|Year : 2019 | Volume
| Issue : 3 | Page : 370-375
Frequencies of regulatory subsets of CD4+ TH cells in peripheral blood in Mycobacterium Tuberculosis-Infected individuals and healthy contacts in a high-burden setting from Assam, Northeast India
Lahari Saikia1, Trinayan Deka2, Pranjal Deori3, Rasmi Roy4, Md Ezaz Hussain2, Reema Nath2
1 Department of Microbiology, Gauhati Medical College and Hospital, Guwahati; Department of Microbiology, Assam Medical College and Hospital, Dibrugarh, Assam, India
2 Department of Microbiology, Assam Medical College and Hospital, Dibrugarh, Assam, India
3 Department of TB and Chest Disease, Assam Medical College and Hospital, Dibrugarh, Assam, India
4 Department of Pathology, Assam Medical College and Hospital, Dibrugarh, Assam, India
|Date of Submission||25-Dec-2018|
|Date of Decision||26-Sep-2019|
|Date of Acceptance||29-Nov-2019|
|Date of Web Publication||29-Jan-2020|
Dr. Lahari Saikia
Department of Microbiology, Gauhati Medical College and Hospital, Guwahati, Assam
Source of Support: None, Conflict of Interest: None
Background: Mycobacterium tuberculosis (Mtb) adapts many strategies to persist and replicate inside human tissue. One such strategy is the manipulation of CD4+ TH cells for subset interconversion to regulatory subsets. The aim of the present study is to get an insight of dynamic changes of CD4+ TH cells to regulatory subsets, CD4+ CD25+ forkhead box P3 (Foxp3)+ T-cells and CD4+ CD25+ Foxp3+ programmed death molecule-1 (Foxp3+) T-cells, in peripheral blood in Mtb-infected individuals and healthy contacts in a high-burden setting from Assam, Northeast India. Materials and Methods: A case–control study was conducted in newly diagnosed active pulmonary tuberculosis (APTBs) patients and 2 sets of controls: (i) individuals infected with latent tuberculosis infection (LTBI) and (ii) healthy close tuberculosis healthy contacts (HCs). The frequencies of different subsets of CD4+ cells with regulatory markers were measured in peripheral blood in 3 groups of study participants. Results and Observations: Frequencies of CD4+ CD25+ Foxp3+ T-cells (1.84 ± 1.40 vs. 4.32 ± 1.82 vs. 11.30 ± 3.66), CD4+ CD25+ Foxp3+ PD1+ T-cells (0.37 ± 1.28 vs. 2.99 ± 3.69 vs. 14.54 ± 5.10) and ligand (PD-L1)-positive CD4+ TH cells (0.80 ± 0.45 vs. 2.28 ± 0.95 vs. 7.13 ± 2.02) were significantly increased from HCs to LTBIs to APTB patients, respectively (P < 0.0001). No significant changes in frequencies of total CD4+ cells were observed between APTBs (29.51 ± 11.93), LTBIs (29.23 ± 8.16) and HCs (28.16 ± 9.73) whereas the mean ratios of CD4+ to CD4+ CD25+ FoxP3+ were significantly decreased from 34.34 ± 47.56 in HCs to 7.96 ± 5.8 in LTBIs to 3.12 ± 2.58 in APTBs (P < 0.0001). Significant decrease in mean ratios of CD4+ CD25+ FoxP3+ to CD4+ CD25+ FoxP3+ PD1+ were also observed from 4.97 ± 1.09 in HCs to 1.44 ± 0.49 in LTBIs to 0.78 ± 0.72 in APTBs. Conclusion: CD4+ TH cells change dynamically to regulatory subsets depending on the status of infection and a shift of response towards excessive regulatory T-cells, and PD-1/PD-L1 production may help in the development of active infection in latently infected individuals. These immunological parameters may be used, as potential biomarkers to see the changing dynamics of Mtb infection.
Keywords: CD4+ TH cells, programmed death molecule-1/programmed death molecule ligand 1 pathway, Treg cells, tuberculosis
|How to cite this article:|
Saikia L, Deka T, Deori P, Roy R, Hussain ME, Nath R. Frequencies of regulatory subsets of CD4+ TH cells in peripheral blood in Mycobacterium Tuberculosis-Infected individuals and healthy contacts in a high-burden setting from Assam, Northeast India. Indian J Med Microbiol 2019;37:370-5
|How to cite this URL:|
Saikia L, Deka T, Deori P, Roy R, Hussain ME, Nath R. Frequencies of regulatory subsets of CD4+ TH cells in peripheral blood in Mycobacterium Tuberculosis-Infected individuals and healthy contacts in a high-burden setting from Assam, Northeast India. Indian J Med Microbiol [serial online] 2019 [cited 2020 Jul 13];37:370-5. Available from: http://www.ijmm.org/text.asp?2019/37/3/370/277062
| ~ Introduction|| |
Tuberculosis (TB) is a chronic disease caused by intracellular bacterial pathogen Mycobacterium TB (Mtb). It is estimated that one-third of world human populations are infected with this intracellular pathogen among which 5%–15% only develop active TB during their lifetime. Bacterial, host and environmental factors all influence in the development of active disease. A commonly adaptive immune response mediated by antigen-specific CD4+ TH1 cells is believed to be the major defensive host immune response against Mtb infection. However, the exaggerated immune response is always detrimental to the host tissue and causes tissue injuries through immunopathological reaction. CD4+ TH1 cells are under tight regulation of multiple mechanisms to limit exaggerated immune response and keep the balance between a protective and immunopathological effect of an immune response. One such mechanism of regulation is the differentiation 'plasticity' of CD4+ TH1 subsets with dynamic functional subset interconversion depending on the status of infection and cytokine microenvironment.,
Regulatory T cells (Tregs), a subset of CD4+ TH cells, constitutes 1%–5% of all circulating CD4+ cells and the main function is to prevent autoimmunity and exaggerated immune response to infection to prevent excessive tissue destruction., However, by limiting the immune response, Tregs may give the pathogens opportunity to persist inside tissue and establish chronic infection. A prominent marker of Tregs is the interleukin-2 receptor α-chain, CD25, but more precise marker identified with most immunosuppressive functional activity is the expression of transcription factor forkhead box P3 (Foxp3) in CD4+ TH cells.
Recently programmed death molecule-1(PD-1) and its ligand (PD-L1) have emerged as a major immune regulatory mechanism. Studies showed that expression of PD-L1 increased in malignant cells and promote tumour progression through inhibition of PD-1 expressing immune effectors., Furthermore novel role of PD-1/PD-L1 pathway in modulation of cell-mediated immune response was observed in many infectious diseases., Few recent studies indicated the role of PD-1/PD-L1 pathway in shifting of CD4+ TH1 cells towards Tregs and in sustaining Foxp3 expression in Tregs.,
The aim of present study was to get an insight in dynamic changes in frequencies of CD4+ TH cells and its regulatory subsets, CD4+ CD25+ Foxp3+ T-cells and CD4+ CD25+ Foxp3+ PD1+ T-cells in peripheral blood in newly diagnosed active pulmonary TB (APTBs) patients in Assam, Northeast India.
| ~ Materials and Methods|| |
This was a case–control observational study.
Between November 2015 and March 2017, all consecutive newly diagnosed treatment naïve adult APTBs patients attending TB unit of Assam Medical College (AMC) were enrolled in the study. Initially, a total of 57 patients of APTBs and age-sex matched 52 individuals infected with latent tuberculosis infection (LTBIs) and 44 individuals of healthy contacts (HCs) were recruited for this study. Age-sex matched LTBIs and HCs were recruited from household contacts and individuals working in TB unit. Due to some technical difficulties such as weak surface staining and sample failure, analysis of Tregs and PD-1/PD-L1 expression, study could be done on 101 individuals (42 patients of APTBs, 26 individuals of LTBIs and 33 individuals of HCs). Patients with positive sputum smears or one positive sputum smear and chest radiograph compatible with TB and treatment naive were considered eligible for the study. Liquid culture in BACTEC MGIT 320-automated TB culture system (Becton and Dickinson, BD, New Jersey, USA) was done to confirm diagnosis of APTBs, to do sensitivity test for the first line of antitubercular drugs and to exclude active infection in LTBIs and HCs. APTBs patients resistant to the first line of anti-tubercular drugs and negative culture were excluded from the study. Mantoux and/or interferon gamma (IFN-γ) release assay (IGRA) tests were used to differentiate between LTBIs and HCs. All apparently HCs were subjected for IGRA test and were performed according to the manufacturer's instruction using ImmuCheck TB Platinum version 3T (P-positive control, N-negative control and t-testing culture tube) (Immunoshop India Pvt. Ltd, Mumbai, Maharashtra, India). One ml of heparinised whole blood was added to each of the three tubes (P, N and T) containing TB antigen in 'P' tube, background control culture tube” and positive control culture 'tube'. Tubes were incubated at 37°C for 20–24 h, centrifuged and plasma removed and stored at −20°C. The amount of IFN-γ in plasma was quantified by enzyme-linked immunosorbent assay. For interpretation, the IFN-γ level in the 'N' tube is subtracted from the IFN-γ level in the 'T' tube and value ≥14 and ≥25% of 'N' value was interpreted as positive. Mantoux test was performed when requested by treating physician. Those individuals showed positive IGRA test were labelled as LTBIs and IGRA negative individuals were labelled as HCs. Mantoux test was performed after collection of blood for IGRA test. According to the standard procedures, test was performed with 0.1 ml of 5 TU of tuberculin PPD RT 23 and read after 72 h. An induration of more than 10 mm was considered as positive test.
Individuals of <15 years of age, apparently unhealthy, the presence of any malignant or autoimmune diseases and HIV positive were excluded from the study. Institutional human ethical clearance was obtained from the institute (no AMC/EC/PG 3519 dated 10th March 2014) and after taking informed written consent; all epidemiological and clinical data were collected in patient's information sheets.
Six millilitre of venous blood was drawn aseptically from anterior cubital vein and added into 3 vacutainers, 2 ml in an Ethylene Diamine Tetraacetic Acid (EDTA) vial for total leucocytes count and differential leucocytes count, 2 ml in clot activator vial for HIV testing and 2 ml in heparin vials for immunophenotyping and IGRA test. All samples were processed immediately and analysed on the same day. For every sample, duplicate reading was taken.
Twenty microliters of each of antigen presenting cells (APC)-Cy7 tagged monoclonal antibody (MoA) against CD4, fluorescein isothiocyanate (FITC) tagged MoA against Foxp3 and CD14, phycoerythrin Cy7 (PE-Cy7) tagged MoA against PD1, APC tagged MoA against PDL1 and PE-Cy5 tagged MoA against CD25 were used for staining. All these antibodies were purchased from BD Bioscience (San Diego, CA, USA). Two panels were used. One panel (panel-1) was used for CD4-APC-Cy7, PD1-PE-Cy7, CD25-PECy5 and Foxp3-FITC. One panel (panel-2) used for CD4-APC-Cy7, PDL1-APC, PD1-PE-Cy7 and CD14-FITC. Lyse-wash sample preparation method using whole blood was performed. For staining 100 μl of whole blood of each individual was placed into 2 FACS tubes for each panel. One tube was used for staining and another for fluorescence minus one (FMO) control. Gating of surface and intracellular markers were determined using control samples by the FMO approach, i.e., controls containing all markers except the one of interest were used to set gates. For compensation control, BD FACS™ 7-color setup beads along with BD Diva software (Becton and Company, Becton Drive, Franklin Lakes) were used. The software automatically calculates the accurate compensation value.
Tubes were mixed and incubated in dark at room temperature for 15 min. Two ml of BD FACS Lyse (<15% formaldehyde and <50% diethylene glycol, was diluted 1:10 in deionised water immediately before use) was added to each tube. Tubes were reincubated in dark for 10 min. Centrifuged at 1200 rpm for 10 min and supernatant was discarded. Pellet was broken and the cells were washed twice by adding 2 ml of sheath fluid, mixed, centrifuged and the supernatant was discarded. Cells were re-suspended in 0.5 ml of sheath fluid with 2% paraformaldehyde. For intracellular staining, 0.5 ml of 1X BD permeabilisation buffer was used and keep it for 30 min and centrifuged at 1200 rpm for 5 min and supernatant was discarded. Pellet was broken and 20 μL of Foxp3-FITC monoclonal antibodies were added and incubated at room temperature for 30 min. The tubes were washed twice by adding 2 ml of sheath fluid, mixed, centrifuged and the supernatant was discarded. Cells were resuspended in 0.5 ml of sheath fluid with 2% paraformaldehyde.
The flow cytometry analysis was done on BD FACS Canto-II (BD Bioscience, San Jose, California, USA). The percentage of positive cells and the median fluorescent intensity (arbitrary units) for a specific marker was calculated using BD FACS Diva software. For analysis, 50,000 events were recorded.
All data were analysed in GraphPad Prism version 6.0.5 GraphPad Software, 2365 Northside Suite, San Diego, CA using the tests: one-way ANOVA, t-test, Kruskal–Wallis test, Mann–Whitney and Chi-squire test. The difference was considered significant at P < 0.05.
| ~ Results|| |
The baseline characteristics of enrolled individuals
Demographic and general characteristics of the enrolled patients are summarised in [Table 1]. There was no difference in mean age and sex between three groups. The mean duration of illness in APTBs patients was 44.09 days. Persons with APTBs were more likely malnourished with body mass index (BMI) <18.5 (P < 0.0001) and haemoglobin level <10.27 and lymphocytopenia (P < 0.0001).
|Table 1: Summary of demographic and general characteristic of the enrolled patients|
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Flow cytometry analysis
Percentage of CD4+ T cells and its regulatory subsets (Tregs) within CD4+ TH cells and frequencies of cells expressing PD-1 and PD-L1 in peripheral blood in three groups of individuals are shown in [Table 2].
Treg cells were gated and frequencies were analysed as a percentage of CD4+ TH cells. CD4+ CD25+ Foxp3+ Tregs frequency were analysed as a percentage of CD4+ CD25+ cells. No statistical difference was observed in the total percentage of CD4+ TH cells among the three groups (P = 0.8420).
Statistically significant differences were observed among the three groups in the analysis of regulatory subsets of CD4+ TH cells. Frequencies of CD4+ CD25+(4.58 ± 2.46 vs. 6.45 ± 1.71 vs. 19.05 ± 4.57), CD4+ Foxp3+ (1.84 ± 1.5 vs. 4.90 ± 1.70 vs. 16.74 ± 6.91), CD4+ CD25+ Foxp3+ T-cells (1.84 ± 1.40 vs. 4.32 ± 1.82 vs. 11.30 ± 3.66) [Figure 1], CD4+ CD25+ Foxp3+ PD1+ T-cells (0.37 ± 1.28 vs. 2.99 ± 3.69 vs. 14.54 ± 5.10) [Figure 2] and PD-L1-positive CD4+ TH cells (0.80 ± 0.45 vs. 2.28 ± 0.95 vs. 7.13 ± 2.02) were significantly increased from HCs to LTBIs to APTBs, respectively (P < 0.0001). The frequency of CD4+ CD25+ Foxp3+ has increased 6 times more in APTBs than in HCs and 2.6 times more than in LTBIs.
|Figure 1: Percentage of CD4+ CD25+ forkhead box P3+ significantly higher in active pulmonary tuberculosis patients compared to latent tuberculosis infections P < 0.0001 and healthy contacts P < 0.0001; which is also higher in latent tuberculosis infections than in healthy contacts P < 0.0001|
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|Figure 2: The percentage of PD-1expressing CD4+ cells were significantly higher in APTBs than in LTBIs and HCs subjects, p<0.0001 for both|
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The mean percentage of PD-1 (27.95 ± 14.95 vs. 33.99 ± 13.44 vs. 41.93 ± 16.05) and PD-L1 expressing (0.80 ± 0.45 vs. 2.28 ± 0.95 vs. 7.13 ± 2.02) CD4+ T-cells were also increased significantly from HCs to LTBIs to APTBs, respectively.
To get an insight into the subset conversion among CD4+ TH cells in Mtb-infected individuals, we determined the mean ratios of CD4+ TH cells to Tregs. The mean ratios of CD4+ to CD4+ CD25+ FoxP3+ were significantly decreased from 34.34 ± 47.56 in HC to 7.96 ± 5.8 in LTBI to 3.12 ± 2.58 in APTB ((P < 0.0001) [Figure 3]. Significant decreased in mean ratios of CD4+ CD25+ FoxP3+ to CD4+ CD25+ FoxP3+ PD1+ were also observed from 4.97 ± 1.09 in HCs to 1.44 ± 0.49 in LTBIs to 0.78 ± 0.72 in APTBs.
|Figure 3: The ratios of CD4+ cells to CD4+CD25+, CD4+CD25+Foxp3+ and CD4+Foxp3+ were significantly decreased in APTBs patients compared to LTBIs |
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Because PD-1-mediated inhibition of effector T-cells function required engagement with the expression of its ligands on APC, we further examined the expression of PD-L1 on CD14+ monocytes. Significantly increases the frequency of PD-L1+ CD14+ cells from 0.90 ± 0.51 in HCs to 1.95 ± 0.59 in LTBIs to 6.78 ± 1.86 in APTBs (P < 0.0001).
Impact of bacillary load, age and sex on frequencies Tregs was not observed in the present study (P > 0.05), but a negative correlation between BMI and Tregs was observed [Figure 4].
|Figure 4: A negative correlation between BMI and percentage of CD4+CD25+, CD4+Foxp3+ and CD4+CD25+Foxp3+ cells |
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| ~ Discussion|| |
The present study was undertaken to get an insight in the changes of CD4+ T-helper cells and frequency of regulatory T-cells in peripheral blood in APTBs and latently infected individuals in a high TB burden setting. To the best of our knowledge, this is the first such type of study conducted on APTBs and latently infected individuals and HCs from Assam, Northeast India.
As expected, a significant lymphocytopenia was observed in APTBs than in LTBIs and HCs. No change in frequencies of CD4+ TH cells was observed among the groups. CD4+ TH cells play an important role in immunity against TB. There are different results from different studies about the CD4+ TH cells level in peripheral blood in active pulmonary TB cases. He et al. reported increased in the percentage of CD4+ TH cells in APTBs compared to healthy subjects. In contrast, Davoudi et al. reported that a significantly lower percentage of CD4+ TH cells in APTBs than in healthy controls. In accord with our study, Herzmann et al. reported no significant difference in CD3+ CD4+ TH cells in PBMC between IGRA+ and IGRA-individuals. Another study was done by de Almeida et al. who reported no difference in frequencies of CD4+ between APTBs, LTBIs and HCs.
Within the compartment of CD4+ TH cells, we found a significantly higher percentage of circulating CD4+ CD25+ Foxp3+ and CD4+ CD25+ Foxp3+ PD1+ T cells along with CD14+ PD-L1+ cells in patients with TB compared with uninfected healthy controls and our results are generally consistent with previous reports from other regions.,,, Moreover, there was a gradual decreased in ratios of CD4+ Cells to CD4+ CD25+, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ and CD4+ CD25+ Foxp3+ to CD4+ CD25+ Foxp3+ PD1+ from baseline level in HCs to LTBIs and APTBs which indicates that CD4+ T-cell is not a stable phenotype and a rise in trend of CD4+ TH cells with regulatory markers in parallel with ongoing Mtb infection.
Balance between protective T-helper cells and differentiation of T-Helper subsets to Regulatory T-cells during Mtb infection decides the extent of host susceptibility. Previous studies had revealed that during the active TB, Fox p3+ regulatory T-cells are over represented in active TB and supress the effector T-cell response against Mtb infection and over expression of PD-1 and its ligands and their interaction is essential for suppression activity of Treg cells.,
Amarnath et al. usingin vivo model demonstrated that PD1-PD-L1 pathway through SHP1/SHP2 signalling pathway plays a special role in modulation of T-helper cells plasticity resulting in Treg phenotype that severally impaired cell-mediated immunity. Francisco et al. demonstrated that in the absence of transforming growth factor beta (TGF-β), PD-L1 alone can enhance and sustain Foxp3 expression and the suppressive function of Treg cells. Furthermore, he observed a profound defect in conversion to Foxp3+ Treg cells when naive T-cells were cultured with PD-L1−/−APC, anti-CD3 and the same range of TGF-β concentrations. The continuous increased and maintenance of Tregs by PD1−/PDL1 may be attributed to the enhanced inhibition of immune response and subsequent development of active disease in latently infected individuals. However, few studies indicated that increased Tregs cells and PD-1/PD-L1 expression were due to exaggerated general inflammation in latently infected individuals due to other factors such as age, sex, chronic disease, co-infection with other agents, exposure to environmental mycobacteria and reduced BMI., In the present study, we have found a significant negative correlation between the frequency of Tregs and PDL1-expressing cells with BMI but no significant correlation with age and sex was observed.
| ~ Conclusion|| |
These results suggest that there is a relationship between Tregs and TB infection. The protective T-helper cells are subjected to convert to regulatory phenotypes depending on the stage of infection and malnutrition play an important role in plasticity. It is anticipated that the Mtb antigenic persistence in the individuals with LTBI induces a continuous generation of T-regs resulting increasing their number in active TB. The plasticity of antigen-experienced human T-cell subsets is highly relevant for translational medicine, since it opens new perspectives for immunomodulatory therapies to prevent development of active pulmonary TB in latently infected individuals. Monitoring PD1-PDL1 expression in LTBIs and manipulation of PD1/PD-L1 pathway may help to evaluate and restore host protective immunity and prevent progression of LTBI to active pulmonary TB in high burden setting. However, the dynamic changes of T-helper cells at the site of infection need further study to correlation with changes that happen in peripheral blood.
This work was supported by financial grants from Department of Biotechnology, Government of India. The authors gratefully acknowledge the help and support given by Principal of Assam Medical College and Hospital and the thank all the laboratory staff of AMLC and MRU for their technical assistance.
Financial support and sponsorship
This work was supported by Department of Biotechnology, Government of India (Grant No BT/530/NE/TBP/2013, DTD-03.03.14).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]