Background

Human tuberculosis (TB) is a contagious infection caused by an intracellular pathogen, Mycobacterium tuberculosis. Due to lack of accurate diagnostic techniques and vaccines, TB has become the second leading cause of death from an infectious disease worldwide and it was declared as ‘global emergency’ by WHO in 1993. This deadly infection leads to 8.5–9.2 million new TB cases with 1.1 million deaths annually; more over 2 billion people are latently infected with M. tuberculosis[1]. This situation is further complicated by the emergence of multidrug-resistant strains of bacteria and HIV co-infection.

Cell mediated immune response includes different T cells (i.e. CD4+, CD8+ and γ/δ T cells) and T cell derived cytokines play a pivotal role in the protection against M. tuberculosis[2, 3]. Among the existing cytokines, IFN-γ is a key cytokine in the control of M. tuberculosis infection, and was confirmed by using IFN-γ knockout mice, which were more susceptible to virulent M. tuberculosis[4]. Individuals defective in genes for IFN-γ or the IFN-γ receptor are prone to serious mycobacterial infections including M. tuberculosis[5]. IFN-γ is predominantly secreted by T cells, as well as Natural Killer (NK) cells which play an important role in macrophage activation which in turn will contain and kill intracellular mycobacteria [6], control mycobacteria replication and granuloma formation both in humans and mice [7].

The ability of T cells to proliferate in response to antigen has been used as an indicator for the presence of antigen-specific T cells. Thus, measuring T cell functions in terms of lymphocyte proliferation and quantification of IFN-γ, in vitro, against M. tuberculosis specific antigens are useful immunological markers, particularly phase I and II vaccine trials and the diagnosis of tuberculosis [8].

Two different assay methods exist to analyze the T-lymphocyte functions, namely Peripheral Blood Mononuclear Cell (PBMC) assays and Whole Blood (WB) assays. The conventional PBMC assays require large volume of blood samples when compared to WB assays. Thus, WB assays gain importance in screening of large number of antigens with the same blood sample; and paediatric and field studies. So, we compared WB assay with PBMC assay to know whether WB assays can be used as an alternative or not.

In this study, we compared the lymphocyte proliferation and IFN-γ levels in 1:5 and 1:10 diluted blood and PBMC against MPT51 recombinant antigen of M. tuberculosis, Purified Protein Derivative (PPD) and Phytohemagglutinin (PHA).

Study subjects

The study was approved by the Institutional Ethics committee of National Institute for Research in Tuberculosis (NIRT) and written consent was obtained from the volunteers before recruitment into the study. Eight healthy laboratory volunteers were recruited in the present study from NIRT (formerly Tuberculosis Research Centre), Chennai, India. These individuals had no history of tuberculosis on the basis of personal history; physical examination, chest x-ray, and negative acid fast bacilli sputum smear microscopy. All the study subjects were TST positive. All participants aged from 25 to 45 years. All subjects were HIV negative as determined by Tridot (J.Mitra & co, India) and Retroquic (Qualprodiagnostics, India) assays with serum.

Antigens and mitogens

M. tuberculosis PPD was obtained from Statens Serum Institute, PHA was purchased from Sigma Chemical Company, MO, USA and MPT51 recombinant protein was cloned, over expressed and purified as described previously [9].

Lymphocyte proliferation assay

Briefly, 10 ml of heparinized venous blood was drawn from each study subject. For WB assay, 1:5 and 1:10 dilutions were made with sterile RPMI 1640 medium (Sigma Chemical Company, MO, USA), supplemented with penicillin (100 IU/ml), streptomycin (0.1 mg/ml), L-glutamine (0.29 gm/l) and amphotericin B (5 mg/ml) and was seeded in 96-well flat bottom plates at 200 μl/well.

PBMC were isolated by Ficoll-Hypaque density centrifugation. A total of 2x10⁵cells/well were cultivated in complete culture medium, supplemented with 10% Human AB serum. Cultures were stimulated either with MPT51 recombinant protein of M. tuberculosis (5 μg/ml), or PHA (5 μg/ml) or PPD (5 μg/ml). Cells cultured under similar conditions without any stimulation served as the negative control. The cultures were set up in triplicates and incubated for 6 days at 37°C in 5%CO₂ atmosphere.

Sixteen hours before termination of cultures, 1 μCi of tritiated (³H) thymidine (Board of Radiation and Isotope Technology, MA, USA) was added to each well. The cells were then harvested onto glass fiber filters on a cell harvester and allowed to dry overnight. 2ml of scintillation fluid (0.05 mg/ml POPOP and 4 mg/ml PPO in lit. of toluene) was added to each tube containing the dried filter discs and counted by using a liquid scintillation beta counter.

Interferon-γ measurement

For quantification of IFN-γ, in all 1:5 and 1:10 diluted blood and PBMC cell-free culture supernatants from lymphocyte proliferation assay were harvested after 6 days of in vitro stimulation with or without antigen stimuli and stored at −80°C until assayed. IFN-γ production was determined by standard ELISA technique using commercially available BD opt-EIA Kit (BD Biosciences, Franklin Lakes, NJ, USA) as per the manufacturer’s instructions.

Statistical analysis

Statistical analysis was performed by using GraphPad Prism software version 5.0 (GraphPad software, CA, USA). One way ANOVA was used for comparing lymphocyte proliferative responses and cytokine levels in all 1:5, 1:10 diluted blood and PBMC assays against the antigens and mitogen.

IFN-γ secretion in response to MPT51 recombinant antigen of M. tuberculosis, PPD and PHA

T lymphocyte function was studied by measuring IFN-γ levels in 1:5, 1:10 diluted WB and PBMCs stimulated with the antigens and mitogen. One way ANOVA was used to compare the differences among the three assay conditions.

The mean values of IFN-γ levels in 1:5, 1:10 diluted WB and PBMC’s stimulated with MPT 51 recombinant antigen were 55.71pg/ml, 166.3pg/ml and 54.86pg/ml respectively. Though there was a difference in IFN-γ secretion in all the three assay conditions, the difference was not statistically significant (p = 0.39) (Figure 1).

In PPD stimulated 1:5, 1:10 diluted WB and PBMCs cultures, the mean values of secreted IFN-γ levels were 1333pg/ml, 1333pg/ml, and 438.7pg/ml respectively. Here also, the difference was not statistically significant (p = 0.20) (Figure 2).

Whereas the IFN-γ levels in 1:5, 1:10 diluted WB and PBMCs in response to PHA, showed statistically significant difference (p = 0.017) (Figure 2). The mean values of IFN-γ in 1:5, 1:10 diluted WB and PBMC cultures stimulated by PHA were 3375pg/ml, 3513pg/ml and 2064pg/ml respectively.

Lymphocyte proliferation response against MPT51 recombinant antigen of M. tuberculosis, PPD and PHA

Apart from IFN-γ measurement, T lymphocyte function was also studied by its proliferative ability in 1:5, 1:10 diluted WB and PBMC’s stimulated with the antigens and mitogen. The differences among the three assay conditions were compared by using one way ANOVA.

Here, proliferative response of lymphocytes was measured in terms of thymidine incorporation and expressed as stimulation index (SI).

In all the assay conditions i.e. in 1:5, 1:10 diluted WB and PBMC, lymphocyte proliferation response against MPT 51 recombinant antigen of M. tuberculosis was similar (Figure 3). On doing one way ANOVA for comparison, there was no statistically significant difference (p = 0.58) among the three conditions. Although, in all the three assay conditions, high thymidine uptake values were observed in response to PPD and PHA (represented positive controls), the difference was not statistically significant (p = 0.45 & 0.60 respectively) (Figure 3 & Figure 4).

These results suggest that the IFN-γ secretion and lymphocyte proliferation patterns were similar in 1:5 and 1:10 diluted WB and in PBMC assays against the antigens and mitogen.