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 Table of Contents  
Year : 2020  |  Volume : 9  |  Issue : 4  |  Page : 405-410

Investigating role of Mycobacterium tuberculosis secretory antigens in altering activation of T cell signaling events in Jurkat T cells

Department of Immunology, ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, Uttar Pradesh, India

Date of Submission10-Sep-2020
Date of Acceptance06-Oct-2020
Date of Web Publication15-Dec-2020

Correspondence Address:
Beenu Joshi
Department of Immunology, ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Dr. M. Miyazaki Marg, Tajganj, Agra - 282 004, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_172_20

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Background: Mycobacterium tuberculosis is able to survive and persist as an intracellular pathogen by modulating its own metabolism and host immunity. The molecules and mechanisms utilized to accomplish this modulation are not fully understood. The present study elucidates the effects of M. tuberculosis secretory antigens on T-cell-receptor (TCR)/CD28-triggered signaling in Jurkat T-cells. Method: In the present study, intracellular calcium mobilization was investigated in CD3-activated cells in response to M. tuberculosis antigens, Ag85A, early secretory antigenic target-6 (ESAT-6), and H37Rv. The activation of mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p-38 was also analyzed in CD3- and CD28-activated cells by western blotting. Results: Our results showed CD3-triggered modulations in free intracellular calcium levels in Jurkat T-cells in response to M. tuberculosis antigens. In addition, we also noted M. tuberculosis antigens induced downregulation in phosphorylation of ERK1/2 and p-38. Overall, our results proposed that M. tuberculosis secretory antigens, particularly ESAT-6, impede TCR/CD28-induced signaling events which could be responsible for T-cell unresponsiveness, implicated in the progression of disease. Conclusion: The present study demonstrated M. tuberculosis secretory antigens induced alteration of T-cell signaling pathways in unsensitized Jurkat T-cell line which might be implied in T-cell dysfunctioning during the progression of the disease.

Keywords: T cells, T cell receptor, IFN-γ, IL-2, Phosphorylation

How to cite this article:
Sharma B, Dua B, Joshi B. Investigating role of Mycobacterium tuberculosis secretory antigens in altering activation of T cell signaling events in Jurkat T cells. Int J Mycobacteriol 2020;9:405-10

How to cite this URL:
Sharma B, Dua B, Joshi B. Investigating role of Mycobacterium tuberculosis secretory antigens in altering activation of T cell signaling events in Jurkat T cells. Int J Mycobacteriol [serial online] 2020 [cited 2022 Jan 23];9:405-10. Available from: https://www.ijmyco.org/text.asp?2020/9/4/405/303446

  Introduction Top

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and remains an important global health problem. Its resurgence is promoted by a large population of immunocompromised human immunodeficiency virus (HIV)-infected persons and also by an alarming increase in the prevalence of drug-resistant strains of M. tuberculosis. Globally, there were an estimated 10.0 million new (incident) TB cases in 2017.[1] The influence of current efforts to moderate the global problem of TB is not sufficient and therefore improved diagnostic and therapeutic efforts need to be combined with additional preventive efforts, including the development of new vaccines. Bacille Calmette–Guérin (BCG) vaccine is the single available vaccine for TB and with variable efficacy.[2] Therefore, it is important to develop an improved vaccine against TB and it mainly depends on a comprehensive understanding of the host-pathogen interactions during M. tuberculosis infection. During infection with M. tuberculosis, innate mechanisms facilitate to control the bacillary spread but T-lymphocyte mobilization to the lung is needed to restrict the infection in granulomas.[3]

T-cell-mediated immunity amplifies macrophage capacities to kill and digest the bacilli.[4] Cell-mediated immunity regulated by Th1 cytokines is considered important to eradicate the intracellular pathogens, whereas Th2 response has been found to have an important role in clearing the extracellular infectious agents. Various T-cell subsets play important role in disease outcomes. Mononuclear cells (lymphocytes and monocytes) are responsible for both humoral and cellular immunity. Ahmed et al. recently evaluated the validity of the automated blood cell counter with the gold standard of a manual count.[5] CD4+ T-lymphocytes play a central regulatory role. The number of CD4+ T-cells in circulation provides significant information about the immune competence of an individual. The decrease in numbers of CD4+ T-cell can compromise the normal immune functions of the body. CD4+ lymphocytes, a subset of these cells may also influence the progression of immunologic diseases such as TB and HIV.[6] Recruitment of various effector T-cell subsets at the sites of TB pathology is assumed to play a vital role in translating the host immunity at the local disease sites. Saha et al. in 2019 studied the functionality of effector T-cells at the disease site and observed that distinct effector T-cells with definitive phenotype and chemokine receptor expression actively infiltrate the pathologic sites of PTB.[7] Other T-cell subsets also play a crucial role in TB and recently elevated levels of Treg cells and NKT cells were observed in the peripheral blood mononuclear cells in TB patients.[8]

The elucidation of the molecular mechanisms that regulate the production of cytokines by T-cells is therefore critical for the understanding of the pathogenesis of the diseases and the development of novel targeted therapeutic strategies. Host immune responses are known to target proteins that are secreted by M. tuberculosis; consequently, these proteins have been targeted for the development of vaccines and immunodiagnostics.

T-cell-receptor (TCR) signaling in response to antigen recognition has a central role in the adaptive immune response. TCR stimulation leads to profound changes in gene expression. T-cell responses to pathogens are regulated by T-cell receptor affinity, co-receptor engagement (e.g., CD28 and cytotoxic T-lymphocyte antigen-4), and cytokine receptor signaling. In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest.[9],[10] Mitogen-activated protein kinases (MAPKs) phosphorylate and activate downstream molecules, resulting in T-cell activation, proliferation, and differentiation into T-helper phenotypes.[11],[12] Although there is compelling evidence for the role of extracellular signal-regulated kinase (ERK) and p-38 protein kinases in the antimycobacterial activity of antigen-presenting cells,[13],[14] little is known about the contribution of these kinases to human T-lymphocyte responses to mycobacteria. Inhibition of CD4+ T-cell activation by cell wall glycolipids has been reported in antigen-specific murine CD4 + T-cells[15] and in primary human T-cells also[16]. It is also reported in a previous study that activation of p-38 and ERK mediates interferon-gamma (IFN-γ) production in T-cells in response to M. tuberculosis and defective p-38 and ERK activation in TB patients has been also reported in the same study[17]. Inhibition of early secretory antigenic target-6 (ESAT-6) mediated IFN-γ production through p-38 MAPK pathway has been also observed in T-cells from healthy individuals.[18] Recently, we have also reported alterations in TCR- and TCR/CD28-induced upstream and downstream signaling events of T-cell activation in purified protein derivative (PPD) +ve healthy individuals and TB patients[19]; therefore, the current study was done to decipher the effect of secretory M. tuberculosis antigens on signaling events leading to T-cell activation in unsensitized Jurkat T-cells, which could give us leads to understand the mechanism of T-cell activation. Our objective was to study calcium mobilization and activation of MAPKs in Jurkat T-cells in the presence or absence of M. tuberculosis antigens.

  Method Top

The study was performed on Jurkat T-cell leukemic line (E6.1) procured from the National Cell Repository, Pune, India. Cells were suspended in RPMI-1640 medium supplemented with 10% FBS (Hyclone, Utah, USA) with 2 mM L-glutamine and 1× antibiotic–antimycotic cocktail (Sigma, St. Loius, MO, USA). Cultures were maintained with 5% CO2 at 37°C in a humidified chamber. Experiments were performed with cell viability =95% as determined by Trypan blue exclusion test.

Chemicals and antigens

Ionomycin, goat anti-mouse-IgG (GAM), phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, anti-protease cocktail, and Bradford reagent were purchased from Sigma, St. Louis, MO, USA. Fura-2/AM was bought from Calbiochem, La Jolla, USA. Anti-human CD3 (clone HIT3-α) and anti-human CD28 (clone CD28.2) were procured from BD Biosciences, CA, USA. Phospho-ERK1/2 and phospho-p-38MAPK antibodies were from Cell Signaling Technology, MA, USA. Anti-Erk-2, β-actin, and peroxidase-conjugated goat anti-mouse/goat anti-rabbit secondary antibodies were purchased from Santacruz Technologies, CA, USA. Enhanced chemiluminescence (ECL) reagents were procured from Millipore, MA, USA.

Lyophilized M. tuberculosis antigens (Ag85A, ESAT-6, and whole cell lysate of H37Rv), used in this study, were provided by J. Belisle (Colorado State University, Denver, CO, USA) through a TB Research Materials and Vaccine Testing Contract (NIH Contract HHSN266200400091C/ADB Contract NOI-AI-40091). PPD (RT-49) was procured from Statens Serum Institute, Copenhagen, Denmark. PPD and whole cell lysate of H37Rv were used as positive antigen control. All antigens were dissolved in filtered phosphate-buffered saline (PBS) pH 7.4 to make 1 mg/ml concentration.

Measurement of Ca2+ mobilization

Jurkat T-cells (5 × 106/ml) were washed with PBS, pH 7.4. Cells were incubated with Fura-2/AM at 1 μM for 30 min at 37°C in loading buffer ([in mM]: NaCl, 110; KCl, 5.4; NaHCO3, 25; MgCl2, 0.8; KH2PO4, 0.4; HEPES, 20; Na2HPO4, 0.33; and CaCl2, 1.2. pH 7.4). After loading, cells were washed three times (500 × g for 5 min) and remained suspended in the identical buffer.[Ca2+]i was measured as reported elsewhere.[20],[21]. Fluorescence intensities were measured in ratio mode using Varian ECLIPSE spectrofluorometer equipped with fast filter accessory (Varian Incorporation, St. Helens, Australia) at 340 and 380 nm (excitation filters) and 510 nm (emission filter). Cells were stirred continuously throughout the experiment. Test molecules were added into the cuvettes in small volumes with no interruptions in recordings. The intracellular concentrations of free Ca2+ [Ca2+]i were calculated using the following equation: [Ca2+]i = Kd × (R - Rmin)/(Rmax - R) × (Sf2/Sb2). A value of 224nM for Kd was added into the calculations. Rmax and Rmin values were obtained by the addition of ionomycin (5%M) and MnCl2 (2 mM), respectively. All experiments were performed at 37°C.

Treatment of cells and western blot analysis of mitogen-activated protein kinase activation

Cells were stimulated as per Kim and White.[22] Briefly, 5 × 106/ml serum-starved Jurkat T-cells were stimulated or not with appropriate doses of M. tuberculosis antigens: 5 μg/ml Ag85A, 10 μg/ml of ESAT-6, 5 μg/ml PPD, and 5 μg/ml of H37Rv for 2 h, then stimulated or not with anti-CD3 antibodies (10 μg/ml) alone or with CD28 (5 μg/ml) at 4°C for 15 min. After washing once with chilled PBS, prewarmed PBS (37°C) containing GAM (5 μg/ml) was added, and cells were further incubated at 37°C for 10 min. Reaction was stopped with chilled PBS and Jurkat T-cells were lysed with 50 μl of buffer (HEPES, 20 mM pH 7.3; EDTA, 1 mM; EGTA, 1 mM; NaCl, 0.15 mM; Triton X-100, 1%; glycerol, 10%; PMSF, 1 mM; sodium orthovanadate, and 2 mM; anti-protease cocktail). After centrifugation (13,000 × g for 5 min), cell lysates were used immediately or stored at -20°C. The protein contents were determined with Bradford reagent. Denatured proteins (30 μg) were separated by SDS-PAGE (10%) and transferred to polyvinylidene difluoride (PVDF) membranes. Immunodetection of phosphorylated forms of ERK1/2 and p-38 MAPK was performed using 2 μg/ml of phospho-specific antibodies in tris-buffered saline (TBS) with 1% bovine serum albumin with overnight incubation at 4°C. After washing with TBS + 0.05% Tween-20, PVDF membranes were treated with peroxidase-conjugated goat anti-mouse/anti-rabbit secondary antibodies, and peroxidase activity was detected with ECL reagents. Equal loading of the proteins was confirmed after striping the blot and reprobing for total forms of Erk-2/β-actin. Densitometric analysis of bands was performed using Quantity OneTM software (Bio-Rad, Hercules, USA).

Statistical analysis

Data were analyzed using Graphpad Prism-5.0 software (San Diego, CA, USA) and mean ± standard error of mean (SEM) were calculated. Data comparisons were made using a Mann–Whitney nonparametric t-test and P < 0.05 was considered as statistically significant.

  Results Top

Lymphocyte transformation test for dose optimization of M. tuberculosis antigens

The concentration of antigens corresponding to the log phase of T-cell proliferation by lymphocyte transformation test (LTT) in Jurkat T-cells was considered as optimum working concentration for further experiments. Optimum working doses (5 μg/ml for Ag85A and H37Rv and 10 μg/ml for ESAT-6) were found to have stimulation indices in the log phase in T cells [Supplementary Figure 1]a, [Supplementary Figure 1]b, [Supplementary Figure 1]c. All the concentrations of antigens (2.5, 5, 7.5, 10, and 15 μg/ml) were used in LTT and the optimal dose of antigen found by LTT results was observed to show the maximum effect. The concentration of antigens also showed optimum proliferative index in LTT in peripheral blood mononuclear cells of healthy individuals.[19]

Increased T-cell-receptor triggered intracellular calcium mobilization in response to Mycobacterium tuberculosis antigens

We measured intracellular calcium concentration by spectrofluorometer to find the effect of M. tuberculosis antigens on intracellular calcium mobilization. We assessed TCR-triggered calcium mobilization by adding M. tuberculosis antigens (Ag85A, ESAT-6, PPD, and H37Rv) on T-cells after stimulation with anti-CD3 antibody. We observed that TCR-triggered calcium mobilization in T cells was significantly increased by Ag85A, ESAT-6, PPD, and H37Rv. The most enhanced transmembrane calcium mobilization was observed by PPD stimulation, followed by Ag85A, ESAT-6, and H37Rv [Figure 1]a. Thapsigargin (Tg) was used to further elucidate whether these antigens influence the opening of calcium channels as a result of internal store depletion. Significant inhibition by Ag85A, PPD, and H37Rv was noted in the refilling of the cytosolic stores from the extracellular environment [Figure 1]b.
Figure 1: M. tuberculosis antigens alter free intracellular calcium concentration in T cells. The experiments were performed on Fura-2AM loaded Jurkat T cells and fluorescence intensities were measured using Varian ECLIPSE spectrofluorometer. Bar diagrams show changes in [Ca2+ ]i in CD3 and Tg treated cells. (a) Effect of M. tuberculosis antigens on CD3 stimulated calcium influx, whereas (b) effect of M. tuberculosis antigens on Tg stimulated calcium mobilization in T-cells. Values in the histogram are mean ± SEM. *P < 0.05; **P < 0.005; ***, P < 0.0001. Tg: Thapsigargin, SEM: Standard error of mean, M. tuberculosis: Mycobacterium tuberculosis, [Ca2+]i: Free intracellular calcium concentrations

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Alteration in T-cell-receptor/CD28-induced mitogen-activated protein kinases activation in response to Mycobacterium tuberculosis antigens

We measured phosphorylation of ERK½ and p-38 by western blot to study whether M. tuberculosis antigens modulate TCR and TCR/CD28 induced MAPK activation. Activation of MAPKs was studied in Ag85A, ESAT-6, PPD, and H37Rv stimulated T-cells. Ag85A, ESAT-6, and PPD significantly reduced TCR and TCR/CD28 induced phosphorylation of ERK1/2. ESAT-6 curtailed the activation of ERK1/2 the most significantly, followed by Ag85A and PPD [Figure 2]. Phosphorylation of p-38 in TCR/CD28-induced cells was also analyzed after M. tuberculosis antigens stimulation. All antigens Ag85A, ESAT-6, PPD, and H37Rv reduced phosphorylation of p-38 but not significantly [Figure 3].
Figure 2: M. tuberculosis antigens inhibit TCR- and TCR/CD28-induced phosphorylation of ERK1/2 MAPK. Densitometric analysis of phosphorylation of ERK½. Arbitrary units of band densities values are expressed as mean ± SEM. Representative blots of independent experiments repeated at least three times are shown where Lane 1 - control, Lane 2 - anti-CD3 stimulated, Lane 3 - anti-CD3+ anti-CD28 stimulated, Lane 4 - anti-CD3+ anti-CD28 stimulated in presence of Ag85A, Lane 5 - anti-CD3+ anti-CD28 stimulated in presence of ESAT-6, Lane 6 - anti-CD3+ anti-CD28 stimulated in presence of PPD and Lane 7-anti-CD3+ anti-CD28 stimulated in presence of H37Rv. Densitometry was performed and the ratios of phosphorylated to β-Actin protein expression were expressed as arbitrary units. *P < 0.05, **P < 0.005; ***P < 0.001W by the Mann–Whitney U-test. ESAT-6: Early secretory antigenic target-6, SEM: Standard error of mean, MAPKs: Mitogen-activated protein kinases, TCR: T-cell-receptor, M. tuberculosis: Mycobacterium tuberculosis

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Figure 3: M. tuberculosis antigens inhibit TCR- and TCR/CD28-induced phosphorylation of p-38 MAPK. Densitometric analysis of phosphorylation of p-38. Arbitrary units of band densities values are expressed as mean ± SEM. Representative blots of independent experiments repeated at least three times are shown where Lane 1 - control, Lane 2 - anti-CD3 stimulated, Lane 3 - anti-CD3+ anti-CD28 stimulated, Lane 4 - anti-CD3+ anti-CD28 stimulated in presence of Ag85A, Lane 5 - anti-CD3+ anti-CD28 sWtimulated in presence of ESAT-6, Lane 6 - anti-CD3+ anti-CD28 stimulated in presence of PPD and Lane 7 - anti-CD3+ anti-CD28 stimulated in presence of H37Rv. Densitometry was performed, and the ratios of phosphorylated to β-Actin protein expression were expressed as arbitrary units. ESAT-6: Early secretory antigenic target-6, SEM: Standard error of mean, MAPKs: Mitogen-activated protein kinases, TCR: T-cell-receptor, M. tuberculosis: Mycobacterium tuberculosis

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  Discussion Top

The pathogenesis of TB is driven by a complex interplay between the host immune system and the survival strategies of the bacterium. The inflammatory response to M. tuberculosis infection is tightly regulated by both the host and the bacterium and protection against TB is based on cell-mediated immune responses. Appropriate T-cell activation is vital for the effective cell-mediated immune response against M. tuberculosis. Therefore, the present study was carried out to explore the effect of secretory antigens of M. tuberculosis on T-cell activation and their role in the modulation of T-cell physiology. In the present study, we investigated calcium mobilization and activation of MAPKs which are important regulatory signal-transduction stages of T-cell activation in Jurkat T-cells in the presence or absence of M. tuberculosis antigens.

Intracellular second messenger, calcium has vital physiological roles in muscle contraction, synaptic transmission and plasticity, cell motility, fertilization, cell growth, cell proliferation, and gene expression.[23] Our investigation on the effect of secretory M. tuberculosis antigens on this downstream second messenger of T-cell activation showed that Ag85A and ESAT-6 both significantly increased TCR-triggered calcium influx. Furthermore, we found that mycobacterial antigens Ag85A, PPD, and H37Rv inhibit store-operated calcium channels by inhibiting Tg stimulated Ca2+ release in cytosol from intracellular stores (ER and Golgi body), which is vital to mediate transmembrane calcium mobilization through plasma membrane-associated CRAC channels and hence the influx of calcium is reduced. On the other hand, ESAT-6 increased Tg mediated calcium release, but it was not significant. The present study confirms that mycobacterial antigens, Ag85A, ESAT-6, PPD, and H37Rv, altered calcium signaling and consequently might play a critical role in the pathogenesis of the disease. Altered calcium signaling and other signaling events have been earlier reported in leprosy patients and T-cell lines and modulation by lipid and soluble fraction of Mycobacterium leprae lysates were shown which could play a major role in the pathogenesis.[24-26] Curtailment of [Ca2+]i levels by M. tuberculosis in macrophages has been shown which could be linked with reduced phagosome-lysosome fusion, thus for increased survival of mycobacteria.[27] Talreja et al. reported intracellular calcium modulation by H37Rv in newly diagnosed and treated TB patients and PPD +ve and PPD -ve healthy controls.[28] In our previous study, we observed that ESAT-6 significantly decreased TCR triggered intracellular calcium levels in PPD +ve healthy individuals, but it was increased by Ag85A, PPD, and H37Rv, though it was not statistically significant, Ag85A, ESAT-6, PPD, and H37Rv significantly curtailed calcium mobilization in pulmonary TB patients[19]. On the contrary, it has been previously reported by Wang et al. that ESAT-6 had no effect on phosphorylation of ZAP-70 and intracellular calcium levels.[29] MAPK pathways regulate Th1 development[30] and could mediate M. tuberculosis-induced IFN-γ production. The importance of the MAPKs in controlling many aspects of immune-mediated inflammatory responses has made it a priority for research related to many human diseases. Here, we observed that ESAT-6 significantly curtailed TCR/CD28 induced phosphorylation of ERK1/2 in T-cells. We also observed reduced activation of p-38 in TCR/CD28 induced T-cells after treatment with M. tuberculosis antigens, though it was not significant. We have previously reported that activation of ERK1/2 and p-38 was curtailed by M. tuberculosis antigens in TB patients, whereas inhibition of only ERK1/2 not p-38 phosphorylation was observed in PPD +ve healthy individuals,[19] which is similar to our findings of the present study with Jurkat T-cells. Recently, Pasquinelli et al. (2013) observed that IFN-γ production is regulated by ERK1/2 and p-38 MAPK signaling pathways in TB patients, and it also involves CREB activation.[17] Peng et al. (20111) reported that ESAT-6 induced inhibition of IFN-γ by activation of p-38 MAPK activity in T-cells but did not affect the activation of ERK1/2 or JNK.[18] Further, reduced TCR-mediated activation of ERK1/2 and p-38 MAPKs has also been observed in T-cell lines after stimulation with M. leprae antigens.[25-26] Previously, the inhibitory effect of ESAT-6 in macrophage signaling pathways particularly the ERK1/2 MAPK pathway has also been reported.[31]

Our previous study has indicated that M. tuberculosis secretory antigens downregulate TCR mediated calcium signaling and MAPKs activation in PPD + ve healthy individuals and TB patients.[19] Interestingly, the present study using unsensitized Jurkat T-cells also corroborates the finding with downregulated TCR-mediated MAPKs activation of Jurkat T-cells. Our study indicates that M. tuberculosis has the ability to modulate host's immune system for its own survival using several approaches besides what is formerly investigated. Although prior research on the T-cell activation mechanism in TB has intensified our knowledge about protective immune responses in TB up to some extent, still few critical questions are unanswered. Research into the development of TB vaccines and immunodiagnostics has focused on the proteins released by M. tuberculosis because these antigens are thought to induce protective cell-mediated immunity and immune responses of diagnostic value. Ag85A and ESAT-6 are widely studied for their potential to trigger effective host immune responses against TB, but very little is known regarding their role in the T-cells signaling mechanisms underlying pro-inflammatory cytokine secretion by T-cells.

  Conclusion Top

Our data demonstrate that the stimulation of unmodulated T-cells of Jurkat T-cell line with the Ag85A and ESAT-6 antigens of M. tuberculosis markedly downregulated TCR-mediated activation of T-cells by affecting upstream and downstream signaling events. Altogether, our findings establish that secretory M. tuberculosis antigens preferentially inhibit TCR-mediated downstream signaling events. This raises the possibility that these signal transduction pathways may be used as targets for the screening and development of therapeutics of mycobacterial diseases.

Further study of the dysfunctional activation of T-cells during the progression of TB could identify new targets to increase protective immune response and improve patient outcomes particularly in those with drug-resistant disease. The understanding of cell-mediated immune responses to M. tuberculosis may facilitate the evaluation of the efficacy of new anti-TB vaccines. Further studies to decipher the pathways by which secretory M. tuberculosis antigens mediate these effects will provide insight into the mechanisms for pathogen-mediated inhibition of T-cell responses and facilitate the development of immunomodulatory therapy to reverse these dysfunctioning effects.

Additional file 1: Figure S1(A-C). Lymphocyte transformation test for dose optimisation of Ag85A, ESAT-6 and H37Rv by using Jurkat T cells by H3-thymidine uptake assay. Lymphoproliferative responses of T cells were calculated using different concentration (2.5, 5, 7.5, 10, 15, μg/ml) of antigens. Bar diagram showing mean ± SEM of stimulation indices (S.I) of stimulated T cells with different doses of A85A, ESAT-6 and H37Rv.

S.I was calculated according to the formula:


IFN-γ: Interferon gamma; ESAT-6 Early secretory antigenic target -6, PPD- Purified protein derivative; [Ca+2]i: Free intracellular calcium concentrations.

Conflicts of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Authors' contributions

BS conceived, executed study and performed all the experiments, analysed data and wrote manuscript BD helped experiments and data analysis and BJ* conceived and executed the study. All authors read and approved the final manuscript.

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  [Figure 1], [Figure 2], [Figure 3]


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