|Year : 2019 | Volume
| Issue : 2 | Page : 80-86
Role of CXCR3+CCR5+ Th1 cells in pulmonary tuberculosis patients: At pathogenic site
Pradip K Saha1, Prabhat K Sharma2, Binit Kumar Singh1, Chaitenya Verma3
1 Department of Medicine, All India Institute of Medical Sciences, New Delhi, India
2 Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, India
3 Dr. B R Ambedkar Centre for Biomedical Research, University of Delhi (North Campus), Delhi, India
|Date of Submission||05-May-2019|
|Date of Decision||07-May-2019|
|Date of Acceptance||05-Jun-2019|
|Date of Web Publication||17-Jun-2019|
Dr. Pradip K Saha
Department of Medicine, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
Objective: Tuberculosis (TB) still remains a major global public health problem followed by drug-resistant TB. Understanding of deeper immunopathogenesis into the pathogen–host interactions is required so that it can be translated into effective tools at a public health level. Methods: We recruited 40 pulmonary TB (PTB, sputum smear-negative) patients and studied their peripheral blood (PBL) and bronchoalveolar lavage (BAL) fluid. Of 40 patients, BAL fluid from 10 patients was collected from normal lung and diseased lung in the same patients. Immunophenotyping and intracellular cytokines were performed by flow cytometry. Results: We observed a significant increased expression of CXCR3+CCR5+T-cells (P = 0.002), CXCR3+ T-cells (P = 0.002), CCR5+ T-cells (P = 0.002), CXCR3+CD11ahigh T-cells (P = 0.002), and CCR5+CD11ahigh T-cells (P = 0.002) in BAL (n = 10) over PBL (n = 40). Increased frequency of CXCR3+CCR5+ T-cells (P = 0.02), CXCR3+ (P = 0.034), CCR5+T-cells (P = 0.039), CXCR3+CD11ahigh T-cells (P = 0.05), and CCR5+CD11ahigh T-cells (P = 0.05) was also observed in BAL of disease lung as compared to normal lung (n = 10). There was a significant decrease of inflammatory cytokine (P = 0.028) in the interferon-gamma level, whereas interleukin-4 level was significantly increased (P = 0.028) in BAL as compared to PBL. Conclusion: We conclude that in spite of significant enrichment of CXCR3+CCR5+ Th1-cells with the ability to produce inflammatory cytokines and CD11ahigh T-cells which play an important role in the recruitment of these T-cells, they are not able to control the TB infection. Therefore, our data provide insight into the functionality of effector T-cells at the disease site.
Keywords: CXCR3, CCR5, pulmonary tuberculosis, tuberculosis, T-cells
|How to cite this article:|
Saha PK, Sharma PK, Singh BK, Verma C. Role of CXCR3+CCR5+ Th1 cells in pulmonary tuberculosis patients: At pathogenic site. Biomed Biotechnol Res J 2019;3:80-6
|How to cite this URL:|
Saha PK, Sharma PK, Singh BK, Verma C. Role of CXCR3+CCR5+ Th1 cells in pulmonary tuberculosis patients: At pathogenic site. Biomed Biotechnol Res J [serial online] 2019 [cited 2020 Jun 4];3:80-6. Available from: http://www.bmbtrj.org/text.asp?2019/3/2/80/260489
| Introduction|| |
Tuberculosis (TB) still remains a major threat to human health with 6.3 million new cases and 1.3 million estimated deaths annually worldwide, despite the use of the Bacille–Calmette–Guerin vaccine and other treatment modalities. In addition, the emergence of drug-resistant forms of Mycobacterium tuberculosis severely compromises control of the global TB epidemic. M. tuberculosis enters the host via airborne nasal droplets and alveolar macrophages are the first cells to encounter the pathogen. Survival of M. tuberculosis and their later reactivation leading to the onset of the active disease is believed to result from the defect or deficit of host immune response directed at containment of the infection., Animal and human studies of intracellular pathogens have been demonstrated that showed induction of innate immune response is initial immune phenomena at the onset of infection,, but T-cell-mediated immunity plays a critical role in conferring protection against TB bacilli.,, Recruitment of various effector T-cell subsets at the sites of TB pathology under the influence of homing molecules, including selectins, chemokines, and adhesion molecules, is believed to play a critical role in translating the host immunity at the local disease sites.,
Granuloma is the immunological hallmark in TB immunopathology and acts as the powerhouse of the potent cellular immune response to the chronic inflammatory reaction of the host. Varieties of T-lymphocytes that are recruited proliferate within the early lesions and subsequently activate macrophages to kill the intracellular Mycobacterium. Mycobacteria-specific CD4+ T-cells are dominant of the Th1 type, in that they are potent interferon-gamma (IFN-γ) producers. IFN-γ is a central factor in the activation of antimycobacterial activities of macrophages and hence is considered crucial for protection against TB., Th1 cells acquire the capacity to produce IFN-γ and preferentially express chemokine receptors such as CCR5 and CXCR3 that drive them to the sites of the pathogenic onslaught. The final outcome of infection with M. tuberculosis infection depends on the balance between (i) growth or survival and killing of M. tuberculosis and (ii) the nature or extent of the host T-cells response elicited at the pathologic sites and local immune response elicited at the site of pathology appear to be a key role in determining the fate of M. tuberculosis infection; a shift toward the Th2 type of cytokine production such as interleukin-4 (IL-4) may lead to the dissemination of disease. We have earlier reported on the hierarchic role of chemokine receptors in the selective infiltration of CXCR3 and CCR5 expressing effector T-cells in local disease sites of in TB pleural effusion and miliary TB patients. In the present study, we planned to observe the role of CXCR3 and CCR5 expressing T-cells in the bronchoalveolar lavage (BAL) fluid in pulmonary TB (PTB)-negative patients as well as normal lung and diseased lung in the same patients. Information in this direction would plausibly allow the development of therapeutic interventions that may be broadly applicable to large sets of chemokine receptors and the role of cytokines, thereby producing better clinical outcomes in complex inflammatory diseases like TB.
| Materials and Methods|| |
After obtaining approval from Institutional Ethics Committee (Ref: B-01:07/12/2005:A-65:12/08/2006), and written informed consents were taken from patients. This study includes 40 PTB (sputum smear-negative) patients (mean age 32 ± 12.43 years; 28 males and 12 females). Peripheral blood (PBL) was collected from 40 patients, and of 40 patients, BAL fluid from 10 patients was collected from diseased lung as well as the normal lung in the same patients as normal controls. All the patients responded by anti-TB treatment as per the RNTCP guidelines. Further, all the specimens were examined to exclude for the presence of M. tuberculosis, fungi, and malignancy. Details of inclusion and exclusion criteria are given as supplementary data (Text E1).
Pulmonary tuberculosis (smear negative)
PTB was diagnosed based on the following criteria: (a) presence of constitutional symptoms; (b) presence of parenchymal lesions at classical sites on chest radiographs and/or on computed tomography (CT) films; (c) demonstration of M. tuberculosis by smear, culture (Lowenstein–Jensen or BACTEC 460), or polymerase chain reaction in the BAL fluid; and (d) a satisfactory response to anti-TB treatment. While the diagnosis of TB was confirmed in 31 patients, nine patients showed satisfactory response to anti-TB treatment. For controls, another unaffected lungs (chest radiograph and CT scan confirmed) of PTB-positive patients were considered. BAL sample was collected carefully from both the lungs (separately) to avoid any contaminations. Percentage of CD3+ T-lymphocytes was satisfactory, confirmed by quick fluorescence-activated cell sorting (FACS) staining.
Serological test for HIV infection
All patients underwent to exclude HIV infection using the enzyme-linked immunosorbent assays (Genelavia-Mixt; Sanofi Diagnostics Pasteur and Genetic System Corporation).
Cell isolation and immunophenotyping
Ten milliliters of PBL was collected from PTB (sputum smear-negative) patients in heparinized vacutainers (BD Bioscience, USA). PBL mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and suspended in complete RPMI-1640 (Caisson Laboratories, USA) supplemented with 2 mM glutamine (Sigma, USA), HEPES (Sigma, USA), antibiotics (Biological Industries, Israel), and 10% heat-inactivated fetal calf serum (Biological Industries, Israel) as per previously published protocol from the laboratory. The viability of the cells was measured by a trypan blue exclusion test and was more than 98%.
Surface and intracellular cytokine staining
Cell surface staining was done using anti-CXCR3, CCR5, and CD11a with conjugated monoclonal antibodies (mAbs, BD Biosciences, USA). For intracellular cytokines, cells were cultured (0.5 × 106 cells/ml)in vitro in the presence of phorbol myristate acetate (PMA, 1 ng/ml, Sigma, USA), ionomycin (2 mM, Sigma, USA), and monensin (1 μM, Sigma, USA) for 6 h and M. tuberculosis antigen whole cell lysate (H37RV obtained from JALMA, Agra, India) at dose of 20 μg/ml after optimizing the dose titration was done for 24 h at 5% CO2 at 37°C. Intracellular staining was performed with anti-IFN-γ and anti-IL-4 mAbs (BD Biosciences, USA). The stained cells were acquired on BD flow cytometer BD FACS Calibur (BD Biosciences, USA).
Individual patients' consent form, clinical details, laboratory parameters, and experimental results were recorded on a predesigned pro forma, and subsequent data were managed on excel spreadsheet. Statistical significance of the results was determined with the program with SPSS version 11.5 (IBM Corp. Armonk, NY) and GraphPad Prism V. 5.03 software (La-Jolla California, USA). The analysis was performed using independent sample t-test, paired sample Student's t-test, Mann–Whitney test and Wilcoxon signed-rank test. A P < 0.05 was considered as statically significant. Experimental data were analyzed by Flow Jo Software (Tree Star Inc., Ashland, OR, USA).
| Results|| |
Overexpression of CXCR3+CCR5+, CXCR3+, and CCR5+ T-cells in bronchoalveolar lavage (n = 10) compared to peripheral blood in pulmonary tuberculosis-negative (n = 40)
To determine the frequency of effector T-cells on the phenotypic basis of expression of CXCR3+ and CCR5+, we performed FACS analysis of the lymphocytes derived from PBL and BAL fluid from patients with PTB negative. We observed a significant increase (P = 0.002, 19 folds) in the levels of CXCR3+ CCR5+ T-cells in the BAL in comparison to that of PBL [Figure 1]a. We also observed a significant increase (P = 0.002, 1.4 folds) in the levels of CXCR3+ T-cells [Figure 1]b and CCR5+ T-cells (P = 0.002, 6.7 folds) in BAL of PTB negative as compared to PBL [Figure 1]c.
|Figure 1: Overexpression of CXCR3+CCR5+, CXCR3+, and CCR5+ T-cells in bronchoalveolar lavage (n = 10) of pulmonary tuberculosis negative over peripheral blood (n = 40). (a) Frequency of CXCR3+CCR5+T-cells was significant increased (P = 0.002, 19 folds) at the local site of pathology (bronchoalveolar lavage) as compared to peripheral blood of same patients. (b) Frequency of CXCR3+T-cells was significantly increased (P = 0.002, 1.4 folds) in bronchoalveolar lavage as compared to autologous peripheral blood. (c) Overexpression of CCR5+T-cells was observed significantly higher (P = 0.002, 6.7 folds) in bronchoalveolar lavage as compared to peripheral blood. Horizontal bars represent the median values|
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In vivo overexpression of CXCR3+CCR5+, CXCR3+, and CCR5+ T-cells in bronchoalveolar lavage fluid of diseased lung versus normal lung in pulmonary tuberculosis negative (n = 10)
After observing the enrichment of CXCR3+CCR5+ T-cells at disease site (BAL) in comparison to PBL, next, we sought to gain insight in the selective enrichment of these highly polarized T-cells were higher in both affected lung (diseased) & unaffected (normal) lung in comparison to PBL. [Figure 2]a shows the representative plot. Interestingly, we observed significant increase in the frequency of CXCR3+ CCR5+ T-cells (P = 0.02, 3.1 folds), CXCR3+ (P = 0.034, 2.6 folds) and CCR5+ T-cells (P = 0.039, 8.6 folds) in BAL of disease lung (affected lung) as compared to normal lung (unaffected lung) [Figure 2]b. This observation provides clear evidence toward the homing of highly activated distinct phenotype-bearing T-cells indicative of immune focus at the diseased lung compared to the normal lung.
|Figure 2: Overexpression of CXCR3+CCR5+, CXCR3+, and CCR5+ T-cells in bronchoalveolar lavage fluid of normal (unaffected) versus diseased (affected) lung in pulmonary tuberculosis negative (n = 10). (a) Representative fluorescence-activated cell sorting plot showing frequency of CXCR3+CCR5+ T-cells among gated lymphocytes isolated from bronchoalveolar lavage of pulmonary tuberculosis-negative patient. Increased frequency of CXCR3+CCR5+ T-cells (2.9% vs. 9.1%) was observed. (b) Significant increase in frequency of CXCR3+CCR5+ T-cells (P = 0.02, 3.1 folds), CXCR3+(P = 0.03, 2.6 folds), and CCR5+ T-cells (P = 0.04, 8.6 folds) was observed, respectively. Data are shown as median with standard deviation bars|
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Overexpression of CXCR3+CD11ahigh and CCR5+CD11ahigh T-cells in bronchoalveolar lavage (n = 10) over peripheral blood in pulmonary tuberculosis negative (n = 40)
After demonstrating the selective enrichment of CXCR3 CCR5 dual- and single-positive T-cells at the local disease site in PTB patients, next, we sought to examine the expression profile of adhesion molecule which could potentially play a crucial role in the preferential localization of these highly polarized T-cells at the pathologic site. We observed a significant (P = 0.002, 3.7 folds) increment of CXCR3+ CD11ahigh T-cells [Figure 3]a and CCR5+ CD11ahigh T-cells (P = 0.002, 3.4 folds) [Figure 3]b in BAL over PBL in PTB negative. Therefore, our data observed that CD11ahigh T-cells could be playing an important role in the enrichment of these T-cells at local disease site in PTB.
|Figure 3: Overexpression of CXCR3+CD11ahigh and CCR5+CD11ahigh T-cells in bronchoalveolar lavage (n = 10) of pulmonary tuberculosis negative over peripheral blood (n = 40). (a) Cumulative frequency of CXCR3+CD11ahigh T-cells in bronchoalveolar lavage and peripheral blood of pulmonary tuberculosis negative showing a significant increase (P = 0.002, 3.7 folds) was observed at local disease site (bronchoalveolar lavage) as compared to peripheral blood in same patients. (b) Cumulative expression of CCR5+CD11ahigh T-cells was significantly increased (P = 0.002, 3.4 folds) in bronchoalveolar lavage as compared to peripheral blood of same patients. Horizontal bars represent the median values|
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Overexpression of CXCR3+CD11ahigh and CCR5+CD11ahigh T-cells in bronchoalveolar lavage of affected versus unaffected lung in pulmonary tuberculosis negative (n = 10)
Next, we sought to evaluate expression profile of CXCR3, CCR5, and CD11a in affected (disease) versus unaffected (normal) lung in PTB negative, we provided the representative plot [Figure 4]a. Overall, we observed a significant increase in the frequency of CXCR3+ CD11ahigh T-cells (P = 0.05, 2.5 folds) and also in CCR5+ CD11ahigh T-cells (P = 0.05, 5.6 folds) [Figure 4]b as compared to the normal lung in the same patients. Therefore, our findings demonstrate that CD11ahigh T-cells plays an important role in the homing of CXCR3+ and CCR5+ T-cells to the pathologic site.
|Figure 4: Overexpression of CXCR3+CD11ahigh and CCR5+CD11ahigh T-cells in bronchoalveolar lavage fluid of affected versus unaffected lung in pulmonary tuberculosis negative (n = 10). (a) Increased frequency of CXCR3+CD11ahigh T-cells in the affected lung (67.8%) versus unaffected lung (0.9%). Further, increased frequency of CCR5+CD11ahigh T-cells in the affected lung (32%) versus unaffected lung (4.2%). (b) Increase (P = 0.05, 2.5 folds) in the frequency of CXCR3+CD11ahigh T-cells was observed in disease lung versus normal lung. In case of CCR5+CD11ahigh T-cells, also increase (P = 0.05, 5.6 folds) in the frequency was observed in disease lung against normal lung|
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Mycobacterium tuberculosis antigen-specific intracellular interferon-gamma, interleukin-4, and tumor necrosis factor-alpha production by CD3+T-cells in bronchoalveolar lavage (n = 10) over peripheral blood of pulmonary tuberculosis negative (n = 40)
To gain insights into the functional ability of these effector T-cells, we evaluated their intracellular cytokine production. The ability of the PBL and BAL PTB-negative-derived cells to produce specific cytokines was evaluated by FACS-based intracellular cytokine assay following stimulation with PMA and M. tuberculosis antigen. We observed a significant decrease (P = 0.028, 5.0 folds) in the IFN-γ level, whereas IL-4 level was significantly increased (P = 0.028, 11.8 folds) in BAL as compared to PBL [Figure 5]a, in fact, possibly tilting of the Th1 and Th2 type of immune response at the site of pathology dictates containment and progression of disease. Possibly in more disease progression, there is shifting of Th1 toward the Th2 type of immune reaction. A significant increase of tumor necrosis factor (TNF)-α in BAL of PTB-negative patients was observed (P = 0.031, 5.9 folds) as compared to PBL [Figure 5]b.
|Figure 5: Mycobacterium tuberculosis antigen-specific intracellular interferon-gamma and interleukin-4 production by CD3+T-cells in bronchoalveolar lavage (n = 10) over peripheral blood (n = 40) in pulmonary tuberculosis negative. (a) CD3+T-cells gated, interferon-gamma level was decrease (P = 0.028, 5.0 folds) in observed in bronchoalveolar lavage as compared to peripheral blood, whereas interleukin-4 level was increased (P = 0.028, 11.8 folds). (b) CD3+T-cells gated, in bronchoalveolar lavage, the level of interferon-gamma was also significantly increased (P = 0.031, 5.9 folds) as compared to peripheral blood. Horizontal bars represent median values|
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| Discussion|| |
Immunopathogenesis of M. tuberculosis is a complex phenomenon, and it has been repeatedly reported that host immunity in the presentation of Th1 effector immune response is critical for containment and dissemination of TB infection.,, Understanding the mechanism of immunopathogenesis of chronic inflammatory diseases like TB in terms of local immune responses is more important than that in the peripheral compartment and is the hallmark. The local immune response of inflammatory diseases involves the generation of cytokine producing polarized Th1-like effector T-cells and their preferential recruitment under the influence of host tissue chemokine and their receptors response induced at the pathologic sites. In various inflammatory and autoimmune diseases, the important role of the Th1 type of chemokine receptors and INF-γ-producing T-cells has been reported.
Despite recent advances in the immunology of various diseases, immunological understanding of varied clinical manifestation and progression of TB disease remains fragmentary. Several recent studies have highlighted the tight correlation between Th1/Th2-like effector responses in the containment of TB disease., Selective trafficking of effector T-cells driven by the chemokines and other homing molecules decides the type and magnitude of the T-cell migration at the disease sites and thereby dictates the immunopathology and clinical manifestation of human TB., The dynamics of chemokine receptor expression during T-cell activation is thought to play a critical role in shaping the immune response because these are primary molecules responsible for the trafficking of the cells with defined phenotype. Chemokine receptors, CXCR3 and CCR5, are associated with the recruitment of effector T-cells with Th1-like immune response.,,
Lin et al., 2006 in their study on early events in M. tuberculosis infection in cynomolgus macaques demonstrated caseous granuloma in the lungs at early time points, indicating a rigorous initial response. Authors demonstrated localization of T-cells (CD 29 and CD69) and chemokine receptors (CXCR3 and CCR5) expression to different anatomic sites. Further, they demonstrated the priming of M. tuberculosis antigen-specific T-cells characterized by the production of IFN-γ only after 4 weeks of infection. Although these responses were observed from T-lymphocytes in the blood, airways, and hilar lymph nodes, they were predominantly localized to the site of infection. This was a landmark study demonstrating that increased expression of chemokine receptor CXCR3 and CCR5 on T-cells is associated with the progression of disease enabling them to speculate that localization of CXCR3 and CCR5-bearing T-lymphocytes is important to process in the pathogenesis of tuberculosis. Based on the above discussion, it can be inferred that the role of chemokine receptors, CXCR3 and CCR5, is well documented in various disease models, whereas in human PTB reports are scarce. Recently, it was demonstrated the hierarchic role of CXCR3+ and CCR5+ effector T-cell recruitment among patients with TB pleural effusion and MTB patients., Involvement of other homing receptors such as CD11a and CCR5 has also been documented by the same author.
In the present study, we extend our study in the pathogenesis of PTB in humans. The present study is an attempt to understand the local effector's immunity in human PTB in terms of phenotypic identity, their functional polarization, and their homing markers recruitment with respect to the TB infection and ability to produce intracellular cytokine which is out most important for immunity to control TB infection.
Our results demonstrate the predominant enrichment of CXCR3+ CCR5+ T-cells in the pathologic site samples (BAL) of PTB patients. In light of previously reported studies, this indicates that these chemokine receptors are involved in preferential trafficking of T-cells to the pathologic milieu in human PTB. Moreover, present data also indicate that on the part of the host, dual-positive (CCR5+ CXCR3+) effectors T-cells infiltrate to the pathologic site of PTB. Enrichment of dual-positive T-cells (CCR5+ CXCR3+) occurs in the local disease milieu in PTB along with that of CCR5+ and CXCR3+ single-positive cells when compared to the PBL. It is well known that BAL fluid of PTB patients represents strong inflammatory milieu containing an abundance of immune activation cytokines such as IFN-γ. We also thought to evaluate intracellular TNF-α production, which is a critical component in anti-bacterial, protective, and the inflammatory immune response to M. tuberculosis by lymphocytes derived from PBL and BAL of PTB-negative patients. It is already reported that in the process of containment of M. tuberculosis infection, TNF-α seems to have a synergistic action with IFN-γ, stimulating the production of reactive nitrogen intermediates, thus mediating the anti- TB immune function of macrophages contributing to granuloma formation, which turns the disease localization. However, it has also reported that surprisingly, in treatment of patients, for example, those with rheumatoid arthritis with biological agents interfering with TNF-α activity have shown an increased risk of reactivating TB. However, conceivably, TNF-targeting biological agents can be developed that because of their particular mode of action, and their specific pharmacodynamics may be less likely to have this side effect. Our result showed a significant increase in TNF-α in BAL as compared to PBL in PTB-negative patients. It may confirm that TNF-α plays an important role at the site infection in PTB.,
PTB is a disease entity where definitive damage to the host lung is observed and such damage is believed to result from the local immune-inflammatory response. Therefore, we studied diseased as well as a normal lung for better understanding of polarization Th1 effector cells. It was evident from the results of this study that in PTB, a definitive enrichment of CCR5+ CXCR3+ (both dual as well as single positive) effector T-cells occurs particularly in the disease (affected) as compared to normal (unaffected) lungs. This supports a distinct role of effector T-cells and chemokine receptors in their focused homing to the pathologic sites of PTB. This study also indicates that CD11a probably plays a crucial role in the selective recruitment of T-cells in this form of TB.
Therefore, it is concluded that distinct effector T-cells with definitive phenotype and chemokine receptor expression actively infiltrate the pathologic sites of PTB. Although the precise immunologic basis for this variation of local immunity and thus in disease manifestation still remains poorly understood. In the present study, an attempt was made to unravel the cellular and functional basis for variation in the local immune response in human PTB. Taken together, all these findings point toward a conclusive enrichment of CXCR3, CCR5 dual-positive and single-positive effector T-cells, as well as homing molecule CD11a at the pathological site over PBL in PTB patients.
We also conclude that in spite of significant enrichment of CXCR3+ CCR5+-expressing Th1 cells with the ability to produce inflammatory cytokine at the local disease site such as BAL in PTB patients, they are not able to control the TB immune pathogenesis due to the coexistence of T-reg cells. Our study also suggests that CD11ahigh T-cells are playing an important role in the recruitment of these T-cells. Therefore, our data provide insight into the functionality of effector T-cells at the disease site in PTB patients.
We are primarily thankful to our patients who willingly participated in this study. Thankful to Dr. Bindu Dey, Advisor, Department of Biotechnology, Government of India for funding. We are also thankful to Dr. V M Katoch, (Ex-DG of ICMR), Government of India, for providing M. tuberculosis antigen whole cell lysate (H37RV) for this study. Many thanks and respect to Dr. D. K. Mittra, Prof. and Head, Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, for experimental and logistic support. We acknowledge the support of Dr. Sanjeev Sinha, Prof. Department of Medicine, All India Institute of Medical Sciences, New Delhi, for providing BAL sample for this experiment.
Financial support and sponsorship
Department of Medicine, All India Institute of Medical Sciences, New Delhi, India, supported the study.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]