MAIT cells and TCRγδ T cells, mediates cytokines and cytotoxicity after immune stimulation [16,17].
Tuberculosis (TB), caused by the highly infectious intracellular pathogen Mycobacterium tuberculosis (M.tb), remains a highly leading cause of infectious rates and mortality worldwide [18].It has shown that the frequencies of MAIT cells are severely reduced in circulation but high levels in the lungs of patients with active TB, suggesting that MAIT cells contribute to protection against M.tb infection in humans [19,20].Although these T cell clusters are important for the persistence of MAIT cells resident in lungs, it is not clear whether these MAIT cells in pleurisy effusion are long term resident and what proportion of MAIT cells in pleurisy effusion are resident versus recirculating.What's more, much less is known about what phenotypes and functions of these MAIT cells and how these cells provide protection.The role of MAIT cells at the local sites of M.tb infection is currently unknown.
In the current study, we examined the frequencies, phenotypes and functions of MAIT cells in a relatively large number of patients with active tuberculosis pleuritis and healthy donors.

Human Subjects
Fifty-seven patients with diagnosed Tuberculosis Pleurisy (TBP) were enrolled in this study from the Chest Hospital of Guangzhou, China (Table 1).The diagnosis of TBP has been described in our previous publication [21].The peripheral blood and pleural fluid samples were obtained from the patients with TBP during therapeutic thoracentesis operated by Dr. Suihua Lao according to the strict medical operation rules and methods.All participating patients had received less than one week of antituberculosis therapy.Patients who had a history of autoimmune diseases or co-infected HIV, HBV or HCV were excluded from the study.Fifty Healthy Donors (HD) were recruited from the blood center of Guangzhou, China (Table 1).The peripheral blood samples were obtained from volunteers.The written consents were obtained from all the subjects and this study was approved by the ethics committee of the Zhongshan School of Medicine, Sun Yatsen University and the Chest Hospital of Guangzhou, China.

Isolation and Preparation of Pleural Fluid Mononuclear Cells (Pfmcs) And Peripheral Blood Mononuclear Cells (Pbmcs)
The PFMCs and PBMCs were isolated and prepared according to the previously described protocols [21].Briefly, Pleural Fluid (PF) collected from TBP patients were centrifuged at 2500 rpm for 20 min, and the cell pellets were collected.The mononuclear cells were isolated and obtained through Ficoll-Hypaque (Tianjin Hao Yang Biological Manufacture, Tianjin, China) density gradient centrifugation at 2000 rpm for 20 min.PFMCs and PBMCs were collected and washed twice with Hank's balanced salt solution, then re-suspended at a final concentration of 2×10 6 cells/ml in complete RPMI 1640 medium (Life Technologies, Grand Island, USA) supplemented with 10% heatedinactivation fatal calf serum (Sijiqing, Hangzhou, China), 100 μg/ Volume 6; Issue 02 J Vaccines Immunol, an open access journal ISSN: 2575-789X ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine and 50 μM 2-mercaptoethanol (Life Technologies, Grand Island, USA).When freezing cells, the cells were isolated and re-suspended in cell freezing medium supplemented with 90% heated-inactivation fatal calf serum (Sijiqing, Hangzhou, China) and 10% Dimethyl Sulfoxide (DMSO) (Zhanchen biological technology co., LTD, Guangzhou, China), and were cryopreserved in liquid nitrogen.When thawing cells, the microtubes were thrown into 37 ° C warm water, and gently shaking them to melt as soon as possible.The cell suspension was transferred into 15 ml tube and was washed twice in the complete RPMI 1640 medium.Finally, the cells were re-suspended at a final concentration of 2×10 6 cells/ml in complete RPMI 1640 medium.

Mycobacterium tuberculosis Antigens
Mycobacterium bovis Bacillus Calmette-Guérin (BCG) was purchased from Chengdu Institute of Biological Products, Chengdu, China.Mycobacterium tuberculosis strain H37Rv (M.tb) was provided by laboratory of Baiqing Li from deparment of Immunology, research center of immunology, Bengbu Medical College, Bengbu, PR China.

Statistical Analysis
Significant differences between data sets were performed with either the unpaired Student's t-test when comparing two groups, one-way ANOVA for more than two groups or two-way ANOVA for two variables (Graph Pad Software Inc, San Diego, CA, USA).

The tissue-tropism chemokine receptors expressed by MAIT cells in PFMCs from TBP patients and PBMCs from HD
To better understand the accumulation of MAIT cells into pleurisy effusion, we analyzed the expression of tissue-tropism chemokine receptors on MAIT cells by flow cytometry.Compared with conventional T cells, the pattern of chemokine receptor expression was, however, very specific, as MAIT cells exhibited high levels of CXCR4, CCR6 and CXCR6, heterogeneous levels of CCR4, and intermediate expression of CXCR3, CXCR5 and CCR5, but did not express CCR10.These chemokine receptors (CXCR3 and CXCR4) were involved in trafficking to local infection sites, especially the airways and lungs (Figure 2A).Altogether, these results clearly indicated that MAIT cells were circulating lymphocytes with tissue tropism, unlike other conventional T cells.Importantly, the MAIT cells in PF (TB) exhibited higher levels of CXCR3, CXCR4, CCR5, CXCR5, CCR6 and CXCR6 than those in PB (HD), but scarcely expression of CCR4 and CCR10 (Figure 2A,B).Specially, the expression of CXCR3 and CXCR4 on MAIT cells in PF (TB) were significantly higher than those in PB (HD) (Figure 2B).Next, we analyzed the indicated chemokine receptors on both subsets CD161 + Vα7.2 + and CD161 -Vα7.2 + T cells.It showed that the CD161 + Vα7.2 + T cells expressed the extremely higher levels of CXCR3 and CXCR4 than counterparts in PF (TB) (Figure 2C).The chemokine IP-10 and SDF-1 for chemokine receptor CXCR3 and CXCR4 in pleural fluid from TBP patients were higher than serum from healthy donors and pleural fluid from cancerous patients (Figure 2D).It suggested that high levels of chemokine receptors on MAIT cells and chemokines in tuberculous pleural fluid were accounting for the accumulation of MAIT cells from peripheral blood into local infection sites, and to be a group of tissue resident T cells.

The memory phenotypes of MAIT cells in PFMCs from TBP patients and PBMCs from HD
To better understand the physiology of MAIT cells in tuberculous pleurisy effusion, MAIT cells were stained with memory surface markers, anti-CD45RO, anti-CCR7 and anti-CD62L, and divided into CD45RO + CCR7 -CD62L -effector memory T cells (T EM ) and CD45RO + CCR7 + CD62L + central memory T cells (T CM ).CCR7 is involved in homing of T cells to various secondary Lymphoid organs such as Lymph nodes.CD62L (L-selectin) also slows lymphocyte trafficking through the blood, and facilitating entry into a secondary lymphoid organ at that point.In PF (TB) and PB (HD), as conventional CD8 + T cells, CD8 + Vα7.2 + T cells expressed CD45RO + and CD45RO − , however, rarely expressed CCR7 and CD62L (Figure 3A).Higher levels of MAIT cells with T EM phenotypes (CD45RO + CCR7 -CD62L -) in PF (TB) were expressed compared to those in PB (HD; P<0.001), however, the frequencies of MAIT cells with T CM phenotype in PF (TB) were similar to those in PB (HD; P>0.05) (Figure 3B), suggesting those MAIT cells were accumulated into pleurisy effusion and uncirculated during infection.Next, we also analyzed the T EM phenotypes in both subsets CD161 + Vα7.2 + and CD161 -Vα7.2 + T cells.In PF (TB), the CD161 + Vα7.2 + T cells were higher frequencies of T EM phenotypes than the counterparts CD161 -Vα7.2 + T cells (P<0.001).However, In PB (HD), the CD161 + Vα7.2 + T cells with T EM phenotypes were similar to the CD161 -Vα7.2 + T cells (P>0.05).Meanwhile, higher frequencies of CD161 + Vα7.2 + with T EM phenotype in PF (TB) were observed compared to those in PB (HD; P<0.001) (Figure 3C,D).Together these data, it suggested that MAIT cells especially the CD161 hi MAIT cells in pleural fluid experienced M.tb infection and displayed an effector/memory potential.

The profile of tissue resident memory phenotype on MAIT cells in PFMCs from TBP patients and PBMCs from HD
The previous explanation of the expression of CD69 was that they were in an activated state, perhaps as a result of retained antigen.However, it was well known that CD69 + expression was a generic characteristic of resting Tissue-Resident Memory (TRM) T cells in the infectious lungs.Thus, we next investigated the expression of tissue resident memory T cell (CD69 + ; TRM) markers of MAIT cells in PF (TB) compared to PB (HD).We found that in PF (TB) and PB (HD), resting MAIT cells expressed higher levels of CD69 and CD25 than conventional CD8 + T cells (P<0.001).In addition, MAIT cells in PF (TB) expressed significantly higher levels of CD69 and CD25 than those in PB (HD; P<0.001) (Figure 4A,B).Next, we also found that the CD161 + Vα7.2 + T cells had a significantly higher expression of CD69 and CD25 in PF (TB) and PB (HD) compared to counterparts CD161 -Vα7.2 + T cells, respectively (Figure 4C,D).We found that TRM (CD69 + ) T cells in PF (TB) were very high, however, rarely in circulation PB (HD).Clearly, almost the majority of CD161 hi MAIT cells expressed CD69, and being tissue resident memory T cells in local infection sites.To examine whether MAIT cells in PF (TB) could respond to TB-Ags, MAIT cells were stimulated with M.tb or BCG.CD161 hi MAIT cells in PF (TB) had higher level of expression of CD69 when responded to M.tb or BCG stimulation compared to un-stimulated control cells.However, in PB (HD), M.tb and BCG showed less effect on MAIT cells (Figure 4E,F).Together those data, it is suggesting that CD161 helped the accumulation of MAIT cells in pleural fluid after infection and being the tissue resident memory T cells in local infection site.

The expression of Tc1-type cytokines and cytotoxic molecules on MAIT cells in PFMCs from TBP patients after polyclonal stimulation
To better understand the effector functions of MAIT cells, we analyzed the secretion patterns of cytokines, cytotoxic molecules and transcription factors after polyclonal stimulation.We found that pleural fluid MAIT cells exhibited high ability of cytokine and cytotoxic molecules expression after the stimulation with PMA and ionomycin.MAIT cells expressed Tc1-type cytokines IFN-γ, TNF-α and IL-2, and also shared cytotoxic molecules CD107a/b with cytotoxic CD8 + T cells (Figure 5A).The majority of MAIT cells were a polyfunctional cell subset in PF (TB) when stimulated with PMA and ionomycin (Figure 5B).Next, we detected the transcription factor in regulate the expression of cytokines on MAIT cells.It showed that the transcription factor T-bet regulated the IFN-γ production of MAIT cells but less effect on the expression of TNF-α and IL-2.The T-bet + MAIT cells hold higher expression of IFN-γ not TNF-α and IL-2 than T-bet -MAIT cells (Figure 5C,  D).Also, the CD161 + Vα7.2 + T cells expressed higher levels of Tc1-type cytokines IFN-γ and TNF-α and transcription factor T-bet than their counterparts CD161 -Vα7.2 + T cells (Figure 5E).We found that the expression of IFN-γ in MAIT cells regulated by T-bet and CD161, and CD161 + Vα7.2 + T cells expressed more IFN-γ and TNF-α compared to their counterparts CD161 -Vα7.2 + T cells.Together these data, it suggested that CD161 + MAIT cells exhibited stronger and more cytokines and cytotoxicity after polyclonal stimulation.

Discussion
MAIT cells are highly conserved between biological species and are very abundant in humans [23].MAIT cells play a critical role in the host defense against a variety of bacterial infections, including Shigella dysenteriae, Klebsiella pneumonia, Francisella tularensis, M.tb bovis BCG and M.tb abscessus infections, and the cytokines and cytotoxic effector molecules secreted by MAIT cells, such as IFN-γ, TNF-α and Granzyme B are probably related to the functions of anti-infection immunity [15].The functional roles of MAIT cells during human M.tb infection remains to be elucidated.Those non-classically restricted M.tb-reactive T cells are MR1restricted nonconventional T cells, and make up a substantial proportion of the M.tb-reactive non-classically restricted CD8 + T-cell responses [19].This suggested that Vα7.2 + MR1-restricted MAIT cells could detect M.tb-infected cells regardless of prior exposure to the microorganisms.MAIT cell levels have been reported to be lower in blood and enriched in lung tissues in active pulmonary TB patients [24,25].Moreover, recent studies have provided in vivo evidence demonstrating that MAIT cells could migrate into infected lung tissues during bacterial infection and that MAIT cells play a protective role against TB [26].The observation that MAIT cells are nearly absent from the peripheral blood in those with TB, and concomitantly enriched in the lungs suggests a dynamic relationship between the presence of M.tb and its associated metabolites and the localization of MAIT cells.
The frequencies of MAIT cells were much lower in the peripheral blood of M.tb infected patients compared to healthy controls, but appeared to be better preserved in pleurisy effusion.This observation can be explained by several hypotheses, such as impaired development, promoted apoptosis stimulated by M.tb or the migration and recruitment of these cells into lungs [27,28], which suggested that human MAIT cells had a significant role in the anti-bacterial response in the infected lesions.The levels of MAIT cells in peripheral blood might be associated with the levels of MAIT cells in infected lung lesions.The lower frequency of MAIT cells represents a specific decrease in numbers and is not diluted by M.tb-reactive mainstream T cells.One possibility for the serious loss of MAIT cells in the peripheral blood of M.tb infected patients is that after infection, MAIT cells infiltrate into lungs and the large accumulation into the pleurisy effusion, which would directly decrease their numbers in peripheral blood.This hypothesis was tested by tissue immunofluorescence with anti-CD3 and anti-Vα7.2(defined as MAIT cells), and we observed that MAIT cells in lung lesions from patients infected with M.tb [29].Human MAIT cells seemed to migrate into infected lungs and were better preserved, which suggested that the M.tb-reactive MAIT cells were involvement in anti-tuberculosis defense.During the early innate phase of infections, MAIT cells were required for prompt production of pro-inflammatory cytokines and timely recruitment of activated conventional CD4 + and CD8 + T cells in the lungs.During later phase of infection, when conventional CD4 + and CD8 + T cells were activated, MAIT cells continued to infiltrate into the lungs of infected mice and produced foremost cytokines such as IFN-γ, TNF-α and cytotoxic granules [30].Potential MAIT cell contributed to the acquisition of M.tb-specific T-cell responses.A wide variety of mycobacteria including M.tb have the capacity to infect lung epithelial cells [31].Moreover, lung epithelial cell lines as well as primary lung epithelial cells infected with M.tb efficiently process and present bacterially derived antigens to both classically and non-classically restricted T cells [32].In humans, the majority of Vα7.2 + T cells directly isolated from lungs were pathogen reactive T cells.MR1-restricted MAIT cells have been shown to produce IFN-γ and TNF in response to M.tb infected lung epithelial cells [33].Additionally, MAIT cells could induce target cell lysis of epithelial cells and contain granulizing previously shown to have anti-microbial properties [34].Given this Th1-like cytotoxic effector phenotype, MAIT cells in the lung tissues have the potential to provide pro-inflammatory cytokines and potentially kill M.tb-infected cells in the infected lungs.
In Humans, MAIT cells are capable of producing IFN-γ in M.tb-infected lung tissues [35].MAIT cells isolated from peripheral blood and lung lesions respond rapidly to M.tb-infected cells.Functionally, this response is characterized by the release of IFN-γ and TNF-α, which is critical for the control of M.tb infection, and also involves the granule exocytosis pathway, which results in target cell apoptosis and delivery of anti-microbial peptides [36].In this case, activation of MAIT cells by APCs producing IL-12 would induce the secretion of cytokines such as IFN-γ, which increases the adaptive and innate immune response to the infection [37].In infected patients, MAIT cells have an anti-bacterial function that could be attributed to the production of IFN-γ.The production of IFN-γ by MAIT cells is moderate compared with that of conventional memory CD4 + and CD8 + T cells stimulated with TB-Ags.In response to infected APCs, MAIT cells share functional characteristics with cytolytic CD8 + effector T cells [34].The cytotoxic capacity of MAIT cells, with a wide microbial reactivity, could have major impacts on many infectious diseases caused by microbial expressing MAIT specific ligands.The resting MAIT cells are uniquely characterized by a lack of Granzyme B and low perforin expression, key granule proteins required for efficient cytotoxic activity.However, bacterial activation of MAIT cells rapidly induced Granzyme B and perforin, licensing these cells to kill their cognate target cells.The lysis of infected target cells could supplement the immune control of microorganism infections by limiting the spread of the microorganism throughout the host.
MAIT cells kill M.tb-infected target cells contributing to the control of intracellular infections.In mouse models, MR1deficient mice, after aerosol infection with M. bovis BCG, showed a lower ability to control the M. bovis BCG than wild-type mice, as evidenced by a higher M. bovis BCG burden in the lung lesions [38].In this case, MR1-dependent protection occurs within the first few days of a M. bovis BCG infection, consistent with the interpretation that MAIT cells are unconventional innate-like lymphocytes [39].The capacity to react rapidly to bacterial challenge enables MAIT cells to display anti-microbial function in vivo.Recently, Hiroshi Wakao reported an Induced Pluripotent Stem Cell (IPSC)based reprogramming approach for the expansion of functional MAIT cells in anti-mycobacterial activity [40,41].Because of the prevalence, location, and effector functions of MAIT cells in conjunction with their ability to detect nearly all intracellularly infected cells, MAIT cells could be targeted to aid in the clearance or control of M.tb.To some extent, the potential use for MAIT cell targeting vaccination or therapy will depend on whether or not vaccination can elicit long-lived memory [42].Alternately, the presence of MAIT cells in the lungs and other tissues, and their inherent effector function could suggest a role for these cells as a target of host-directed therapy.It is also possible that MAIT cell ligands could be used as adjuvants in the delivery of traditional antigens [43].MAIT cells will be as a diagnostic or as a target of vaccination to fight against M.tb infection.
In the current study, we provided evidence that MAIT cells exerted unique innate functions in immune responses to M.tb.The results presented here have shown that MAIT cells represented an evolutionarily conserved innate-like lymphocyte population that sensed and participated in immune responses to M.tb.Given the abundance of this cell type in humans, their wide microbial specificity, their protective capacity and the manipulation of MAIT cells could have a considerable effect on the development of vaccines and therapeutic pathways for infectious diseases.

Figure 3 :
Figure 3: Detection of the memory phenotypes of MAIT cells in pleural fluid from TBP patients and peripheral blood from HD. PFMCs from TBP (n = 12) and PBMCs from HD (n = 12) were harvested and stained with fluorochrome-conjugated monoclonal antibodies, and analyzed for the lineage differentiation and memory phenotype expression with flow cytometry.(A): Representative FACS data.(B): Statistical analysis showed that the expression of effector memory (T EM ) phenotype and central memory (T CM ) phenotype in CD3 + CD8 + Vα7.2 + T cells from PFMCs and PBMCs.(C): Representative FACS data.(D): Statistical analysis showed that the expression of effector memory (T EM ) phenotype in both CD161 + Vα7.2 + and CD161 -Vα7.2 + subsets of CD3 + CD8 + Vα7.2 + T cells from PFMCs and PBMCs.Data were shown as mean ± SD.Each dot represented one patient.ns, not significant; *** P<0.001.

Figure 4 :
Figure 4: Detection of the profile of tissue resident memory phenotype on MAIT cells in pleural fluid from TBP patients and peripheral blood from HD. PFMCs from TBP (n = 8) and PBMCs from HD (n = 8) were harvested and stained with fluorochrome-conjugated monoclonal antibodies, and analyzed for the lineage differentiation and profile of tissue resident memory phenotype expression with flow cytometry.(A): Representative FACS data.(B): Statistical analysis showed that the expression of CD69 and CD25 on CD3 + CD8 + Vα7.2 + T cells versus conventional CD3 + CD8 + T cells from PFMCs and PBMCs.(C): Representative FACS data.(D): Statistical analysis showed that the expression of CD69 and CD25 in both CD161 + Vα7.2 + and CD161 -Vα7.2 + subsets of CD3 + CD8 + Vα7.2 + T cells from PFMCs and PBMCs.(E): Representative FACS data.(F): Statistical analysis showed that the expression of CD69 in both CD161 + Vα7.2 + and CD161 -Vα7.2 + subsets of CD3 + CD8 + Vα7.2 + T cells from PFMCs and PBMCs after stimulation with or without BCG or M.tb in the presence of anti-CD28 for 1 day, respectively.Data were shown as mean ± SD.Each dot represented one patient.ns, not significant; * P<0.05; ** P<0.01; *** P<0.001.

Figure 5 :
Figure 5: Detection of the Tc1-type cytokines and cytotoxic molecules by MAIT cells in pleural fluid from TBP patients after polyclonal stimulation.PFMCs from TBP (n = 8) and PBMCs from HD (n = 8) were stimulated with PMA (20 ng/ml) and Ionomycin (1 μg/ml) for 6 hours.Cells were harvested and stained with fluorochrome-conjugated monoclonal antibodies, and analyzed for the lineage differentiation, intracellular cytokine and transcription factor expression with flow cytometry.(A): Representative FACS data showed that the expression of IFN-γ, TNF-α, IL-2 and CD107a/b by MAIT cells after polyclonal stimulation.(B): The statistical analysis showed that the polyfunctional of MAIT cells in PFMCs.(C): Representative FACS data and (D): Statistical analysis showed that the expression of IFN-γ regulated by T-bet in CD3 + CD8 + Vα7.2 + T cells from PFMCs.(E): Representative FACS data showed that the expression of IFN-γ, TNF-α and T-bet in both CD161 + Vα7.2 + and CD161 -Vα7.2 + subsets of CD3 + CD8 + Vα7.2 + T cells from PFMCs.Data were shown as mean ± SD.Each dot represented one patient.** P<0.01; *** P<0.001.

Table 1 :
The study was conducted in a total of fifty-seven patients with Tuberculosis Pleuritis (TBP) that were recruited from the Chest Hospital of Guangzhou, China.A total of fifty Healthy Donors (HD) were recruited from the blood center of Guangzhou, China.Analysis of the demographic and clinical characteristic of patients with tuberculosis pleuritis and healthy donors.