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Influence of HLA-DR antigens on lymphocyte response to Mycobacterium tuberculosis culture filtrate antigens and mitogens in pulmonary tuberculosis
Tubercle and Lung Disease (1999) 79(4), 199–206 © 1999 Harcourt Publishers Ltd Article no. tuld.1998.0211
Influence of HLA-DR antigens on lymphocyte response to Mycobacterium tuberculosis culture filtrate antigens and mitogens in pulmonary tuberculosis H. Uma, P. Selvaraj, A. M. Reetha, T. Xavier, R. Prabhakar, P. R. Narayanan Tuberculosis Research Centre (ICMR), Chennai 600 031, India
Summary Setting: Influence of HLA-DR antigens and lymphocyte responses in pulmonary TB patients. Objective: To elucidate the role of HLA-DR genes/gene products on lymphocyte responses to Mycobacterium tuberculosis antigens and mitogens, the present study was carried out in pulmonary tuberculosis during active and cured stage of the disease. Design: Serological determination of HLA-DR antigens was carried out in 50 active TB patients, 44 cured TB patients and 58 normal healthy control subjects. The influence of HLA-DR antigens on peripheral blood lymphocyte responses to M. tuberculosis culture filtrate antigens and mitogens such as phytohaemagglutinin (PHA) and concanavalin-A (Con-A) was studied in the patients as well as normal healthy control subjects. Results: Of all the DR antigens studied, patients (active TB and cured TB) with DR2 antigen showed an increased lymphocyte response (stimulation index) to a higher dose of antigenic (10 µg/ml) stimulation. A significantly lower lymphocyte response to antigen and mitogens was seen in HLA-DR3 positive normal healthy subjects than non-DR3 (DR3 negative) subjects. Conclusion: The present study suggests that HLA-DR genes/gene products may be playing an immunoregulatory role in eliciting an immune response against M. tuberculosis antigens and mitogens induced lymphocyte response in pulmonary TB patients and normal healthy subjects. © 1999 Harcourt Publishers Ltd
INTRODUCTION Tuberculosis is a chronic infectious disease which is predominantly regulated by the cell-mediated immune (CMI) response of the host against the causative organism, Mycobacterium tuberculosis.1 Disease susceptibility to tuberculosis is multifactorial. Host factors such as the immune status and the genetic make up of the individual play a major role in the susceptibility to tuberculosis. Whether the host will resist or succumb to tuberculosis greatly depends on specific T lymphocytes against M. tuberculosis antigens. It is well established that the immune status of an individual is influenced by human Correspondence to: Dr P. Selvaraj, Department of Immunology, Tuberculosis Research Centre (ICMR), Spurtank Road, Chetpet, Chennai 600 031, India. Fax: +91 44 82621371; E-mail: [email protected] [email protected] Received: 29 July 1998; Revised: 25 January 1998; Accepted: 1 February 1999
leucocyte antigens (HLA). The HLA antigens, especially the class II antigens on the antigen presenting cells, present the processed antigens to the CD4+ T lymphocytes, which in turn through their lymphokines, help in subsequent immune responses.2 The secretion of cytokines, such as tumor necrosis factor-α and interleukin-1,3 as well as the lymphocyte response to antigens,4,5 are influenced by HLA genes and their gene products. The HLA class II genes/gene products, otherwise known as immune response (Ir) genes,6 encode for inter-individual differences in antigen specific immune reactivity.7 It has been suggested that the HLA-DR locus has been associated with T-cell helper activity, in human immune responses to various antigens and pathogens.8 Several studies have been carried out to understand whether the susceptibility and/or immune response to M. tuberculosis is associated with HLA phenotype and/or 199
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controlled by genes linked to the Major Histocompatibility Complex (MHC).4,5,9–14 The role of HLA-Class II genes/gene products on delayed type hypersensitivity skin responses, and lymphocyte responses to purified protein derivative of M. tuberculosis has been studied.4,7,10,15–17 The influence of HLA-class-II genes on lymphoproliferative response to mycobacterial antigens has been studied in leprosy patients.18 Recently, the role of HLA-DR polymorphism on the cytokine profile has been studied in leprosy patients.19 Studies have also been carried out to find relevant T-cell epitopes of M. tuberculosis antigens and their peptides in the context of HLA-DR molecules (Mustafa et al., unpublished), and to define their usefulness for diagnosis or vaccine design5,20–23 against tuberculosis in the HLA heterogenous population. Though a number of studies have been carried out in depth to understand the immune response against M. tuberculosis antigens during the active stage of the disease, attempts have not been made to evaluate the immune status (cell mediated immunity) of the patients after treatment (cured patients). Further, most of the studies on the genetic control of cell mediated immune response has been focussed on delayed type hypersensitivity to various mycobacterial antigens and little attention has been paid to the lymphocyte response in leprosy and tuberculosis. Since, a comprehensive study on HLA-DR and lymphocyte response in active and cured pulmonary TB patients and healthy control subjects has not been explored completely, the present study was carried out. MATERIALS AND METHODS Study subjects
Active tuberculosis patients (ATB) Patients attending the Tuberculosis Research Centre (TRC), Chennai, India, with respiratory symptoms and radiographic abnormalities suggestive of pulmonary-TB were studied. These patients had sputum positive for M. tuberculosis by smear and culture. Blood samples were taken before the start of chemotherapy. Among 50 ATB patients studied, 45 were male and five were female. The mean age with SE was 37.7±1.8 for males and 27.8±4.6 for females. Inactive tuberculosis patients (ITB) (Cured patients) Patients classified as suffering from active pulmonary tuberculosis were given anti-tuberculosis treatment at our centre. All of these patients had received a short course of chemotherapy of 6–8 months duration. These patients were treated 10–15 years previously. At the time of blood sample collection, all of these cured patients were in the quiescent stage of the disease. Of the 44 studied, 35 were male and nine were female. The mean age with SE was 42.2±1.7 for males and 36±1.9 for females. Tubercle and Lung Disease (1999) 79(4), 199–206
Controls Spouses of cured TB patients living together for 10–15 years (family contacts; n=19) and clinicians, social workers, health visitors, laboratory volunteers and other staff (n=39) working at TRC for more than 3 years were studied. Among the 58 control subjects studied, 25 were male and 33 were female. The mean age with SE was 38.6±1.7 for males and 36.9±1.5 for females. All of the family contacts and other control subjects were clinically normal at the time of blood sample collection. The patients and spouses were not consanguineous. The other control subjects were not related to any of the patients. The patients and the control subjects were the same ethnic origin and were randomly selected from the same ethnic group. They were the Tamil-speaking South Indian population (belonging to different communities) living in and around Chennai. HLA typing Peripheral blood mononuclear cells (PBMC) were separated from heparinized (20 units/ml) blood samples using Ficoll-hypaque density gradient centrifugation as described by Boyum.24 T- and B-lymphocytes were separated from PBMC using nylon wool column and nylon wool adherent cell population (enriched B-lymphcoytes) was used for HLA-DR typing using a two-stage microlymphocytotoxicity assay.25 Commercial sources of antisera (Biotest, Frankfurt, Germany) were used for HLA-DR typing and at least three antisera were used for each specificity. M. tuberculosis culture filtrate antigen The H37Rv strain of M. tuberculosis was grown for 6 weeks (late logarithimic phase) as a surface pellicle on Sauton’s medium at 37°C. The culture was centrifuged at 3000 rpm for 30 min to remove the bacilli and the supernatant was filtered through a Seitz filter (0.45 µm) (SeitzFilter-Werke GmbH und Co., Badkreuznack, Germany) to ensure complete removal of the bacilli. The culture filtrate antigens were precipitated with 80% ammonium sulphate and dissolved in sterile phosphate buffered saline (PBS) (pH 7.4). The excess salt was removed by dialysis using 0.2 M, 0.1 M and 0.02 M PBS (pH 7.4) at 4°C. The dialysed protein was lyophilized and resuspended in minimal volume of sterile PBS. The protein concentration was estimated by Lowry’s procedure,26 against bovine serum albumin as standard. The culture filtrate protein antigens were aliquoted and stored at –20°C until required. The antigen concentrations used for lymphocyte response were 0.1, 1.0 and 10.0 µg/ml of culture. Mitogens Three different concentrations (0.1, 1.0 and 10.0 µg/ml) of either Phytohaemagglutinin (PHA) (Wellcome Diagnos© 1999 Harcourt Publishers Ltd
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tics, Dartford, UK) or concanavalin-A (Con-A) (Sigma Chemical Co., St Louis, USA) were used to stimulate the lymphocytes.
Lymphocyte Transformation Test PBMC of active-TB, cured-TB (inactive-TB) and control subjects were suspended in RPMI tissue culture medium (Sigma Chemical Co., St Louis, USA) supplemented with 2 mM L-glutamine (Flow Laboratories, Irvine, UK), gentamycin (10 µg/ml) (M. A. Bio Products, Walkerville, MD, USA), fungizone (5 µg/ml) (Squibb, Princeton, NJ, USA) and 10% autologous plasma for controls and cured patients. Autologous serum was used instead of plasma in the cultures set up with active tuberculosis patients samples to avoid clotting. No difference in the counts was observed when autologous plasma or serum was used. 0.1 × 106 PBMC in 200 µl were cultured with either M. tuberculosis culture filtrate antigen or mitogens at 0.1, 1.0, 10 µg/ml. Cultures without any stimulation served as controls. Cultures with M. tuberculosis culture filtrate antigen were kept for 144 h (6 days) in a 96 well ‘U’ bottom tissue culture plate (Costar, Cambridge, MA, USA), at 37°C and 5% carbon dioxide in a CO2 incubator (Flow Laboratories Ltd., Rickmansworth, Herts, UK). Similarly, proliferation assays with PHA and Con-A mitogens were also carried out. The mitogen-stimulated cultures were maintained for 72 h (3 days). The lymphocyte cultures were set up at least in triplicates for each concentration of antigen and mitogens. Tritiated thymidine (3H) (BARC, Trombay, Mumbai, India) was added to each well (1 µCi/well) 16 h before termination. The cells were harvested onto glass fibre filters using a PHD cell harvester (Cambridge Technology Inc, Water Town, MA, USA). The lymphocyte response to M. tuberculosis antigens and mitogens were measured by tritium labelled thymidine uptake by the cells in a scintillation system using Beta counter (LKB, Wallac, Oy, Turku, Finland) and expressed as counts per minute (cpm). The mean cpm value of the triplicate culture was calculated and the stimulation index (SI) was derived using the formula: SI:
mean cpm of antigen stimulated cell cultures mean cpm of cell cultures without antigen
Statistics The frequencies of HLA-DR antigens in patients and controls were determined by direct allelic count. The results on lymphocyte response are expressed as arithmetic mean±S.E. Student’s t-test was used to see the significance of the data. RESULTS HLA-DR2 positive active and cured-TB (inactive-TB) patients showed an increased proliferative response to © 1999 Harcourt Publishers Ltd
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M. tuberculosis antigen stimulation at a higher dose. ATB: DR2: mean SI±SE = 1.4±0.2; 3.0±0.7; 6.2±1.8 and nonDR2 ATB = 1.3±0.1; 2.0±0.3; 3.1±0.5; (0.1; 1.0; 10 µg concentration of antigen respectively) (10 µg/ml: ATB: DR2 vs non-DR2 P > 0.05). ITB: DR2: 8.5±1.8; 21.2±4.3; 39.9±8.1/non-DR2 = 8.2±2.8; 13.0±2.8; 22.8±4.3 at 0.1 µg/ ml, 1 µg/ml and 10 µg/ml concentrations of antigen, respectively (10 µg/ml: ITB: DR2 vs non-DR2 P < 0.05) (Figs 1 and 2). A significantly suppressed response or low response was seen against M. tuberculosis antigen in DR3 positive control subjects compared to non-DR3 individuals, whereas no such difference was observed in the patient group (Controls: DR3 positive – mean SI±SE = 1.4±0.4; 2.9±0.8; 5.9±2.4 and DR3 negatives – 4.3±0.8; 7.9±1.3; 15.0±2.3 at 0.1 µg/ml, 1 µg/ml and 10µg/ml concentrations of antigen respectively; (P < 0.01 at all the three concentrations of antigen) (Fig. 3). Further, a significantly lower response to 1 µg and 10 µg of M. tuberculosis antigen was also seen in DR1 positive inactive TB patients compared with non-DR1 patients (1 µg: P < 0.01; 10 µg: P < 0.01). Such a low response was not seen in control subjects and active TB patients (Fig. 1). The lymphocyte response to antigenic stimulation in different heterozygous combination of HLA-DR antigens of controls, active TB and inactive TB patients were analysed and compared. DR 2/7 and 3/7 heterozygous combinations were present in all the three groups of subjects. Among the different DR7 heterozygous combinations in control subjects (DR 1/7, 2/7, 3/7 and 6/7), DR 3/7 heterozygotes showed a suppressed response at all the doses of antigen stimulation (mean SI±SE in DR3/7 positives = 1.3±0.3; 1.9±0.8; 2.3±0.8 and DR3/7 negatives = 3.8±0.7; 7.3±1.2; 14±2.1) when compared to other heterozygous combinations studied. DR 2/4, 2/6, 2/7, 3/7, 4/6 and 6/7 combinations were analysed in ATB patients. No difference was observed in the response among the different DR antigen combinations. DR2/3, 2/4, 2/5, 2/6, 2/7, 3/7 and 4/7 antigen combinations were analysed in ITB patients DR 3/7 combination showed a trend towards a suppressed response compared to all of the other combinations at all three doses of antigen (DR 3/7 positives = 3.7±2.2; 9.0±4.9; 21.2±15.2 and DR 3/7 negatives = 8.0±1.5; 18.2±3.1; 33.3±5.7). ITB patients with DR 2/7 heterozygous combination showed a trend towards an increased lymphocyte response at higher doses (1 µg and 10 µg/ml) of antigen stimulation than DR 3/7 combination. (ITB: Mean SI±SE: 1 µg 17.9±7.9, 10 µg 49.0±13.5; DR 3/7: 1 µg 9.0±4.9; 10 µg 21.2±15.2). DR3 positive individuals in patients as well as control subjects showed lower lymphocyte response to PHA than DR3 negative individuals. A suppressive/non-responsive effect was seen in control subjects compared to the Tubercle and Lung Disease (1999) 79(4), 199–206
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Fig. 1 Influence of HLA-DR antigens on lymphocyte response to M. tuberculosis culture filtrate antigens (10 µg/ml). Results are expressed as arithmetic mean±S.E. HLA-DR1 +ve vs DR1 –ve: ITB: 0.1 µg; P < 0.05; 1 µg P < 0.01 HLA-DR2 +ve vs DR2 –ve: ATB; P > 0.05; ITB; P < 0.05 n = number of subjects studied Controls: DR1 (n=11); non-DR1 (n=34) DR2 (n=18); non-DR2 (n=40) DR3 (n=9); non-DR3 (n=36) DR4 (n=12); non-DR4 (n=35) DR5 (n=14); non-DR5 (n=34) DR6 (n=10); non-DR6 (n=36) DR7 (n=20); non-DR7 (n=26) DR8 (n=5); non-DR8 (n=39) ATB: DR1 (n=6); non-DR1 (n=39) DR2 (n=23); non-DR2 (n=27) DR3 (n=11); non-DR3 (n=34) DR4 (n=12); non-DR4 (n=33) DR5 (n=5); non-DR5 (n=41) DR6 (n=15); non-DR6 (n=30) DR7 (n=15); non-DR7 (n=31) DR8 (n=2); non-DR8 (n=43) DR9 (n=3); non-DR9 (n=43) DR10 (n=3); non-DR10 (n=42) ITB: DR1 (n=5); non-DR1 (n=36) DR2 (n=23); non-DR2 (n=21) DR3 (n=7); non-DR3 (n=33) DR4 (n=14); non-DR4 (n=27) DR5 (n=6); non-DR5 (n=34) DR6 (n=6); non-DR6 (n=34) DR7 (n=13); non-DR7 (n=27) DR8 (n=3); non-DR8 (n=37) DR9 (n=6); non-DR9 (n=35)
Tubercle and Lung Disease (1999) 79(4), 199–206
Fig. 2 Influence of HLA-DR2 antigen on M. tuberculosis antigen (0.1, 1.0 and 10 µg/ml) induced lymphocyte response in controls, active TB (ATB) and inactive TB (TIB) patients. Results are expressed as arithmetic mean±S.E. Controls: DR2 (n=18); non-DR2 (n=40) ATB: DR2 (n=23); non-DR2 (n=27) ITB: DR2 (n=23); non-DR2 (n=21)
patients (Controls – DR3 positive – mean SI±SE = 4.7±1.7; 56.1± 11.4; 50.7±16.2 and DR3 negative = 11.9±2.8; 97.2±13; 88.2±12 at 0.1 µg/ml, 1 µg/ml and 10 µg/ml concentrations of PHA). However the values were not significant. DR8 positive individuals also showed a trend towards a decreased proliferative response to PHA compared to DR8 negative individuals in the control subjects only, (Controls – DR8 positive – 12.9±6.0; 50.3±15.1; 34.2±12.1 and DR8 negative – 9.9±2.5; 94.2±12.1; 87.3±11.3; P > 0.05 (not significant) at 10 µg/ml of PHA). No significant differences in the stimulation indices were observed for other DR antigens (Table 1). Irrespective of the Con-A concentrations used (0.1, 1.0, 10.0 µg/ml) the lymphocyte response in DR3 positive control subjects was significantly decreased when compared © 1999 Harcourt Publishers Ltd
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Fig. 3 Influence of HLA-DR3 antigen on lymphocyte response to M. tuberculosis culture filtrate antigen in controls, active TB (ATB) and inactive TB (ITB) patients. Results are expressed as arithmetic mean±S.E. Controls: DR3 (n=9); non-DR3 (n=36); ATB: DR3 (n=11); non-DR3 (n=34) ITB: DR3 (n=7); non-DR3 (n=33) Controls: DR3 +ve vs non-DR3: P < 0.01 (all concentrations studied)
to non-DR3 (DR3 negative) controls (Controls: DR3 +ve vs DR3 –ve, 1 µg < 0.05; 10 µg; P < 0.05) (Fig. 4 and Table 1). DR1 positive control subjects showed a trend towards a higher proliferative response than DR1 negative individuals. However, this increase was not significant. The lymphocyte response to Con-A in DR1 positive ATB patients was higher than non-DR1 (DR1 negative) subjects at 10 µg/ml concentration (P < 0.01) (Table 1). The proliferative responses to Con-A was decreased in DR8 positive control subjects compared to DR8 negative individuals (Controls – DR8 positive – mean SI±SE = 2.3±0.7; 15.2±10.8; 31.1± 6.4 and DR8 negative – 4.1±1.1; 23±5.2; 56.8±7.6 at 0.1 µg/ml, 1 µg/ml and 10 µg/ml). However, the differences were not significant. On the other hand DR9 positive ITB patients showed an increased lymphocyte response compared to DR9 negatives (non-DR9), the values being significant at 1 µg/ml and 10 µg/ml doses of ConA (ITB – DR9 positive – mean SI±SE = 54.3±28.4; 115±46.8 and DR9 negative 20.9±3.5; 61.1±7.9 at 1 µg/ml and 10 µg/ml concentrations of Con-A respectively;
Table 1 Influence of selected HLA-DR antigens on lymphocyte response to the mitogens (PHA and Con-A) HLA DR
Mean stimulation index±S.E. PHA stimulation CON-A stimulation (10 µg/ml) (10 µg/ml) Controls ATB ITB Controls ATB ITB
DR1+
57.3±8.9 (n=9) 87.4±13.1 (n=28) 99.1±18.6 (n=16) 74.6±10.6 (n=33) 50.7±16.2 (n=8) 88.2±12 (n=29) 34.2±12.1 (n=5) 87.3±11.3 (n=32) 29.3 (n=1) 80.1±10.9 (n=36)
DR1– DR2+ DR2– DR3+ DR3– DR8+ DR8– DR9+ DR9–
35.1±34.1 (n=3) 69.3±13.2 (n=16) 75.5±25.4 (n=7) 54.7±11.5 (n=14) 81.6±40.6 (n=3) 60.6±13 (n=16) 116±0 (n=2) 57.8±11.9 (n=17) 22.8 (n=1) 63.9±12.4 (n=19)
124±82.1 (n=5) 75.9±9.4 (n=34) 82.6±12.4 (n=23) 76.4±22.9 (n=19) 107±36 (n=6) 76.5±14.2 (n=32) 67.7±43 (n=3) 82.5±13.9 (n=35) 127±80 (n=5) 73.4±9.7 (n=34)
60.1±16.8 (n=10) 51.5±7.6 (n=30) 68.1±12.7 (n=17) 51.2±7.6 (n=35) 30.9±7.5 (n=8) 58±8.2 (n=32) 31.1±6.4 (n=5) 56.8±7.6 (n=35) 20.5±14.8 (n=2) 52.3±6.5 (n=38)
4.9±4.5 (n=3) 22.9±3.4 (n=20) 20.6±3.5 (n=10) 21.4±4.4 (n=17) 20.4±16.3 (n=3) 20.6±3.2 (n=20) 47.5±0 (n=2) 18±3.1 (n=21) 10.2 (n=1) 20.6±3.3 (n=23)
78.5±58.5 (n=5) 68.8±8.5 (n=35) 77.5±11 (n=24) 56.4±15.7 (n=20) 61.8±25 (n=7) 72.6±11 (n=33) 73.8±37.4 (n=3) 70.5±10.5 (n=37) 115±46.8 (n=6) 61.1±7.9 (n=35)
(i) Con-A: DR1 +ve vs DR1 –ve: ATB: P < 0.01. (ii) Con-A: Controls: DR3 +ve vs DR3 –ve; P < 0.05. (iii) Con-A: ITB DR9 +ve vs DR9 –ve 1 µg and 10 µg concentrations; P < 0.05. (iv) n=number of subjects studied.
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Fig. 4 Influence of HLA-DR3 on Con-A induced lymphocyte response in controls, ATB and ITB patients. Results are expressed as arithmetic mean±S.E Controls: DR3 (n=8); non-DR3 (n=32) ATB: DR3 (n=3); non-DR3 (n=20) ITB: DR3 (n=7); non-DR3 (n=33) Controls: DR3 +ve vs non-DR3: 1.0 µg P < 0.05; 10.0 µg P < 0.05
P < 0.05). There was no significant difference in the responses elicited by other DR antigens in patients as well as in control subjects. DISCUSSION In the present study, HLA-DR1, DR2 and DR3 were associated with an increased or decreased lymphocyte response to M. tuberculosis culture filtrate antigens and mitogens in healthy control subjects, active TB and inactive TB patients. A trend towards an increased lymphocyte response (SI), to M. tuberculosis antigens (1.0 and 10 µg/ml) was seen in Tubercle and Lung Disease (1999) 79(4), 199–206
HLA-DR2 positive compared to non-DR2 active and inactive TB patients. However, such an increase was not seen in control subjects. An increase in PPD induced lymphoproliferative response has been observed in DR2 positive active TB patients.11 Further, it has been shown that DRB1 *1501 is associated with a high PPD responder status compared with DRB1 *1502 in PTB patients. Moreover, the cytokine profile to Mycobacterium leprae heat shock proteins HSP65, HSP18 and their tryptic fragments has been shown to be influenced by HLA-DR2 in association with other DR antigens in leprosy and control subjects.19 Our study on tuberculin skin test response revealed that HLA-DR2 is associated with both low and high responder status to the tuberculin response.17 Bothamley et al.16 have shown that tuberculin responses were higher in individuals with DR15 (a sub-type of HLA-DR2). HLADR2 is also associated with increased antibody response in pulmonary tuberculosis.14 It has been shown that the response to M. tuberculosis 38 kDa antigen is HLA-DR2 restricted and higher antibody response to some of the epitopes of 38 kDa antigen has been seen in Indonesian PTB patients positive for HLA-DR15, (a sub-type of DR2).9 Our recent study14 revealed a trend towards an increased recognition of the immunodominant antigens such as 38, 32/34, 30/31 kDa antigens by the plasma of DR2 positive active and inactive pulmonary TB patients. In general, HLA-DR2 is associated with an increased humoral and cell mediated immune response to M. tuberculosis antigens. Though HLA-DR2 is associated with a high responder status in Southern Asian Indian patients with PTB, HLA-DR2 was more strongly associated with far-advanced smear positive cases than with cases of minimum and moderate radiographic lung lesions.11 Further, extensive disease occurred significantly more frequently in patients with drug failure in whom HLA-DR2 was overwhelmingly present.12 This suggests that in pulmonary tuberculosis, association of HLA-DR2 with tuberculo-immunity and the immunopathogenic mechanism may be regulated by the M. tuberculosis antigen reactive T-cells and the different T-cell sub-types and their cytokines. In the present study, HLA-DR3 was associated with a decreased lymphocyte response to M. tuberculosis culture filtrate antigen as well as mitogens such as PHA, Con-A in the control subjects. Other studies suggested that HLA-DR3 was associated with both responder and non-responder status. A decrease in the frequency of DR3 antigen in Mexican-American patients with TB as compared with healthy persons who were PPD skin-test positive has been shown.4 HLA-DR3 seems to protect against non-responsiveness to mycobacterial antigens in healthy British individuals perhaps caused by a DR3 associated genetic factor.27 Further, HLA-DR3 is associated with high responder (tuberculoid) © 1999 Harcourt Publishers Ltd
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leprosy with strong T-cell activity to mycobacterial antigens in vitro and in vivo.28 However, HLA-DR3 associated nonresponder status of T-lymphocyte responses to PPD has also been reported.29 The decreased response that is associated with DR3 has been suggested to be due to imbalanced cytokine production, that is a decrease in IL-2 and IFN-γ production, showing an increased humoral immune response in the face of a low T-cell immune responsiveness.30 In the present study, low responsiveness was seen not only with M. tuberculosis antigens but also with mitogens (PHA and Con-A). In the present study, the observed antigen and/or mitogens induced suppressive response may be due to some factors/cytokines that are produced by the DR3 positive healthy subjects which may be regulating the response of the lymphocytes. A significantly altered memory response to M. tuberculosis antigen was seen in DR1 negative (non-DR1) cured patients when compared to active TB patients. This suggests that DR1 genes/gene products may be associated with a low memory response when compared to non-DR1 genes/gene products. Though this preliminary finding is interesting, this needs further study. The present study suggests that the immune response of the cured patients to M. tuberculosis antigen is similar to that of normal healthy control subjects. Further, the memory response to the antigen is well maintained even 10–15 years after treatment. Moreover, HLA-DR2 is associated with an increased lymphocyte response in pulmonary TB patients and DR3 with a decreased response in control subjects. HLA-DR antigens such as DR1, DR8 and DR9 also influence the lymphocyte responses to antigen and mitogens. This suggests that the influence of one HLA-DR gene/gene product is regulated by the other DR gene(s). Heterozygous combination of various HLA-DR genes/gene products may be associated with other HLA and non-HLA genes in eliciting a good immune response against M. tuberculosis or may be playing a regulatory role in the mechanism of disease susceptibility to tuberculosis.
ACKNOWLEDGEMENTS Mrs H. Uma was a recipient of a Junior Research Fellowship from the Indian Council of Medical Research, New Delhi, India. The authors thank Mrs V. Shanthi for typing the manuscript.
REFERENCES 1. Dannenberg A M Jr. Pathology of pulmonary tuberculosis. Am Rev Respir Dis 1982; 125: 25–29. 2. Unanue E R, Beller D I, Lu C, Allen P M. Antigen presentation: Comments of its regulation and mechanism. J Immunol 1984; 132: 1–5. 3. Bendentzen K, Morling N, Fomsgaard A et al. Association between HLA-DR2 and production of tumour necrosis factor – alpha and interleukin-1 by mononuclear cells activated by lipopolysaccharide. Scand J Immunol 1988; 28: 599–606.
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4. Cox R A, Downs M, Neimes R E, Ognibene A J, Yamashita T S, Ellner J J. Immunogenetic analysis of human tuberculosis. J Infect Dis 1988; 158: 1302–1308. 5. Mustafa A S, Lundin K E A, Oftung F. Human T cells recognize mycobacterial heat shock proteins in the context of multiple HLA-DR molecules: studies with healthy subjects vaccinated with M. bovis BCG and M. leprae. Infect Immun 1993; 61: 5294–5301. 6. Benacerraf B. Role of MHC gene products in immune regulation. Science 1981; 212: 1229–1238. 7. de Vries R R P. Regulation of T-cell responsiveness against mycobacterial antigens by HLA class 2 immune response genes. Rev Infect Dis 1989; 11(Suppl 2): S400–S403. 8. Dausset J. The Major Histocompatibility Complex in man: past, present and future concepts. Science 1981; 213: 1469–1474. 9. Bothamley G H, Beck J S, Schreuder G M T, et al. Association of tuberculosis and M. tuberculosis-specific antibody levels with HLA. J Infect Dis 1989; 159: 549–555. 10. Khomenko A G, Litvinov V I, Chukanova V P, Pospelov L E. Tuberculosis in patients with various HLA phenotypes. Tubercle 1990; 71: 187–192. 11. Brahmajothi V, Pitchappan R M, Kakkanaiah V N et al. Association of pulmonary tuberculosis and HLA in South India. Tubercle 1991; 72: 123–132. 12. Rajalingam R, Mehra N K, Jain R C, Myneedu V P, Pande J N. Polymerase chain reaction based sequence-specific oligonucleotide hybridisation analysis of HLA-Class II antigens in pulmonary tuberculosis: Relevance to chemotherapy and disease severity. J Infect Dis 1996; 173: 669–676. 13. Selvaraj P, Uma H, Reetha A M et al. HLA antigen profile in pulmonary tuberculosis patients and their spouses. Ind J Med Res 1998; 107: 155–158. 14. Selvaraj P, Uma H, Reetha A M, Xavier T, Prabhakar R, Narayanan P R. Influence of HLA-DR2 phenotype on humoral immunity and lymphocyte response to M. tuberculosis culture filtrate antigens in pulmonary tuberculosis. Indian J Med Res 1998; 107: 208–217. 15. Ottenhoff T H M, Torres P, de las Agus J T et al. Evidence for an HLA-DR4 associated immune-response gene for M. tuberculosis. A clue to the pathogenesis of rheumatoid arthritis? Lancet 1986; II: 310–312. 16. Bothamley G H, Gibbs J H, Beck J S. Delayed hypersensitivity and HLA in smear positive pulmonary tuberculosis. Int Arch Allerg Immunol 1995; 106: 38–45. 17. Selvaraj P, Reetha A M, Uma H et al. Influence of HLA-DR and -DQ phenotypes on tuberculin reactive status in pulmonary tuberculosis patients. Tuberc Lung Dis 1996; 77: 369–373. 18. de Vries R R P, Serjeantson S W, Layrisse Z. In: Albert E D et al. eds. Histocompatibility testing. Berlin; Springer Verlag, 1984; 362–367. 19. Mitra D K, Rajalingam R, Taneja V, Bhattacharyya B C, Mehra N K. HLA-DR polymorphism modulates the cytokine profile of M. leprae HSP-reactive CD4+T cells. Clin Immunol Immunopathol 1997; 82: 60–67. 20. Roche P W, Feng C G, Britton W J. Human T-cell epitopes on the Mycobacterium tuberculosis secreted protein MPT64. Scand J Immunol 1996; 43: 662–670. 21. Jurcevic S, Hills A, Pasvol G, Davidson R N, Ivanyi J, Wilkinson R J. T-cell responses to a mixture of M. tuberculosis peptides with complementary HLA-DR binding profiles. Clin Exp Immunol 1996; 105: 416–421. 22. Geluk A, van Meijgaarden K E, de Vries R R, Sette A, Ottenhoff T H. A DR17-restricted T-cell epitope from a secreted Mycobacterium tuberculosis antigen only binds to DR17 molecules at neutral pH. Eur J Immunol 1997; 27: 842–847.
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23. Zhu X, Venkataprasad N. Thangaraj H S et al. Functions and specificity of T cells following nucleic acid vaccination of mice against Mycobacterium tuberculosis infection. J Immunol 1997; 158: 5921–5926. 24. Boyum A. Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest 1968; 21: 31–50. 25. Terasaki P I, McClelland J D. Microdroplet assay of human serum cytotoxins. Nature 1964; 204: 998–1000. 26. Lowry O H, Rosebrough N J, Farr A L, Randall R J. Protein measurement with Folin Phenol reagents. J Biol Chem 1951; 193: 265–275. 27. Van Eden W, de Vries R R P, Stanford J L, Rook G A W. HLA-DR3
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associated genetic control of response to multiple skin tests with new tuberculins. Clin Exp Immunol 1983; 52: 287–292. 28. Ottenhoff T H M, Haanen J B A G, Geluk A et al. Regulation of mycobacterial heat shock protein reactive T-cells by HLA ClassII molecules: lessons from leprosy. Immunol Rev 1991; 121: 171–191. 29. Van Eden E, Elferink B G, Hermans J, De Vries R R P, Van Rood J J. Role of HLA class II products in proliferative T-lymphocyte responses to PPD. Scand J Immunol 1984; 20: 503–510. 30. Candore G, Cigna D, Gervasi F, Colicci A T, Modica M A, Caruso C. In vitro cytokine production by HLA-B8, DR3 positive subjects. Autoimmunity 1994; 18: 121–132.
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Influence of HLA-DR antigens on lymphocyte response to Mycobacterium tuberculosis culture filtrate antigens and mitogens in pulmonary tuberculosis H. Uma, P. Selvaraj, A. M. Reetha, T. Xavier, R. Prabhakar, P. R. Narayanan Tuberculosis Research Centre (ICMR), Chennai 600 031, India
Summary Setting: Influence of HLA-DR antigens and lymphocyte responses in pulmonary TB patients. Objective: To elucidate the role of HLA-DR genes/gene products on lymphocyte responses to Mycobacterium tuberculosis antigens and mitogens, the present study was carried out in pulmonary tuberculosis during active and cured stage of the disease. Design: Serological determination of HLA-DR antigens was carried out in 50 active TB patients, 44 cured TB patients and 58 normal healthy control subjects. The influence of HLA-DR antigens on peripheral blood lymphocyte responses to M. tuberculosis culture filtrate antigens and mitogens such as phytohaemagglutinin (PHA) and concanavalin-A (Con-A) was studied in the patients as well as normal healthy control subjects. Results: Of all the DR antigens studied, patients (active TB and cured TB) with DR2 antigen showed an increased lymphocyte response (stimulation index) to a higher dose of antigenic (10 µg/ml) stimulation. A significantly lower lymphocyte response to antigen and mitogens was seen in HLA-DR3 positive normal healthy subjects than non-DR3 (DR3 negative) subjects. Conclusion: The present study suggests that HLA-DR genes/gene products may be playing an immunoregulatory role in eliciting an immune response against M. tuberculosis antigens and mitogens induced lymphocyte response in pulmonary TB patients and normal healthy subjects. © 1999 Harcourt Publishers Ltd
INTRODUCTION Tuberculosis is a chronic infectious disease which is predominantly regulated by the cell-mediated immune (CMI) response of the host against the causative organism, Mycobacterium tuberculosis.1 Disease susceptibility to tuberculosis is multifactorial. Host factors such as the immune status and the genetic make up of the individual play a major role in the susceptibility to tuberculosis. Whether the host will resist or succumb to tuberculosis greatly depends on specific T lymphocytes against M. tuberculosis antigens. It is well established that the immune status of an individual is influenced by human Correspondence to: Dr P. Selvaraj, Department of Immunology, Tuberculosis Research Centre (ICMR), Spurtank Road, Chetpet, Chennai 600 031, India. Fax: +91 44 82621371; E-mail: [email protected] [email protected] Received: 29 July 1998; Revised: 25 January 1998; Accepted: 1 February 1999
leucocyte antigens (HLA). The HLA antigens, especially the class II antigens on the antigen presenting cells, present the processed antigens to the CD4+ T lymphocytes, which in turn through their lymphokines, help in subsequent immune responses.2 The secretion of cytokines, such as tumor necrosis factor-α and interleukin-1,3 as well as the lymphocyte response to antigens,4,5 are influenced by HLA genes and their gene products. The HLA class II genes/gene products, otherwise known as immune response (Ir) genes,6 encode for inter-individual differences in antigen specific immune reactivity.7 It has been suggested that the HLA-DR locus has been associated with T-cell helper activity, in human immune responses to various antigens and pathogens.8 Several studies have been carried out to understand whether the susceptibility and/or immune response to M. tuberculosis is associated with HLA phenotype and/or 199
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controlled by genes linked to the Major Histocompatibility Complex (MHC).4,5,9–14 The role of HLA-Class II genes/gene products on delayed type hypersensitivity skin responses, and lymphocyte responses to purified protein derivative of M. tuberculosis has been studied.4,7,10,15–17 The influence of HLA-class-II genes on lymphoproliferative response to mycobacterial antigens has been studied in leprosy patients.18 Recently, the role of HLA-DR polymorphism on the cytokine profile has been studied in leprosy patients.19 Studies have also been carried out to find relevant T-cell epitopes of M. tuberculosis antigens and their peptides in the context of HLA-DR molecules (Mustafa et al., unpublished), and to define their usefulness for diagnosis or vaccine design5,20–23 against tuberculosis in the HLA heterogenous population. Though a number of studies have been carried out in depth to understand the immune response against M. tuberculosis antigens during the active stage of the disease, attempts have not been made to evaluate the immune status (cell mediated immunity) of the patients after treatment (cured patients). Further, most of the studies on the genetic control of cell mediated immune response has been focussed on delayed type hypersensitivity to various mycobacterial antigens and little attention has been paid to the lymphocyte response in leprosy and tuberculosis. Since, a comprehensive study on HLA-DR and lymphocyte response in active and cured pulmonary TB patients and healthy control subjects has not been explored completely, the present study was carried out. MATERIALS AND METHODS Study subjects
Active tuberculosis patients (ATB) Patients attending the Tuberculosis Research Centre (TRC), Chennai, India, with respiratory symptoms and radiographic abnormalities suggestive of pulmonary-TB were studied. These patients had sputum positive for M. tuberculosis by smear and culture. Blood samples were taken before the start of chemotherapy. Among 50 ATB patients studied, 45 were male and five were female. The mean age with SE was 37.7±1.8 for males and 27.8±4.6 for females. Inactive tuberculosis patients (ITB) (Cured patients) Patients classified as suffering from active pulmonary tuberculosis were given anti-tuberculosis treatment at our centre. All of these patients had received a short course of chemotherapy of 6–8 months duration. These patients were treated 10–15 years previously. At the time of blood sample collection, all of these cured patients were in the quiescent stage of the disease. Of the 44 studied, 35 were male and nine were female. The mean age with SE was 42.2±1.7 for males and 36±1.9 for females. Tubercle and Lung Disease (1999) 79(4), 199–206
Controls Spouses of cured TB patients living together for 10–15 years (family contacts; n=19) and clinicians, social workers, health visitors, laboratory volunteers and other staff (n=39) working at TRC for more than 3 years were studied. Among the 58 control subjects studied, 25 were male and 33 were female. The mean age with SE was 38.6±1.7 for males and 36.9±1.5 for females. All of the family contacts and other control subjects were clinically normal at the time of blood sample collection. The patients and spouses were not consanguineous. The other control subjects were not related to any of the patients. The patients and the control subjects were the same ethnic origin and were randomly selected from the same ethnic group. They were the Tamil-speaking South Indian population (belonging to different communities) living in and around Chennai. HLA typing Peripheral blood mononuclear cells (PBMC) were separated from heparinized (20 units/ml) blood samples using Ficoll-hypaque density gradient centrifugation as described by Boyum.24 T- and B-lymphocytes were separated from PBMC using nylon wool column and nylon wool adherent cell population (enriched B-lymphcoytes) was used for HLA-DR typing using a two-stage microlymphocytotoxicity assay.25 Commercial sources of antisera (Biotest, Frankfurt, Germany) were used for HLA-DR typing and at least three antisera were used for each specificity. M. tuberculosis culture filtrate antigen The H37Rv strain of M. tuberculosis was grown for 6 weeks (late logarithimic phase) as a surface pellicle on Sauton’s medium at 37°C. The culture was centrifuged at 3000 rpm for 30 min to remove the bacilli and the supernatant was filtered through a Seitz filter (0.45 µm) (SeitzFilter-Werke GmbH und Co., Badkreuznack, Germany) to ensure complete removal of the bacilli. The culture filtrate antigens were precipitated with 80% ammonium sulphate and dissolved in sterile phosphate buffered saline (PBS) (pH 7.4). The excess salt was removed by dialysis using 0.2 M, 0.1 M and 0.02 M PBS (pH 7.4) at 4°C. The dialysed protein was lyophilized and resuspended in minimal volume of sterile PBS. The protein concentration was estimated by Lowry’s procedure,26 against bovine serum albumin as standard. The culture filtrate protein antigens were aliquoted and stored at –20°C until required. The antigen concentrations used for lymphocyte response were 0.1, 1.0 and 10.0 µg/ml of culture. Mitogens Three different concentrations (0.1, 1.0 and 10.0 µg/ml) of either Phytohaemagglutinin (PHA) (Wellcome Diagnos© 1999 Harcourt Publishers Ltd
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tics, Dartford, UK) or concanavalin-A (Con-A) (Sigma Chemical Co., St Louis, USA) were used to stimulate the lymphocytes.
Lymphocyte Transformation Test PBMC of active-TB, cured-TB (inactive-TB) and control subjects were suspended in RPMI tissue culture medium (Sigma Chemical Co., St Louis, USA) supplemented with 2 mM L-glutamine (Flow Laboratories, Irvine, UK), gentamycin (10 µg/ml) (M. A. Bio Products, Walkerville, MD, USA), fungizone (5 µg/ml) (Squibb, Princeton, NJ, USA) and 10% autologous plasma for controls and cured patients. Autologous serum was used instead of plasma in the cultures set up with active tuberculosis patients samples to avoid clotting. No difference in the counts was observed when autologous plasma or serum was used. 0.1 × 106 PBMC in 200 µl were cultured with either M. tuberculosis culture filtrate antigen or mitogens at 0.1, 1.0, 10 µg/ml. Cultures without any stimulation served as controls. Cultures with M. tuberculosis culture filtrate antigen were kept for 144 h (6 days) in a 96 well ‘U’ bottom tissue culture plate (Costar, Cambridge, MA, USA), at 37°C and 5% carbon dioxide in a CO2 incubator (Flow Laboratories Ltd., Rickmansworth, Herts, UK). Similarly, proliferation assays with PHA and Con-A mitogens were also carried out. The mitogen-stimulated cultures were maintained for 72 h (3 days). The lymphocyte cultures were set up at least in triplicates for each concentration of antigen and mitogens. Tritiated thymidine (3H) (BARC, Trombay, Mumbai, India) was added to each well (1 µCi/well) 16 h before termination. The cells were harvested onto glass fibre filters using a PHD cell harvester (Cambridge Technology Inc, Water Town, MA, USA). The lymphocyte response to M. tuberculosis antigens and mitogens were measured by tritium labelled thymidine uptake by the cells in a scintillation system using Beta counter (LKB, Wallac, Oy, Turku, Finland) and expressed as counts per minute (cpm). The mean cpm value of the triplicate culture was calculated and the stimulation index (SI) was derived using the formula: SI:
mean cpm of antigen stimulated cell cultures mean cpm of cell cultures without antigen
Statistics The frequencies of HLA-DR antigens in patients and controls were determined by direct allelic count. The results on lymphocyte response are expressed as arithmetic mean±S.E. Student’s t-test was used to see the significance of the data. RESULTS HLA-DR2 positive active and cured-TB (inactive-TB) patients showed an increased proliferative response to © 1999 Harcourt Publishers Ltd
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M. tuberculosis antigen stimulation at a higher dose. ATB: DR2: mean SI±SE = 1.4±0.2; 3.0±0.7; 6.2±1.8 and nonDR2 ATB = 1.3±0.1; 2.0±0.3; 3.1±0.5; (0.1; 1.0; 10 µg concentration of antigen respectively) (10 µg/ml: ATB: DR2 vs non-DR2 P > 0.05). ITB: DR2: 8.5±1.8; 21.2±4.3; 39.9±8.1/non-DR2 = 8.2±2.8; 13.0±2.8; 22.8±4.3 at 0.1 µg/ ml, 1 µg/ml and 10 µg/ml concentrations of antigen, respectively (10 µg/ml: ITB: DR2 vs non-DR2 P < 0.05) (Figs 1 and 2). A significantly suppressed response or low response was seen against M. tuberculosis antigen in DR3 positive control subjects compared to non-DR3 individuals, whereas no such difference was observed in the patient group (Controls: DR3 positive – mean SI±SE = 1.4±0.4; 2.9±0.8; 5.9±2.4 and DR3 negatives – 4.3±0.8; 7.9±1.3; 15.0±2.3 at 0.1 µg/ml, 1 µg/ml and 10µg/ml concentrations of antigen respectively; (P < 0.01 at all the three concentrations of antigen) (Fig. 3). Further, a significantly lower response to 1 µg and 10 µg of M. tuberculosis antigen was also seen in DR1 positive inactive TB patients compared with non-DR1 patients (1 µg: P < 0.01; 10 µg: P < 0.01). Such a low response was not seen in control subjects and active TB patients (Fig. 1). The lymphocyte response to antigenic stimulation in different heterozygous combination of HLA-DR antigens of controls, active TB and inactive TB patients were analysed and compared. DR 2/7 and 3/7 heterozygous combinations were present in all the three groups of subjects. Among the different DR7 heterozygous combinations in control subjects (DR 1/7, 2/7, 3/7 and 6/7), DR 3/7 heterozygotes showed a suppressed response at all the doses of antigen stimulation (mean SI±SE in DR3/7 positives = 1.3±0.3; 1.9±0.8; 2.3±0.8 and DR3/7 negatives = 3.8±0.7; 7.3±1.2; 14±2.1) when compared to other heterozygous combinations studied. DR 2/4, 2/6, 2/7, 3/7, 4/6 and 6/7 combinations were analysed in ATB patients. No difference was observed in the response among the different DR antigen combinations. DR2/3, 2/4, 2/5, 2/6, 2/7, 3/7 and 4/7 antigen combinations were analysed in ITB patients DR 3/7 combination showed a trend towards a suppressed response compared to all of the other combinations at all three doses of antigen (DR 3/7 positives = 3.7±2.2; 9.0±4.9; 21.2±15.2 and DR 3/7 negatives = 8.0±1.5; 18.2±3.1; 33.3±5.7). ITB patients with DR 2/7 heterozygous combination showed a trend towards an increased lymphocyte response at higher doses (1 µg and 10 µg/ml) of antigen stimulation than DR 3/7 combination. (ITB: Mean SI±SE: 1 µg 17.9±7.9, 10 µg 49.0±13.5; DR 3/7: 1 µg 9.0±4.9; 10 µg 21.2±15.2). DR3 positive individuals in patients as well as control subjects showed lower lymphocyte response to PHA than DR3 negative individuals. A suppressive/non-responsive effect was seen in control subjects compared to the Tubercle and Lung Disease (1999) 79(4), 199–206
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Fig. 1 Influence of HLA-DR antigens on lymphocyte response to M. tuberculosis culture filtrate antigens (10 µg/ml). Results are expressed as arithmetic mean±S.E. HLA-DR1 +ve vs DR1 –ve: ITB: 0.1 µg; P < 0.05; 1 µg P < 0.01 HLA-DR2 +ve vs DR2 –ve: ATB; P > 0.05; ITB; P < 0.05 n = number of subjects studied Controls: DR1 (n=11); non-DR1 (n=34) DR2 (n=18); non-DR2 (n=40) DR3 (n=9); non-DR3 (n=36) DR4 (n=12); non-DR4 (n=35) DR5 (n=14); non-DR5 (n=34) DR6 (n=10); non-DR6 (n=36) DR7 (n=20); non-DR7 (n=26) DR8 (n=5); non-DR8 (n=39) ATB: DR1 (n=6); non-DR1 (n=39) DR2 (n=23); non-DR2 (n=27) DR3 (n=11); non-DR3 (n=34) DR4 (n=12); non-DR4 (n=33) DR5 (n=5); non-DR5 (n=41) DR6 (n=15); non-DR6 (n=30) DR7 (n=15); non-DR7 (n=31) DR8 (n=2); non-DR8 (n=43) DR9 (n=3); non-DR9 (n=43) DR10 (n=3); non-DR10 (n=42) ITB: DR1 (n=5); non-DR1 (n=36) DR2 (n=23); non-DR2 (n=21) DR3 (n=7); non-DR3 (n=33) DR4 (n=14); non-DR4 (n=27) DR5 (n=6); non-DR5 (n=34) DR6 (n=6); non-DR6 (n=34) DR7 (n=13); non-DR7 (n=27) DR8 (n=3); non-DR8 (n=37) DR9 (n=6); non-DR9 (n=35)
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Fig. 2 Influence of HLA-DR2 antigen on M. tuberculosis antigen (0.1, 1.0 and 10 µg/ml) induced lymphocyte response in controls, active TB (ATB) and inactive TB (TIB) patients. Results are expressed as arithmetic mean±S.E. Controls: DR2 (n=18); non-DR2 (n=40) ATB: DR2 (n=23); non-DR2 (n=27) ITB: DR2 (n=23); non-DR2 (n=21)
patients (Controls – DR3 positive – mean SI±SE = 4.7±1.7; 56.1± 11.4; 50.7±16.2 and DR3 negative = 11.9±2.8; 97.2±13; 88.2±12 at 0.1 µg/ml, 1 µg/ml and 10 µg/ml concentrations of PHA). However the values were not significant. DR8 positive individuals also showed a trend towards a decreased proliferative response to PHA compared to DR8 negative individuals in the control subjects only, (Controls – DR8 positive – 12.9±6.0; 50.3±15.1; 34.2±12.1 and DR8 negative – 9.9±2.5; 94.2±12.1; 87.3±11.3; P > 0.05 (not significant) at 10 µg/ml of PHA). No significant differences in the stimulation indices were observed for other DR antigens (Table 1). Irrespective of the Con-A concentrations used (0.1, 1.0, 10.0 µg/ml) the lymphocyte response in DR3 positive control subjects was significantly decreased when compared © 1999 Harcourt Publishers Ltd
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Fig. 3 Influence of HLA-DR3 antigen on lymphocyte response to M. tuberculosis culture filtrate antigen in controls, active TB (ATB) and inactive TB (ITB) patients. Results are expressed as arithmetic mean±S.E. Controls: DR3 (n=9); non-DR3 (n=36); ATB: DR3 (n=11); non-DR3 (n=34) ITB: DR3 (n=7); non-DR3 (n=33) Controls: DR3 +ve vs non-DR3: P < 0.01 (all concentrations studied)
to non-DR3 (DR3 negative) controls (Controls: DR3 +ve vs DR3 –ve, 1 µg < 0.05; 10 µg; P < 0.05) (Fig. 4 and Table 1). DR1 positive control subjects showed a trend towards a higher proliferative response than DR1 negative individuals. However, this increase was not significant. The lymphocyte response to Con-A in DR1 positive ATB patients was higher than non-DR1 (DR1 negative) subjects at 10 µg/ml concentration (P < 0.01) (Table 1). The proliferative responses to Con-A was decreased in DR8 positive control subjects compared to DR8 negative individuals (Controls – DR8 positive – mean SI±SE = 2.3±0.7; 15.2±10.8; 31.1± 6.4 and DR8 negative – 4.1±1.1; 23±5.2; 56.8±7.6 at 0.1 µg/ml, 1 µg/ml and 10 µg/ml). However, the differences were not significant. On the other hand DR9 positive ITB patients showed an increased lymphocyte response compared to DR9 negatives (non-DR9), the values being significant at 1 µg/ml and 10 µg/ml doses of ConA (ITB – DR9 positive – mean SI±SE = 54.3±28.4; 115±46.8 and DR9 negative 20.9±3.5; 61.1±7.9 at 1 µg/ml and 10 µg/ml concentrations of Con-A respectively;
Table 1 Influence of selected HLA-DR antigens on lymphocyte response to the mitogens (PHA and Con-A) HLA DR
Mean stimulation index±S.E. PHA stimulation CON-A stimulation (10 µg/ml) (10 µg/ml) Controls ATB ITB Controls ATB ITB
DR1+
57.3±8.9 (n=9) 87.4±13.1 (n=28) 99.1±18.6 (n=16) 74.6±10.6 (n=33) 50.7±16.2 (n=8) 88.2±12 (n=29) 34.2±12.1 (n=5) 87.3±11.3 (n=32) 29.3 (n=1) 80.1±10.9 (n=36)
DR1– DR2+ DR2– DR3+ DR3– DR8+ DR8– DR9+ DR9–
35.1±34.1 (n=3) 69.3±13.2 (n=16) 75.5±25.4 (n=7) 54.7±11.5 (n=14) 81.6±40.6 (n=3) 60.6±13 (n=16) 116±0 (n=2) 57.8±11.9 (n=17) 22.8 (n=1) 63.9±12.4 (n=19)
124±82.1 (n=5) 75.9±9.4 (n=34) 82.6±12.4 (n=23) 76.4±22.9 (n=19) 107±36 (n=6) 76.5±14.2 (n=32) 67.7±43 (n=3) 82.5±13.9 (n=35) 127±80 (n=5) 73.4±9.7 (n=34)
60.1±16.8 (n=10) 51.5±7.6 (n=30) 68.1±12.7 (n=17) 51.2±7.6 (n=35) 30.9±7.5 (n=8) 58±8.2 (n=32) 31.1±6.4 (n=5) 56.8±7.6 (n=35) 20.5±14.8 (n=2) 52.3±6.5 (n=38)
4.9±4.5 (n=3) 22.9±3.4 (n=20) 20.6±3.5 (n=10) 21.4±4.4 (n=17) 20.4±16.3 (n=3) 20.6±3.2 (n=20) 47.5±0 (n=2) 18±3.1 (n=21) 10.2 (n=1) 20.6±3.3 (n=23)
78.5±58.5 (n=5) 68.8±8.5 (n=35) 77.5±11 (n=24) 56.4±15.7 (n=20) 61.8±25 (n=7) 72.6±11 (n=33) 73.8±37.4 (n=3) 70.5±10.5 (n=37) 115±46.8 (n=6) 61.1±7.9 (n=35)
(i) Con-A: DR1 +ve vs DR1 –ve: ATB: P < 0.01. (ii) Con-A: Controls: DR3 +ve vs DR3 –ve; P < 0.05. (iii) Con-A: ITB DR9 +ve vs DR9 –ve 1 µg and 10 µg concentrations; P < 0.05. (iv) n=number of subjects studied.
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Fig. 4 Influence of HLA-DR3 on Con-A induced lymphocyte response in controls, ATB and ITB patients. Results are expressed as arithmetic mean±S.E Controls: DR3 (n=8); non-DR3 (n=32) ATB: DR3 (n=3); non-DR3 (n=20) ITB: DR3 (n=7); non-DR3 (n=33) Controls: DR3 +ve vs non-DR3: 1.0 µg P < 0.05; 10.0 µg P < 0.05
P < 0.05). There was no significant difference in the responses elicited by other DR antigens in patients as well as in control subjects. DISCUSSION In the present study, HLA-DR1, DR2 and DR3 were associated with an increased or decreased lymphocyte response to M. tuberculosis culture filtrate antigens and mitogens in healthy control subjects, active TB and inactive TB patients. A trend towards an increased lymphocyte response (SI), to M. tuberculosis antigens (1.0 and 10 µg/ml) was seen in Tubercle and Lung Disease (1999) 79(4), 199–206
HLA-DR2 positive compared to non-DR2 active and inactive TB patients. However, such an increase was not seen in control subjects. An increase in PPD induced lymphoproliferative response has been observed in DR2 positive active TB patients.11 Further, it has been shown that DRB1 *1501 is associated with a high PPD responder status compared with DRB1 *1502 in PTB patients. Moreover, the cytokine profile to Mycobacterium leprae heat shock proteins HSP65, HSP18 and their tryptic fragments has been shown to be influenced by HLA-DR2 in association with other DR antigens in leprosy and control subjects.19 Our study on tuberculin skin test response revealed that HLA-DR2 is associated with both low and high responder status to the tuberculin response.17 Bothamley et al.16 have shown that tuberculin responses were higher in individuals with DR15 (a sub-type of HLA-DR2). HLADR2 is also associated with increased antibody response in pulmonary tuberculosis.14 It has been shown that the response to M. tuberculosis 38 kDa antigen is HLA-DR2 restricted and higher antibody response to some of the epitopes of 38 kDa antigen has been seen in Indonesian PTB patients positive for HLA-DR15, (a sub-type of DR2).9 Our recent study14 revealed a trend towards an increased recognition of the immunodominant antigens such as 38, 32/34, 30/31 kDa antigens by the plasma of DR2 positive active and inactive pulmonary TB patients. In general, HLA-DR2 is associated with an increased humoral and cell mediated immune response to M. tuberculosis antigens. Though HLA-DR2 is associated with a high responder status in Southern Asian Indian patients with PTB, HLA-DR2 was more strongly associated with far-advanced smear positive cases than with cases of minimum and moderate radiographic lung lesions.11 Further, extensive disease occurred significantly more frequently in patients with drug failure in whom HLA-DR2 was overwhelmingly present.12 This suggests that in pulmonary tuberculosis, association of HLA-DR2 with tuberculo-immunity and the immunopathogenic mechanism may be regulated by the M. tuberculosis antigen reactive T-cells and the different T-cell sub-types and their cytokines. In the present study, HLA-DR3 was associated with a decreased lymphocyte response to M. tuberculosis culture filtrate antigen as well as mitogens such as PHA, Con-A in the control subjects. Other studies suggested that HLA-DR3 was associated with both responder and non-responder status. A decrease in the frequency of DR3 antigen in Mexican-American patients with TB as compared with healthy persons who were PPD skin-test positive has been shown.4 HLA-DR3 seems to protect against non-responsiveness to mycobacterial antigens in healthy British individuals perhaps caused by a DR3 associated genetic factor.27 Further, HLA-DR3 is associated with high responder (tuberculoid) © 1999 Harcourt Publishers Ltd
Influence of HLA-DR on lymphocyte response in PTB
leprosy with strong T-cell activity to mycobacterial antigens in vitro and in vivo.28 However, HLA-DR3 associated nonresponder status of T-lymphocyte responses to PPD has also been reported.29 The decreased response that is associated with DR3 has been suggested to be due to imbalanced cytokine production, that is a decrease in IL-2 and IFN-γ production, showing an increased humoral immune response in the face of a low T-cell immune responsiveness.30 In the present study, low responsiveness was seen not only with M. tuberculosis antigens but also with mitogens (PHA and Con-A). In the present study, the observed antigen and/or mitogens induced suppressive response may be due to some factors/cytokines that are produced by the DR3 positive healthy subjects which may be regulating the response of the lymphocytes. A significantly altered memory response to M. tuberculosis antigen was seen in DR1 negative (non-DR1) cured patients when compared to active TB patients. This suggests that DR1 genes/gene products may be associated with a low memory response when compared to non-DR1 genes/gene products. Though this preliminary finding is interesting, this needs further study. The present study suggests that the immune response of the cured patients to M. tuberculosis antigen is similar to that of normal healthy control subjects. Further, the memory response to the antigen is well maintained even 10–15 years after treatment. Moreover, HLA-DR2 is associated with an increased lymphocyte response in pulmonary TB patients and DR3 with a decreased response in control subjects. HLA-DR antigens such as DR1, DR8 and DR9 also influence the lymphocyte responses to antigen and mitogens. This suggests that the influence of one HLA-DR gene/gene product is regulated by the other DR gene(s). Heterozygous combination of various HLA-DR genes/gene products may be associated with other HLA and non-HLA genes in eliciting a good immune response against M. tuberculosis or may be playing a regulatory role in the mechanism of disease susceptibility to tuberculosis.
ACKNOWLEDGEMENTS Mrs H. Uma was a recipient of a Junior Research Fellowship from the Indian Council of Medical Research, New Delhi, India. The authors thank Mrs V. Shanthi for typing the manuscript.
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