Mycobacterium tuberculosis secretory proteins downregulate T cell activation by interfering with proximal and downstream T cell signalling events

Abstract

Background: Mycobacterium tuberculosis (M. tuberculosis) modulates host immune response, mainly T cell responses for its own survival leading to disease or latent infection. The molecules and mechanisms utilized to accomplish immune subversion by M. tuberculosis are not fully understood. Understanding the molecular mechanism of T cell response to M. tuberculosis is important for development of efficacious vaccine against TB.

Methods: Here, we investigated effect of M. tuberculosis antigens Ag85A and ESAT-6 on T cell signalling events in CD3/CD28 induced Peripheral blood mononuclear cells (PBMCs) of PPD+ve healthy individuals and pulmonary TB patients. We studied CD3 induced intracellular calcium mobilization in PBMCs of healthy individuals and TB patients by spectrofluorimetry, CD3 and CD28 induced activation of mitogen activated protein kinases (MAPKs) in PBMCs of healthy individuals and TB patients by western blotting and binding of transcription factors NFAT and NFκB by Electrophorectic mobility shift assay (EMSA).

Results: We observed CD3 triggered modulations in free intracellular calcium concentrations in PPD+ve healthy individuals and pulmonary TB patients after the treatment of M. tuberculosis antigens. As regards the downstream signalling events, phosphorylation of MAPKs, Extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 was curtailed by M. tuberculosis antigens in TB patients whereas, in PPD+ve healthy individuals only ERK1/2 phosphorylation was inhibited. Besides, the terminal signalling events like binding of transcription factors NFAT and NFκB was also altered by M. tuberculosis antigens. Altogether, our results suggest that M. tuberculosis antigens, specifically ESAT-6, interfere with TCR/CD28-induced upstream as well as downstream signalling events which might be responsible for defective IL-2 production which further contributed in T-cell unresponsiveness, implicated in the progression of disease.

Conclusion: To the best of our knowledge, this is the first study to investigate effect of Ag85A and ESAT-6 on TCR- and TCR/CD28- induced upstream and downstream signalling events of T-cell activation in TB patients. This study showed the effect of secretory antigens of M. tuberculosis in the modulation of T cell signalling pathways. This inflection is accomplished by altering the proximal and distal events of signalling cascade which could be involved in T-cell dysfunctioning during the progression of the disease.

Fig. 1

M. tuberculosis antigens alter free intracellular calcium concentration in PPD+ve healthy individuals and pulmonary TB patients. Peripheral blood mononuclear cells (PBMCs) from 13 PPD+ve healthy donors and five pulmonary TB patients were stimulated with M. tuberculosis antigens for 4 h. Fluorescence intensities were measured in ratio mode using Varian ECLIPSE spectrofluorometer as mentioned in material and methods. Bar diagrams show changes in [Ca2+]i in anti CD₃ and ionomycin treated cells of (a) PPD+ve healthy individuals (b) Pulmonary TB patients and values in histogram are mean ± SEM. *, P < 0.05; **, P <0.005

Fig. 2

M. tuberculosis antigens modulate TCR- and TCR/CD28-induced phosphorylation of ERK1/2 in PPD+ve healthy individuals and pulmonary TB patients. (a) Peripheral blood mononuclear cells (PBMCs) from six PPD+ve healthy donors and 12 pulmonary TB patients were stimulated with M. tuberculosis antigens for 4 h. Cells were then activated with anti CD3 and anti CD28 antibodies (as mentioned in methodology). Phosphorylated ERK ½, total ERK (p- ERK ½ and tERK, respectively) and β- Actin expression were determined by western blot. One representative blot of healthy individual and TB patient is 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. (b) Densitometry was performed, and the ratios of phosphorylated to total protein expression were expressed as arbitrary units. *P < .05, ** P <0.005; *** P <0.005 by the Mann–Whitney U

Fig. 3

M. tuberculosis antigens modulate TCR- and TCR/CD28-induced p38 phosphorylation in PPD+ve healthy individuals and pulmonary TB patients. (a) Peripheral blood mononuclear cells (PBMCs) from six PPD + ve healthy donors and 12 pulmonary TB patients were stimulated with M. tuberculosis antigens for 4 h. Cells were then activated with anti CD3 and anti CD28 antibodies (as mentioned in methodology). Phosphorylated p38, total p-38 (p- p38 and tp38, respectively) and β- Actin expression were determined by western blot. One representative blot of each study case is 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. (b) Densitometry was performed, and the ratios of phosphorylated to total protein expression were expressed as arbitrary units. *P < .05, ** P <0.005; *** P <0.005 by the Mann–Whitney U test

Fig. 4

M. tuberculosis antigens modulate TCR- and TCR/CD28-induced DNA binding affinity of NFκB to the promoter of IL-2 cytokine in PPD+ve healthy individuals and pulmonary TB patients. (a) Peripheral blood mononuclear cells (PBMCs) from 11 PPD+ve healthy donors and 10 pulmonary TB patients were stimulated with M. tuberculosis antigens for 4 h. Cells were then activated with anti CD3 and anti CD28 antibodies (as mentioned in methodology). Nuclear lysates were prepared and binding affinity of NFκB was determined by EMSA. One representative blot of each study case is 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. (b) Densitometry was performed, Histograms represent mean band intensities. *P < .05, ** P <0.005; *** P <0.005 by the Mann–Whitney U test

Fig. 5

M. tuberculosis antigens modulate TCR- and TCR/CD28-induced DNA binding of NFAT to the promoter of IL-2 cytokine in PPD+ve healthy individuals and pulmonary TB patients. (a) Peripheral blood mononuclear cells (PBMCs) from 11 PPD+ve healthy donors and 10 pulmonary TB patients were stimulated with M. tuberculosis antigens for 4 h. Cells were then activated with anti CD3 and anti CD28 antibodies (as mentioned in methodology). Nuclear lysates were prepared and binding affinity of NFAT was determined by EMSA. One representative example of each study case is 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. (b) Densitometry was performed, Histograms represent mean band intensities. *P < .05,** P <0.005; *** P <0.005 by the Mann–Whitney U test