Evaluation of T cell immune responses in multi-drug-resistanttuberculosis (MDR-TB) patients to Mycobacterium tuberculosis totallipid antigens

A. S. Shahemabadi*,A. Zavaran Hosseini*,S. Shaghsempour†, M. R. Masjedi†,M. Rayani† and M. Pouramiri‡

*Department of Immunology, Faculty of Medical

Sciences, Tarbiat Modares University, Tehran,

Iran, †National Research Institute of Tuberculosis

and Lung Diseases (NRITLD), Masih Daneshvari

Hospital, Beheshti University of Medical Sciences,

Tehran, Iran, and ‡Mycobacteriology Research

Centre, NRITLD, Masih Daneshvari Hospital,

Beheshti University of Medical Sciences,

Tehran, Iran

Summary

Mycobacterium tuberculosis lipid antigens produce significant T cell responsesin healthy tuberculin reactor [purified protein derivative (PPD-positive]individuals. In the present study, proliferation and interferon (IFN)-g/interleukin (IL)-4 responses were analysed to M. tuberculosis total lipidantigens in T lymphocytes from 25 patients with multi-drug-resistanttuberculosis (MDR-TB). The obtained results were compared with those of30 asymptomatic healthy PPD-positive and 30 healthy tuberculin skin testnegative (PPD-negative) subjects. Peripheral blood mononuclear cells(PBMCs) and T cells (CD4+ and CD8+) were stimulated using autologousimmature dendritic cells. Proliferation responses were assessed using3–{4,5-dimethylthiazol-2-yl}–2,5 diphenyl tetrazolium bromide (MTT).IFN-g/IL-4 concentrations in the supernatant of the CD4+ and CD8+T cellswere measured by enzyme-linked immunosorbent assay. Proliferation assayshowed that the peripheral blood mononuclear cells and CD4+ T cells fromthe MDR-TB patients responded significantly less to the M. tuberculosis totallipid antigens than to the CD4+ T cells in the PPD-positive subjects. Total lipidantigen-specific proliferative responses in the CD8+ T cells from the MDR-TBpatients were minimally detected and the responses were similar to those ofthe PPD-positive subjects. IFN-g production by the CD4+ T cells stimulated bytotal lipid antigens from the MDR-TB patients was decreased significantlycompared with the PPD-positive individuals, whereas IL-4 production in thepatients was elevated. IFN-g and IL-4 production in the CD8+ T cells of theMDR-TB patients was similar to those of the PPD-positive subjects. In con-clusion, it is suggested that stimulated CD4+ T cells by M. tuberculosis totallipid antigens may be shifted to T helper 2 responses in MDR-TB patients.

Keywords: interferon-gamma, interleukin-4, multi-drug-resistant tuberculo-sis, T cells, total lipid antigens

Accepted for publication 29 March 2007

Correspondence: Dr Ahmad Zavaran Hosseini,

Department of Immunology, Faculty of Medical

Sciences, Tarbiat Modares University, Jalale-Ale

Ahmad Avenue, Tehran, Iran, PO Box

14115–331.

E-mail: zavarana@modares.ac.ir

Introduction

Mycobacterium tuberculosis is the causative agent of pulmo-nary tuberculosis (TB) in humans and leads to an estimatedof 2–3 million deaths worldwide each year [1,2]. Theappearance of strains of M. tuberculosis resistance to currentantibiotics is a growing problem, both in the third world andin the developed countries. The strains of M. tuberculosisresistance to both isoniazid and rifampicin with or withoutresistance to other drugs have been termed multi-drug-resistant (MDR) strains [3–5]. On the other hand, the highprevalence of HIV-1 infection in the MDR-TB patients dueto the suppressed immune system is of concern [6].

Substantial studies have indicated that antigen-specific Tcells play an important role in developing and maintainingimmunity to M. tuberculosis [7]. It has also been shown thatT cells and major histocompatibility complex (MHC) class Iand class II molecules are involved in the protective immuneresponse to M. tuberculosis protein antigens [8]. However, inrecent years it has been proved clearly that CD1 moleculesare also involved in the generation of cell-mediated immuneresponses to mycobacterial pathogens [9–12]. Human CD1markers are a family of antigen-presenting molecules thatbind lipids and present them to T cells. These molecules areexpressed constitutively on professional antigen-presentingcells and can be induced in immature dendritic cells

Clinical and Experimental Immunology ORIGINAL ARTICLE doi:10.1111/j.1365-2249.2007.03406.x

285© 2007 British Society for Immunology, Clinical and Experimental Immunology, 149: 285–294

derived from peripheral blood monocytes by treatmentwith granulocyte-macrophage colony-stimulating factor(GM-CSF) and interleukin (IL)-4 [13,14]. CD1-restricted Tcells can contribute to protective immunity by production ofhigh levels of interferon (IFN)-g and IL-4 [15,16]. Adminis-tration of M. tuberculosis lipid vaccine to guinea-pigs createda considerable immune response [17]. It has been revealedrecently that the type of lipids contributes to the severity ofM. tuberculosis strain infection [18,19].

The precise role and relative importance of this novelpathway for antigen recognition in generating protectiveimmunity to M. tuberculosis, especially in TB and MDR-TBpatients, remains poorly understood. The present study wasundertaken to determine the role of CD4+ and CD8+ T cellsin MDR-TB patients against M. tuberculosis total lipid andtotal sonicate antigens in comparison with newly smear-positive TB patients and healthy (positive tuberculin reactorand non-reactor) subjects.

Materials and methods

Preparation of total sonicate antigens

M. tuberculosis strain H37Rv was obtained from the Myco-bacteriology Research Centre, Masih Daneshvari Hospital(Tehran, Iran). Preparation of M. tuberculosis total sonicateantigens was carried out using the method described previ-ously [17]. Briefly, the bacteria were resuspended in Trisbuffer and centrifuged. The supernatant was removed and thebacteria pellet resuspended in sterile phosphate-bufferedsaline (PBS). To inactivate the bacteria, the pellet was resus-pended in ethanol and allowed to stand overnight. Ethanolwas removed by evaporation and the remaining bacteria werelyophilized until desiccated. Finally, a suspension of M. tuber-culosis with a concentration of 20 mg/ml in RPMI-1640 wasprepared. This suspension was sonicated using sonicator (DrHielscher, GmbH, Germany; up to 400 s) on ice for 5 minwith 50% maximum power. The total sonicate was centri-fuged and the supernatant was filtered and stored at -20°C asthe total sonicate antigens. The cells were stimulated by20 mg/ml concentration of the total sonicate antigens.

Preparation of total lipid antigens

The Folch procedure, with some modifications, was used toproduce M. tuberculosis total lipid antigens, as described pre-viously [20]. Briefly, the dried bacteria were resuspended inchloroform: methanol (2 : 1) and sonicated with 50%maximum power for 5 min on ice and mixed overnight on aplatform rocker. The suspension was then centrifuged andthe supernatant was removed. The insoluble bacterial par-ticles were removed by centrifugation and re-extracted bychloroform : methanol, as described above. Supernatantsfrom the centrifugation steps containing soluble total lipidantigens were collected and dried under nitrogen gas stream.

The extracted lipid antigens were weighed and appropriateconcentrations were prepared in chloroform : methanol(2 : 1) and stored at -20°C. The cells were stimulated by20 mg/ml concentration of the total lipid antigens.

Evaluation of total lipid antigens

To evaluate the possible contamination of the protein in thelipid extracted solution, the Lamelli sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE)procedure was carried out on the dried lipids. Electrophore-sis was performed under reducing conditions, as describedpreviously [21]. For this purpose, an SDS-PAGE samplebuffer was added to the dried lipids, boiled for 5 min and runon 12% polacrylamide gel. Standard protein marker (Sigma,St. Louis, MO, USA) was also loaded. To visualize the proteinbands, the silver staining method was used according to theprocedure described [22].

The extracted lipid was also analysed by thin-layer chro-matography (TLC), as described previously [23], with modi-fications in which phosphatidylcholine, phosphatidylinositoland phosphatidylethanolamine mixture (Avanti, PolarLipids, Alabaster, AL, USA) was used as standard. TLC wascarried out on a silica gel F60 plate (Merck, Kronbery,Germany) using petroleum benzene : diethyl ether: aceticacid (8 : 2 : 1) as the solvent system. The lipid bands werevisualized by 10% sulphuric acid spray followed by heatingat 140°C.

Limulus amoebocyte lysate (LAL) assay

Endotoxin contamination in M. tuberculosis total sonicateand total lipid antigen extracts was carried out using aLAL assay (Cambrex Bio Science, Walkersville, MD, USA)according to the manufacturer’s instructions. Briefly, lyo-philized purified endotoxin from Escherichia coli strain0111.B4 was used as control standard. After reconstitution ofthe endotoxin vial with 1·0 ml reagent water, a serial twofolddilution up to 0·125 EU/ml concentration was prepared. LALreagent was used as negative control. The LAL clot assay wasperformed in test tubes to which 250 ml of diluted antigensamples was added and incubated for 60 � 2 min at37°C � 1°C. The test tubes were examined by 180 inversionfor the presence of a stable clot, which was consideredpositive.

Human subjects

Four groups, including MDR-TB, TB, healthy tuberculinreactor [purified protein derivative (PPD)-positive] andhealthy tuberculin skin test negative (PPD-negative) sub-jects, were selected from the Masih Daneshvari Hospital,National Research Institute of Tuberculosis and LungDisease (NRITLD), Beheshti University of Medical Sciences(Tehran, Iran). This study was approved by the Institutional

A. Shams Shahemabadi et al.

286 © 2007 British Society for Immunology, Clinical and Experimental Immunology, 149: 285–294

Review Board (IRB) and Ethical Review Board (ERB) ofNRITLD. All the patients and healthy volunteers consentedto take part in the study.

Healthy tuberculin reactor (PPD-positive) donors

Thirty healthy PPD-positive donors (14 females, 16 males,mean age: 35·7 years) were selected from the Masih Danesh-vari Hospital personnel and patients accompanied by familymembers. All individuals had a history of contact with TBpatients. M. tuberculosis infection was confirmed in the sub-jects using the tuberculin skin test. A positive tuberculin skintest was confirmed if the diameter of induration at the site ofinjection was > 10 mm. These individuals did not show anyclinical TB symptoms.

Healthy tuberculin skin test-negative(PPD-negative) subjects

Thirty healthy PPD-negative donors (13 females, 17 males,mean age: 33·5 years) were selected from the controlpopulation. None of them had any history of contact withTB patients in the family or in the workplace. Tuberculinskin tests were carried out according to the standard method[24]. Briefly, the tuberculin skin test was considered negativeif an initial injection of 1 U of PPD (Razi Institute, Karaj,Tehran) and a follow-up injection of 10 U of PPD were bothnegative (defined as � 10 mm of induration) 2 weeks later.Eleven individuals had received bacille Calmette–Guérin(BCG) vaccine and had had a negative PPD skin test, whilethe remaining individuals had not received BCG vaccine.

Active TB patients

Thirty new smear-positive cases (18 males, 12 females, meanage: 47·7 years) were selected from the Masih DaneshvariHospital. Microscopic and culture examinations of theirsputum specimens were positive for acid-fast bacilli. All ofthem were diagnosed clinically as tuberculosis (TB). Theirblood samples were obtained prior to the initiation ofanti-TB drug therapy.

Multi-drug-resistant tuberculosis (MDR-TB) patients

Twenty-five MDR-TB patients (12 males, 13 females, meanage: 44·2 years) were obtained from the Masih DaneshvariHospital. They had the following inclusion criteria: they hada history of at least one previous period of TB treatmentunder the centre’s direct observation (6 months documen-tation), two positive sputum smear tests and a positivesputum culture. Their susceptibility testing showed resis-tance to isoniazid and rifampin, and their chest X-ray andclinical symptoms were compatible with pulmonary TB.Duration of infection in all the patients was less than 3 years.Characteristics of the patients are showed in Table 1.

Exclusion criteria for all the subjects were: human immu-nodeficiency virus (HIV), hepatitis C virus (HCV) antibody-positive, hepatitis B surface antigen (HBsAg)-positive, anyknown concurrent infection, allergy and asthma, graftorgan-implanted individuals and age < 14 and > 70 years.

From all subjects, 15–20 ml of heparinized whole bloodwas obtained by venipuncture at the same time each day.

Preparation of cells

Peripheral blood mononuclear cells (PBMCs) were isolatedby Lymphodex (Inno-Train, Germany) density centrifuga-tion according to the standard protocol. Viability of thePBMCs was determined by Trypan blue (0·4%) andcounted. The cells were resuspended in complete RPMI-1640 medium (10 mM HEPES buffer, 200 mm l-glutamine,50 U of streptomycin-penicillin/ml, all from Gibco, Auck-land, New Zealand) and cultured in plastic tissue cultureflasks (Nunclon, Nunc A/S, Kamstrupvej, Denmark) for 2 hat 37°C to allow firm adherence of the monocytes. Thennon-adherent PBMCs were isolated and the monocytes werecultured in complete medium supplemented with 10% ABserum (Sigma, Germany) and 200 m/ul IL-4 (R&D Systems,Minneapolis, MN, USA) and 400 m/ul GM-CSF (Roche,Mannheim, Germany). On the third day, the same doses ofIL-4 and GM-CSF were added, and on the fifth day theimmature dendritic cells were detached by 5 mM ethylene-diamine teraacetic acid (EDTA) in PBS and irradiated with5000 rads. Fluorescence activator cell sorter (FACSCaliber)flow cytometry (Becton-Dickinson, San Jose, CA, USA) andcellquest software were used for the analysis of CD14,CD1a, -b, -c and human leucocyte antigen D-related(HLA)-DR (DakoCytomation, Glostrup, Denmark A/S)expressions on the immature dendritic cells.

Magnetic cell sorting

For the enrichment of the CD4+ and CD8+ T cells, non-adherent PBMCs were incubated with anti-CD4 and anti-CD8 magnetic microbeads (Miltenyi-Biotec GmbH,Gladbach, Germany). The PBMCs were washed by PBS-0·05% EDTA and loaded onto mini-magnetic cell sorting(MACS) columns (Miltenyi-Biotec), placed in the magneticfield of a MACS Separator (Miltenyi-Biotec). The separatedcells were analysed for CD4 and CD8 markers using anti-CD4, CD8 and anti-CD3 antibodies (DakoCytomation).Purity of the CD4+ and CD8+ T cells was determined by flowcytometry and resuspended in freezing medium (65% com-plete medium with 20% AB serum, 15% dimethylsulphox-ide) and cryopreserved in liquid nitrogen.

3–{4,5-dimethylthiazol-2-yl}–2,5 diphenyl tetrazoliumbromide (MTT) assay

Immature dendritic cells were cultured in flat-bottomedmicrotitre plates at 30 000 cells/well in 0·20 ml of a complete

T cells immune responses in MDR-TB patients to total lipid antigens

287© 2007 British Society for Immunology, Clinical and Experimental Immunology, 149: 285–294

medium containing 10% AB serum. The cells werestimulated by M. tuberculosis total sonicate and total lipidantigens at 20 mg/ml concentration and incubated for 24 h.Autologus PBMCs and CD4+ and CD8+ T cells with a densityof 100 000 cells/well were added to the immature dendriticcells and incubated for 72 h. The supernatants were collectedfor IL-4 and IFN-g measurements. To the remaining cells,0·1 ml of the medium and then 10 ml of labelling reagentMTT (Roche) at a final concentration of 0·5 mg/ml wasadded and incubated for 4 h. The cells were then mixed withsolubilization solution (Roche) and incubated at 37°Covernight. Optical density was read at 570 nm wavelengthusing 650 nm wavelength references.

Cytokine assay

After 72 h incubation, supernatant fluids from the CD4+ andCD8+ T cell cultures were collected and cytokine concentra-tions in pg/ml were measured with Quantikine human IL-4and IFN-g immunoassay kits (R&D Systems) according tothe manufacturer’s instructions. The enzyme-linked immu-nosorbent assay (ELISA) assay was performed in duplicatefor each sample and the cytokine concentrations were calcu-lated using standard curves.

Statistical methods

Statistical analysis was performed by non-parametric analy-sis and Mann–Whitney U-test. A value of P < 0·05 was con-sidered significant.

Results

Antigen evaluation

M. tuberculosis H37Rv total sonicate and total lipid antigenswere prepared according to Materials and Methods. Wholelipid extracts were analysed by SDS-PAGE and silver stainingto assess protein contamination in the extracted solution. Asshown in Fig. 1a, no protein bands were observed. Theextracted lipid was shown by thin-layer chromatography(TLC) (Fig. 1b). Based on the LAL assay with sensitivity of0·125 EU/ml, lipopolysaccharide (LPS) was undetectable inM. tuberculosis total sonicate and total lipid extracts.

Cell analysis

In order to study the effect of conventional T cell subtypes ininduction of immune responses to M. tuberculosis lipid

Table 1. Profiles of the multi-drug-resistant tuberculosis (MDR-TB) patients.

PN§ Sex/age CS* ID**

TD

(months)† CXR†† DS‡ AT‡‡

P1 M/42 3 + 1·5 10 C A 2

P1 M/43 2 + 2 14 I M > 2

P3 M/38 3 + 1 12 I M 2

P4 F/45 1 + 3 16 C A > 2

P5 F/61 2 + 2 14 I M > 2

P6 F/39 2 + 1 6 I M 2

P7 F/46 3 + 3 11 C A > 2

P8 M/34 3 + 2·5 10 I M > 2

P9 M/57 1 + 2 12 I M > 2

P10 M/49 1 + 3 15 C A > 2

P11 F/43 2 + 1 7 I M > 2

P12 M/48 1 + 1 8 I M 2

P13 M/56 2 + 2 11 I M 2

P14 F/34 3 + 1 10 I M 2

P15 M/51 2 + 3 17 I M 2

P16 F/43 2 + 2 14 I M 2

P17 F/41 1 + 1 10 C A > 2

P18 M/44 2 + 1 8 I M > 2

P19 M/38 3 + 2 7 C A > 2

P20 M/56 3 + 3 18 C A > 2

P21 F/42 3 + 2 14 C A 2

P22 F/47 2 + 2 16 I M > 2

P23 F/44 3 + 3 20 I M > 2

P24 M/53 3 + 3 19 C A > 2

P25 M/36 2 + 2 12 C A > 2

§Patient number (PN). *Culture sputum (CS) test result. **Diagnosed infection (years), confirmed by positive sputum culture. †Duration of

treatment (DT) in months. ††Chest X-ray results. C, cavity; I, infiltration without cavity. ‡Disease stage (DS): A, advanced; M, moderate. ‡‡Anti-biogram

test (AT) results: 2, resistance to isoniazid and rifampcin: >2 resistance to other antibiotics in addition to isoniazid and rifamcin.

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288 © 2007 British Society for Immunology, Clinical and Experimental Immunology, 149: 285–294

antigens, CD3+ CD4+ and CD3+ CD8+ T cells were separatedfrom the non-adherent PBMCs by magnetic cell sortingaccording to Materials and Methods. More than 95% of theobtained cells from the magnetic cell sorting expressed CD4and CD8 markers according to the flow cytometry analysisdocuments (Fig. 2).

Proliferation responses

For comparative analysis of proliferation responses in theMDR-TB patients with the other groups, the PBMCs and T(CD4+ and CD8+) lymphocytes were cultured with autolo-gous immature dendritic cells primed by M. tuberculosis total

(a)

Mole

cula

r w

eig

ht (D

al)

Pro

tein

mark

er

Extr

acte

d lip

id

Pro

tein

mark

er

(b)

116 000

84 000

66 000

55 000

45 000

36 000

29 000

24 000

20 000

14 200

6 500

1 2 1 21

Fig. 1. Biochemical analysis of M. tuberculosis total lipid extracts. (a) SDS-PAGE analysis of M. tuberculosis lipid extracts. Lipid extracts from M.

tuberculosis H37Rv were analyzed by SDS-PAGE and silver staining to assess protein contamination. In Lane 1, 10 mg wide molecular protein marker

and in Lane 2, 100 mg lipid extract were loaded. (b) Thin layer chromatography (TLC) analysis. M. tuberculosis total lipid extract was evaluated

using TLC according to the Materials and Methods. In Lane 1, 100 mg M. tuberculosis total lipid extract and in Lane 2, 40 mg phosphadidylcholin

(PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) mixture were loaded.

T cells immune responses in MDR-TB patients to total lipid antigens

289© 2007 British Society for Immunology, Clinical and Experimental Immunology, 149: 285–294

sonicate and total lipid antigens. After 72 h of stimulation,the proliferation responses were assessed using MTT. Prolif-erative responses of the PBMCs to total sonicate antigensrevealed that there was no significant difference in thehealthy donors in this regard, but there was a significantdecrease in the TB and MDR-TB patients (P = 0·00). In

contrast, the PBMC proliferative responses to total lipid anti-gens in the PPD-positive donors were significantly greaterthan in the other groups (P = 0·001), while the responses inthe PPD-negative, TB and MDR-TB patients were similar(Fig. 3). Accordingly, PBMC proliferation responses to thetotal sonicate and total lipid antigens in the MDR-TBpatients were lower than those in the healthy PPD-positivedonors.

For elucidation of T cell subtype importance in M. tuber-culosis infection, the CD4+ and CD8+ T cell proliferativeresponses to the antigens in the four groups were analysed.Our findings showed that proliferative responses ofthe CD4+ T cells to the total sonicate antigens in thePPD-positive (OD = 0·501 � 0·071) and PPD-negative(OD = 0·453 � 0·074) healthy donors were greaterthan in the TB (OD = 0·408 � 0·076) and MDR-TB(OD = 0·393 � 0·060) patients (P = 0·002). On the otherhand, these responses in the TB and MDR-TB patientsshowed no differences (Fig. 3). According to Fig. 3, prolifera-tive responses of the activated CD4+ T cells by the total lipidantigens in the PPD-positive donors (OD = 0·417 � 0·060)showed a significant increase relative to the PPD-negative healthy subjects (OD = 0·228 � 0·066), TB(OD = 0·190 � 0·051) and MDR-TB patients (OD =0·197 � 0·042) (P = 0·001). To elucidate the role of the CD8+

T cells in M. tuberculosis infection, we also investigated theproliferative responses of these cells in all the groups. Theresults showed that proliferative responses of the CD8+ Tcells to the total sonicate antigens had no significant differ-ences in the four groups (P = 0·243), but the responses wereweak compared to those of the CD4+ T cells. Proliferativeresponses of the CD8+ T cells stimulated by the total lipidantigens were very weak in the study groups and, in somecases, no responses were observed (Fig. 3). Taken together,

Data. 115(a)

(b)

1000

SS

C-H

01000

FSC-H

R2

Data. 115

FL2-H

104

104

Quad % Gated

UL

UR

LL

LR

0·20

98·04

0·98

0·77100

101

102

103

104

0FL1-H

Data. 1221000

SS

C-H

01000

FSC-H

Data. 122

FL2-H

Quad % Gated

UL

UR

LL

LR

1·10

98·06

0·16

0·68100

101

102

103

104

0FL1-H

Fig. 2. Analysis of the separated CD3+ CD4+ and CD3+ CD8+ T cells:

Nonadherent PBMCs were separated to the CD3+ CD4+ and CD3+

CD8+ T cells by magnetic cell sorting according to the Materials and

Methods. Purity of the CD4 and CD8 T cells were analyzed by

flowcytomery. (a) CD3+ CD4+ T cells analysis (FL1-H = Anti-CD3

FITC, FL2-H = Anti-CD4 PE). (b) CD3+ CD8+ T cell analysis

(FL1-H = Anti-CD3 FITC, FL2-H = Anti-CD8 PE). Purity of the

CD3+ CD4+ T and CD3+ CD8+ T cells was 98·04% and 98·06%,

respectively.

0·70 Cells without antigenic stimulation

Total sonicate antigens

Total lipid antigens0·60

0·50

0·40

0·30

Pro

life

ratio

n r

esp

on

se

s,

OD

(me

an

± s

.d.)

0·20

0·10

0·70

0·60

0·50

0·40

0·30

0·20

0·10

PPD-pos PPD-neg

PBMCs CD4+ T cells CD8+ T cells

MDR-TBTB PPD-pos PPD-neg MDR-TBTB PPD-pos PPD-neg MDR-TBTB

Fig. 3. Proliferation responses of the PBMCs, CD4+ and CD8+ T cells to M. tuberculosis total sonicate and total lipid antigens. Proliferative responses

of the PBMCs to both the total sonicate and total lipid antigens in the PPD-positive donors were significantly higher than in the other groups

(P = 0·00), while the responses to total lipid antigens in the PPD-negative subjects, TB and MDR-TB patients were similar (P = 0·233). Proliferative

responses of the CD4+ T cells to total sonicate and total lipid antigens in the PPD-positive subjects were higher than in the TB and MDR-TB

patients (P = 0·00), while the responses to total lipid antigens in the PPD-negative, TB and MDR-TB patients were similar (P = 0·033) to each

others. Also proliferative responses of the CD8+ T cells to the total sonicate and total lipid antigens in the all subjects were similar (P > 0·005).

A. Shams Shahemabadi et al.

290 © 2007 British Society for Immunology, Clinical and Experimental Immunology, 149: 285–294

proliferative responses of the CD4+ T cells to the total lipidantigens in the MDR-TB and TB patients were significantlydecreased comparing to the PPD-positive donors. On theother hand, CD8+T cells responses in this regard were similarin all the groups.

IFN-g production by CD4+ and CD8+ T cells

The CD4+ and CD8+ T cells of the MDR-TB, TB patients andhealthy donors were stimulated by M. tuberculosis total soni-cate (20 mg/ml) and total lipid antigens (20 mg/ml). After72 h incubation, the supernatants were collected and theirIFN-g and IL-4 concentrations were measured by ELISA.Mean IFN-g concentrations in each group were comparedwith those of the other groups by Mann–Whitney U-test.

IFN-g

The results showed that mean IFN-g concentrations in theMDR-TB and TB patients were significantly lower than theirvalues in the PPD-positive healthy donors (P = 0·004). Inaddition, IFN-g levels in the PPD-negative donors werelower than in the PPD-positive subjects (P = 0·063) (Fig. 4a).

Evaluation of IFN-g production by the CD4+ T cells inresponse to the total lipid antigens showed that the meanIFN-g concentration in the PPD-positive subjects was sig-nificantly higher than in the PPD-negative healthy donors(P = 0·003). On the other hand, in the TB and MDR-TBpatients, the mean IFN-g concentration was lower than inthe healthy PPD-negative and PPD-positive donors(P = 0·00) (Fig. 4a). Based on our findings, there was nosignificant difference in the mean IFN-g concentration in theCD8+ T cell culture supernatant in response to the totalsonicate antigens among the different groups. Besides, only aweak IFN-g production response to the total lipid antigenwas detected (P = 0·012) (Fig. 4b). According to these results,IFN-g production in the stimulated CD4+ T cells by totallipid antigens in the TB and MDR-TB patients was sup-pressed in comparison to the healthy donors.

IL-4

IL-4 concentration in the supernatants of the CD4+ andCD8+ T cells were assessed simultaneously with IFN-g,according to Methods and methods. In the MDR-TB and TBpatients, production of IL-4 by CD4+ T cells in response tototal sonicate antigens were significantly greater than its cor-responding values in the PPD-positive and PPD-negativehealthy donors (P = 0·004) (Fig. 5a), while it was similarbetween the PPD-positive and PPD-negative subjects. Inresponse to lipid antigens, IL-4 concentration in the TB andMDR-TB patients was higher than in the healthy donors(P = 0·00), while it was similar between the PPD-positiveand PPD-negative subjects (P = 0·233) (Fig. 5a). In response

to the total sonicate antigens, production of IL-4 by the CD8+

T cells in the MDR-TB and TB patients was significantlyhigher in the healthy donors (P = 0·00). The CD8+ T cellsstimulated by total lipid antigens did not produce significantIL-4 in all the groups (Fig. 5b). Collectively, the stimulatedCD4+ T cells by total lipid antigens produced higher levels ofIL-4 in the MDR-TB and TB patients than in the healthydonors.

MDR-TBTBPPD-negPPD-pos

MDR-TBTBPPD-negPPD-pos

4000

(a)

(b)

3000

2000

1000

0IFN

-γ p

rod

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y C

D4

+T

ce

lls (

pg

/ml)

800

600

400

200

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rod

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n b

y C

D8

+T

ce

lls (

pg

/ml)

Fig. 4. IFN-g production by the CD4+ and CD8+ T cells. (a) IFN-gproduction by the CD4+ T cells. The mean of IFN-g concentration in

response to the total sonicate and total lipid antigens in the TB and

MDR-TB patients was significantly lower than in the healthy donors

(P = 0·004). IFN-g titer in the TB and MDR-TB patients also were

similar (P = 0·123). IFN-g production in response to the total lipid

antigens in the PPD-positive donors was higher than in the other

groups (P = 0·00). In the TB and MDR-TB patients, level of IFN-gproduction was lower than in the PPD-negative donors (P = 0·002).

(b) IFN-g production by the CD8+ T cells. IFN-g production in the

CD8+ T cells stimulated by total sonicate antigens was higher in the

PPD-positive subjects than in the other groups, but statistically it was

not significant (P = 0·123). Differences in IFN-g response to the total

lipid antigens in the four groups were not significant (P = 0·134). :

Cells without antigenic stimulation, : Cells stimulated with PHA, :

Cells stimulated by total sonicate antigens, : Cells stimulated with total

lipid antigens.

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291© 2007 British Society for Immunology, Clinical and Experimental Immunology, 149: 285–294

Discussion

MDR-TB is now a serious problem in the control and man-agement of M. tuberculosis infection even in the developedcountries [1,2]. The high prevalence of HIV infection inMDR-TB patients is of great concern. Therefore, furtherresearches into the recognition of immune responses mecha-nisms and aetiology of MDR-TB is essential. In the presentstudy, antigen preparation was carried out with LPS-free

solutions and instruments. On the other hand, in M. tuber-culosis, LPS exists in undetectable amounts [24]. Moreover,the total lipid antigens were used for stimulation of the cellsin the same conditions in all the patients and healthysubjects. Several researchers have investigated the role of M.tuberculosis protein antigens in PBMCs and T cell stimula-tion [25–27]. While presentation of lipids and glycolipidsantigens by CD1 molecules is an important pathway in T cellimmune responses [15,16,28], the function of T lympho-cytes against M. tuberculosis lipid antigens in TB andMDR-TB patients is less investigated. In the present study,for the first time, the function of CD4+ and CD8+ T cells inMDR-TB patients against M. tuberculosis total lipid antigenswas assessed.

In agreement with previous studies [16,29–32], our resultsshow that proliferation responses of the PBMCs cells to M.tuberculosis total sonicate antigens in the PPD-positivedonors were significantly greater than in the TB andMDR-TB patients. It was also elucidated that proliferationresponses to the CD4+ T cells from PPD-positive donors tototal lipid antigens increased significantly in comparison tothe other groups, whereas the same number of CD4+ andCD8+ T cells (100 000 cells/well) was used in MTT andcytokine assays. It seems that, in PPD-positive donors, totallipid antigens recruit T cells memory immune responses. Onthe other hand, it was revealed that such responses in the TBand MDR-TB patients were suppressed. It is possible thatlipid-specific T cells were accumulated in the inflammatorytissues of the lung in TB and MDR-TB patients. Stenger et al.showed that expression of CD1 molecules on monocytesinfected with M. tuberculosis was decreased [33]. Also, ourdata demonstrate (data not shown) that stimulated imma-ture dendritic cells by total lipid antigens in the MDR-TBpatients up-regulated IL-10 production.

Proliferation responses to the total lipid antigens were alsoobserved in PPD-negative subjects who have not beeninfected previously with M. tuberculosis. It seems that therewas cross-reactivity between M. tuberculosis lipid antigensand the other bacteria lipids. Hence, further studies on themitogenic properties of M. tuberculosis lipid antigens aresuggested. Our results, in agreement with the other studies[16], confirmed that CD8+ T cell proliferation responses tototal lipid antigens was very weak, and the role of these cells inthe pathogenesis of the TB and MDR-TB patients was notcritical.

In analysis of IFN-g and IL-4 production, our results showthat IFN-g production by CD4+ T cells in the PPD-positivedonors in response to M. tuberculosis total sonicate and totallipid antigens increased significantly compared to the othergroups. Other researchers have shown that the activatedCD4+ T cells by M. tuberculosis lipid antigens from the PPD-positive healthy donors produced more IFN-g than in thePPD-negative subjects [16]. According to other studies,IFN-g production by the PBMCs in response to the 30 kDalM. tuberculosis antigen in the MDR-TB and TB patients was

MDR-TBTBPPD-negPPD-pos

MDR-TBTBPPD-negPPD-pos

1000

(a)

(b)

800

600

400

200

0

IL-4

pro

duct

ion

by C

D4+

T c

ells

(pg

/ml)

800

600

400

200

0

IL-4

pro

duct

ion

by C

D8+

T c

ells

(pg

/ml)

Fig. 5. IL-4 production by the CD4+ and CD8+ T cells. (a) IL-4

production by the CD4+ T cells. Mean of IL-4 concentration in

response to the M. tuberculosis total sonicate antigens in the TB and

MDR-TB patients was significantly greater than those in the healthy

donors (P = 0·004). IL-4 concentration in the PPD-positive and

PPD-negative donors was similar (P = 0·233). IL-4 production in

response to the M. tuberculosis total lipid antigens in the TB and

MDR-TB patients was higher than in the other groups (P = 0·00). (b)

IL-4 production by the CD8+ T cells: IL-4 production by the CD8+ T

cells stimulated by the M. tuberculosis total sonicate antigenn in the

TB and MDR-TB patients was higher than in the healthy subjects

(P = 0·003). IL-4 response in the M. tuberculosis total lipid antigens in

the four group was not significant (P = 0·154). : Cells without anti-

genic stimulation, : Cells stimulated by PHA, : Cells stimulated by

total sonicate antigens, : Cells stimulated by total lipid antigens.

A. Shams Shahemabadi et al.

292 © 2007 British Society for Immunology, Clinical and Experimental Immunology, 149: 285–294

suppressed [34,35]. Similar studies have confirmed that thedecrease of IFN-g production in MDR-TB and TB patientswas not due to the increase of IL-10 [35,36]. In addition, ahigh frequency of T cell apoptosis, a decrease in IL-12Rb1,IL-12Rb2 expression and up-regulation of TGF-b1 andIL-10 simultaneously in mycobacterial infection could be thecause of IFN-g down-regulation in CD4+ T cells from the TBand MDR-TB patients [30–32,36,37]. However, the obviousreason for the depression of IFN-g production by CD4+ Tcells against M. tuberculosis lipid antigens in TB andMDR-TB patients is not well known. It is postulated thatdefects in CD1 expression on antigen-presenting cells inmycobacterial infection decreased lipid antigen presentationto the T cells.

Our results show that IFN-g production in the stimulatedCD8+ T cells by M. tuberculosis total lipid and total sonicateantigens in the TB and MDR-TB patients had no significantdifferences with the healthy donors. The results of a study bySmith et al. [38] have shown that intracellular IFN-g produc-tion in the CD8+ T cells stimulated by M. tuberculosis in theTB patients was decreased significantly in comparison to thePPD-positive donors.

Based on our findings, CD4+ T cells of the TB andMDR-TB patients activated by M. tuberculosis total sonicateantigens produced high levels of IL-4 compared with thePPD-positive donors. A study by Zhang revealed that IL-4production by T cells infected with live M. tuberculosis in TBpatients and PPD-positive donors were similar [29]. A studyby Bai et al. showed that IL-10 and IL-4 expression on thebiopsies of granuloma tissues in pulmonary TB was reducedcompared to PPD-positive subjects [39]. Based on themajority of studies, expression of IL-4 is elevated in T cellsstimulated by M. tuberculosis protein antigens [36,38,40]. Itseems that M. tuberculosis protein antigens and lipid antigenshave similar behaviour in stimulation of CD4+ T cells.

Further to the present study, the CD8+ T cells from the TBand MDR-TB patients produced prominent IL-4 comparedwith the healthy donors. In agreement with our results,another study showed that CD8+ T cells in the TB patientsshifted to IL-4 production [42]. According to other studies,increased IL-4 expression in the lymphocytes infected to M.tuberculosis promoted CD30 expression which, in turn, sen-sitized the lymphocytes to TNF-a-mediated apoptosis [24].Therefore, IL-4 can suppress cell-mediated immunity in TBand MDR-TB patients. In another study, IL-4 knock-outmice developed large granulomas accompanied by a signifi-cant increase in lung colony-forming units (CFU) of M.tuberculosis [41]. Overall, these data show that IL-4 may alsohave a beneficial effect in the prevention of lung tissuedestruction induced by T helper 1 (Th1) cells. Accordingly,investigation of the exact role of IL-4 in the pathogenesis ofTB and MDR-TB patients is strongly suggested.

It is concluded that proliferation responses and IFN-gconcentrations in T lymphocytes stimulated by total lipidantigens in the TB patients were slightly decreased compared

to MDR-TB patients, but the differences were not statisticallysignificant. IL-4 concentration in the TB patients was alsoincreased in comparison to MDR-TB patients, and not sta-tistically significant (P = 0·01). The responses may be relatedto the clinical status and chemotherapy diet in the patients.Other studies also showed that 2 or more weeks after che-motherapy of the immune system functions were improvedin the TB patients [18].

According to our results, CD4+ T cell proliferationresponses and IFN-g production to M. tuberculosis lipid anti-gens in the PPD-positive healthy donors were significantlyhigher than in the MDR-TB patients. Further, lipid antigensin all the patients promoted IL-4 expression in the CD4+ Tcells. Therefore, it appears that M. tuberculosis lipid antigens,like protein antigens, play an important role in the specificimmune response. Taken together, the role of M. tuberculosislipid antigens should be considered in the designation ofeffective vaccines and treatment protocols.

Acknowledgements

This work was supported by a grant from NIRTLD, MasihDaneshvari Hospital, Shaheed Beheshti University ofMedical Sciences and Tarbiat Modares University (Tehran,Iran). The authors also thank Dr Farnia for the gift ofM. tuberculosis H37Rv.

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