Immunology 1994 81 171-176

Thl/Th2 profiles in tuberculosis, based on the proliferation and cytokine response of blood lymphocytes to mycobacterial antigens
H.-M. SURCEL,* M. TROYE-BLOMBERG,t S. PAULIEt G. ANDERSSON,4 C. MORENO,* G. PASVOL & J. IVANYI* *MRC Tuberculosis and Related Infections Unit, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K. tDepartment of Immunology, Stockholm University, Sweden, $Department of Medical Microbiology and Clinical Immunology, University of Lund, Sweden, and Unit of Infectious Diseases and Tropical Medicine, St Mary's Hospital Medical School, Northwick Park Hospital, London, U.K.

SUMMARY
Proliferation and cytokine production profiles by blood mononuclear cells in response to in vitro stimulation with mycobacterial antigens were compared in patients with active tuberculosis and in sensitized healthy controls. Interleukin-4 (IL-4) and interferon-y (IFN-y) were detected at single-cell level using the ELISPOT assay. Patients showed significantly (P < 0-01) increased numbers of IL-4secreting cells and decreased thymidine incorporation, but no significant difference in IFN-yproducing cells in response to the 38,000 MW or 19,000 MW antigens and their immunodominant peptide epitopes. Pronounced individual variations were found in both patient and control groups, when comparing the responsiveness to the mycobacterial extract, two protein antigens and five synthetic peptides. None of the antigens or peptides tested showed preferential stimulation of either IL-4- or IFN-y-secreting T cells, and proliferation was not correlated with either IL-4 or IFN-y production. In particular, cytokine responsiveness was of similar frequency in subjects who did or did not show positive proliferation, indicating that the latter test was not fully representative of the active T-cell repertoire. It is concluded that the demonstrated Th2 type of profile in response to two prominent mycobacterial antigens may play a role in the mechanisms of defective host resistance in tuberculosis.
INTRODUCTION

Resistance to tuberculosis (TB) depends crucially on antigenspecific T-cell mediated activation of macrophages which are the major effectors of cell-mediated killing of intracellular pathogenic mycobacteria. Lymphocyte proliferation in vitro against the 'purified protein derivative' (PPD), containing a large variety of antigens, is decreased in tuberculosis patients.'A An inverse relationship between T-cell proliferation and antibody levels to Mycobacterium tuberculosis antigens has been considered to be an indicator of defective resistance in tuberculosis

patients.3'5
The profile of secreted cytokines in vitro is taken as indicative of T-lymphocyte function in vivo, but the possible relationships between antigen specificity and T-lymphocyte function have so far not been examined. Murine CD4+ T cells have been divided into at least two different subsets (Thl and Th2), based on
Received 26 July 1993; revised 18 August 1993; accepted 21 October 1993.
Abbreviations: BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate in dimethylformade/p-nitroblue tetrazolium chloride; Fmoc, g-fluorenyl

methoxy carbonyl; HOBT/PyBOP, 1-hydroxybenzotriazone/benzotriazone-l-yl-oxy-TRIS-pyrrolidine-phosphonium hexafluoro phosphate. Correspondence: Dr H.-M. Surcel, National Public Health Institute, Box 310, 90100 Oulu 10, Finland.
17i

cytokine profiles that they secrete upon antigen stimulation.6 Thi cells characteristically secrete interleukin-2 (IL-2) and interferon-y (IFN-y), whereas Th2 lymphocytes produce typically IL-4, IL-5 and IL-10, which enhance antibody synthesis of B cells6 and play a role in allergic diseases.7 CD4+ T cells with an intermediate cytokine profile (ThO) have also been described.8 The secreted cytokines of Th I and Th2 cell types can mutually regulate and inhibit each other's functions.9 Therefore, the fine balance between the secreted cytokines is important for the resulting nature of host resistance against the pathogen. The Thl/Th2 diversification on the basis of cytokine secretion has recently been examined in parasitic and viral diseases9 and in leprosy.'0 IL-2 and IFN-y coding mRNA was most evident in tuberculoid leprosy, whilst mRNA for IL-4, IL-5 and IL-10 was predominant in lepromatous leprosy. These findings indicate that the Th 1 phenotype can be associated with certain forms of the disease as well as with protection. The present study investigated ThI/Th2-like cell profiles in patients with active tuberculosis. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with purified 38,000 MW and 19,000 MW antigens of M. tuberculosis, both representative of lipoproteins" which have previously been found to induce the most frequent and highest titres of serum antibodies in tuberculosis.'2"3 Association of anti-38,000 MW antibody levels with the severity of multibacillary disease'4 made this antigen particularly suitable for the study of Th2-like response profiles in the human.

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Lymphocyte cultures PBMC were isolated from citrated blood by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). The PBMC were suspended in RPMI-1640 (Gibco, Paisley, U.K.), supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 gg/ml streptomycin and 5 % A+ heat-inactivated human serum. The PBMC (1 x 105/well) were cultured in triplicate wells of 96-well round-bottomed microtitre plates, in the presence of the appropriate antigen, in a total volume of 200 p1, for 6 days in a humidified 5% CO2 atmosphere at 37. [3H]thymidine was present in the cultures for the last 18 hr and the proliferation was assayed from the incorporated radioactivity measured by using a liquid scintillation counter. The results are expressed as geometric mean counts per minute (c.p.m.) of triplicate cultures or in terms of the stimulation index (SI). The SI was calculated as a ratio of the mean proliferative response (c.p.m.) in the presence or absence of antigen. SI>3 was considered as a positive response.16 Minimal antigen concentrations, which in preliminary experiments gave maximal lymphocyte proliferative responses, were used throughout this study. Thus, MTSE, 38,000 and 19,000 MW proteins were used in a final concentration of 10 pg/ml, and the synthetic peptides were used at 25 pg/ ml.

The immunogenic and genetically permissive T-cell epitopes of the 38,000 MW and 19,000 MW antigens have been mapped using the proliferative assay.'5-'7 Corresponding synthetic peptides were used to explore the relationships between epitope specificity and T-cell function in this study.
MATERIALS AND METHODS

Subjects Nineteen patients (mean age 39-8+21-5 years) with active tuberculosis were diagnosed by routine clinical, bacteriological and histological parameters. Thirteen of the patients had active pulmonary tuberculosis, of which 10 had been confirmed as smear positive. Six patients had extrapulmonary tuberculosis: pleural (P7 + P1), spinalosteomyelitis (P2), jejunal (P8), cervical abscess (P5) or cervical adenopathy (P23). These patients had neither suspicion nor evidence of human immunodeficiency virus (HIV) infection. Blood was drawn either before or within 2 weeks of the onset of chemotherapy. PPD-positive healthy laboratory donors ranging in age from 23 to 58 years, who had no history of previous clinical tuberculosis, served as controls. Patients and healthy donors had similar sex, race and age distribution, but were not matched.
Antigens Mycobacterium tuberculosis, strain H37Rv (Difco, Detroit, MI), soluble extract (MTSE) was prepared by mechanical disruption. 13 The 38,000 MW protein was isolated from the culture supernatant of M. tuberculosis, strain H37Rv, using 70-90% ammonium sulphate and alcohol precipitation, followed by gel filtration.18 Protein eluted from the DE52 gel bed with 0-1-0-2 M NaCl showed only a single band of 38,000 MW in an overloaded sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue (results not shown). The 19,000 MW recombinant protein expressed in M. smegmatis'9 was obtained as a gift from Dr T. Garbe (MRC, London, U.K.).

Synthetic peptides
Peptides comprising 16-20 amino acids, representing the sequence of the previously determined T-cell immunogenic epitopes of the 38,000 MW and 19,000 MW protein antigens of M. tuberculosis, were synthesized by simultaneous multiple peptide synthesis, as described previously.'6 Briefly, the Fmoc methodology was used, with a trialkoxy-diphenyl-methylester (Rink) resin and HOBT/PyBOP-activated coupling. After cleavage with trifluoroacetic acid (TFA), the crude peptides were purified by gel filtration (Sephadex G-15, Pharmacia, 25% aqueous acetic acid). Homogeneity was confirmed by reversephase high-performance liquid chromatography (HPLC) (Zorbax ODS, Jones Chromatography, Hengoed, U.K., 0-1% TFA in water/acetonitrile) and amino acid sequences were determined by automated Edman degradation. The sequences of peptides were derived from the 38,000 MW antigen: 38-A (1-20) MKIRLHLLAVLTAAPLLLA, 38-I (65-83) FNLWGPAFHERYPNVTITA, 38-G (350-369) DQVHFQPLPPAVVKLSDALI, or 19,000 MW antigen: 196A (50-64) GAASGPKWIDGKDQN and 19-7 (61-80) VTGSVVCTTAAGNVNIAIGG. Each peptide used in these experiments was from a single batch.

Cytokine-producing cells Cytokine production was induced by incubating the isolated PBMC (106/ml) in RPMI-1640 supplemented with 5% fetal calf serum (FCS) in the presence or absence of phytohaemagglutinin (PHA; 10 pg/ml; Wellcome, Beckenham, U.K.) or the appropriate mycobacterial antigen. IFN-y- and IL-4-secreting cells were determined using a modification of an ELISPOT assay described earlier.20 Briefly, nitrocellulose-bottomed 96-well Millititer HA plates (Millipore Co., Bedford, MA) were coated with 100 p1 of monoclonal antibody (mAb) 82-4 for IL-4 (Department of Immunology, University of Stockholm, Sweden) or mAb 7-B6-1 for IFN-y (Chromogenix AB, M6lndal, Sweden) at a concentration of 15 pg/ml in phosphate-buffered saline (PBS), and incubated overnight at 4. Unbound antibodies were removed by four successive washings with PBS. Freshly isolated PBMC, suspended in RPMI-1640 medium containing 5% FCS, were either incubated in round-bottomed 96-well plates for 72 hr before transfer to anti-IFN-y antibodycoated nitrocellulose-bottomed plates, or added directly to the anti-IL-4-coated wells (1 x 105 PBMC/well) in the presence of antigen and incubated for 20 hr in a humidified 5% CO2 atmosphere at 37. After incubation, the cells were removed by washing the wells four times with PBS containing 0-05% Tween80 (PBS-T). Biotin-conjugated mAb 12-1 for IL-4 (Department of Immunology, University of Stockholm, Sweden) or mAb 1DIK for IFN-y (Chromogenix AB, Molndal, Sweden) was added (100 p1) to each well at optimal dilution (1-3 pg/ml) and incubated for 3 hr at room temperature. Then the wells were washed four times with PBS-T and exposed to 100 p1 streptavidin-alkaline phosphatase (Mabtech AB, Stockholm, Sweden) for 1 hr. Unbound conjugate was removed by washing thoroughly with PBS and, finally, 100 p1 of BCIP/NBT substrate solution (Bio-Rad Laboratories, Hercules, CA) was added and incubated until the appearance of blue spots in the wells (30-40 min). The colour development was stopped by extensive washing under running tap water and, after drying, the

T-cell subsets in tuberculosis

number of spots was scored using a dissection microscope (Nikon, SMZ-2B, Tokyo, Japan). The mean error of the duplicated counts for the ELISPOT assay was less than 20% of the counted spot numbers. HLA typing HLA-DR genotypes were determined from genomic DNA isolated from anti-coagulated whole peripheral blood by the RFLP typing system described by Bidwell et al.2'
C.)

173
MTSE 38,000 MW 19,000 MW

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Statistical analysis Statistical analysis of the lymphocyte proliferative responses and cytokine levels was carried out using the Student's t-test. Frequencies of positive responders among total groups were compared between patients and controls using the x2 test with Yates' correction. RESULTS

1000,
100
-

38,000 MW

19,000 MW

AA

Lymphocyte proliferation and cytokine secretion responses to MTSE, 38,000 and 19,000 MW proteins Lymphocyte proliferative reactivity to mycobacterial antigens was studied with PBMC from 19 patients with active tuberculosis and from 15 healthy controls. Figure 1 shows that PBMC from tuberculosis patients had slightly decreased proliferative responses (c.p.m.) against MTSE and 38,000 and 19,000 MW protein compared with healthy individuals, but the differences did not reach statistical significance. The high thymidine uptake values in response to PHA represented the positive control and indicated good viability of the cultured PBMC. T-lymphocyte activation was further studied by analysing IL-4 and IFN-y production at single-cell level using ELISPOT assays in PBMC stimulated with individual mycobacterial antigens. Figure 2 shows the numbers of IL-4- and IFN-ysecreting cells after subtracting the spontaneous production of IL-4 (1-6+2-7/105 for controls and 2-3+2-9/105 for patients) and IFN-y (5-0+11-9/105 for controls and 0-9+1-4/105 for patients). The average numbers of cytokine-secreting cells were generally elevated in TB patients following the stimulation of PBMC with MTSE containing a wide range ofantigens, and this was confirmed using the 38,000 and 19,000 MW proteins. The numbers of IL-4-producing cells were significantly elevated in
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Figure 2. Frequency of cytokine-secreting cells following antigenic stimulation of 105 PBMC in vitro. Each symbol represents individual values determined by ELISPOT assay ofcells from tuberculosis patients (P) and sensitized healthy controls (C). Horizontal bars are mean values of cytokine-secreting cells.

Table 1. Summary of results on anti-mycobacterial T-cell responses in tuberculosis (P) and control (C) subjects
% of subjects with elevated response

Proliferationt
Stimulation with
Extract MTSE P

IL-4:
P

IFN-yt
C
20
7 13

C 70 33 38 25 15 15 38 18

95
53 47 13
7

93
73 80 38 33 53 33 61

79***
58*** 74***
38 33 22 50 40

69
50 49 33 27 13 13 33

Antigens 38,000 MW 19,000 MW Peptides 38-A 38-G 38-I 19-6A 19-7

El Patients 0

Controls

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22 47 40

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*** P < 0-01 was obtained comparing the frequencies of positive responders between tuberculosis patients and controls using x2 test with

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PHA

MTSE 38,000 MW 19,000 MW Medium

Figure 1. Proliferative responses (geometric mean + SD) of PBMC to PHA, MTSE or purified antigens. The ratio of responder/total tested individuals is given at the bottom of each column. Responders were defined when thymidine counts (c.p.m.) in the presence of antigen were three times above their individual values in the absence of antigen.

PBMC from TB patients compared with controls in response to the 38,00 MW antigen (6-8+7-8 versus 2-2+3-0, P<0-025). Moreover, the 19,000 MW antigen induced more prominent activation of IFN-y-producing cells in TB patients compared with controls (mean number of spots 12-9 + 22-7 versus

174
38,000 MW 38-A
Patients P17 P6 P26 P15 P3 P4 P21 P28 P19 P24
P5 P7 P2 P9 P23 P8 Controls C5

H.-M. Surcel et al.

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Figure 3. Individual variation in T-cell responsiveness to immunodominant synthetic peptides. PBMC were cultured in the presence of whole protein (38,000 and 19,000 MW) or peptides (38-A, G, I and 19-6A,7), derived from the 38,000 and 19,000 MW antigens. The results for each stimulant are shown in the sequence of proliferation (LP, stimulation index), IFN-y- and IL-4-producing cells/105 stimulated PBMC. The symbols correspond to high (13), low (U) or no (0) response. ND, not done.

5-9+8-2, P<0005). No correlation was found between proliferation and numbers of IL-4 or IFN-y producing cells following antigenic stimulation. Subsequently, all subjects were classified as either responders or non-responders. The IFN-y or IL-4 response was considered positive when the counted number of spots/l x 105 PBMC in the presence of antigen minus the number of spots/l x 105 PBMC in the absence of antigen was more than
two.

Lymphocyte proliferation and cytokine secretion response to synthetic peptides

In view of the pronounced variations of proliferative responses in both TB patients and controls, it was of interest to evaluate the occurrence of individual variations in the secretion of IL-4 and IFN-y cytokines. It was of particular interest to find out if cytokine profiles would associate with distinct immunodominant epitopes, which have previously been identified for both the 38,000 MW and 19,000 MW antigens.'5"'7 TB patients showed positive proliferation (SI > 3) to several synthetic peptides less frequently than healthy controls (see Fig. 3 and Table 1), but the relatively small size of groups in relation to low frequency of responders did not warrant further statistical evaluation. However, peptide-stimulated IL-4- and IFN-y-producing cell counts did not correlate with proliferation in individual TB patients or control subjects. In response to synthetic peptides, positive proliferation (SI > 3) correlated with significant IL-4 and/or IFN-y production in only five out of 22 tested TB cases (Fig. 3). Thus, IL-4- and/or IFN-y-secreting cells were demonstrably at similar frequencies in patients who did or did not show antigen-stimulated cell proliferation.

The IL-4 response to MTSE was significantly stronger in the

patients than in the controls (79% versus 20%, P < 0-0 1). Of the
TB patients 58% responded to 38,000 MW and 74% to 19,000 MW antigens with a positive activation of IL-4-producing cells, while 7% of the controls showed a positive IL-4 response to the 38,000 MW and 13% to the 19,000 MW (P<0-01, Table 1). MTSE induced a strong IFN-y response, both in the patients and in controls (69% versus 70%, P>0 1). 49-50% of the patients responded to the 38,000 MW and/or 19,000 MW antigens with a positive activation of IFN-y-producing cells, while 33% of the controls showed a positive IFN-y response to the 38,000 MW and 38% to the 19,000 MW antigen (P>0 1,

Table 1).

T-cell subsets in tuberculosis

None of the five peptides tested stimulated PBMC to produce one cytokine (either IL-4 or IFN-y) in preference to the other, and this was the case both in controls and patients. The synthetic peptides induced IL-4 24 times and IFN-y 27 times in all tested subjects.

175

DISCUSSION Defective proliferative responsiveness and deficient IL-2- and IFN-y secreting capacity to mycobacteria have previously been reported in patients with severe tuberculosis.'"3 In the present study, lymphocyte proliferative reactivity to MTSE, the 38,000 MW and the 19,000 MW protein did not significantly differ between patients and controls. Lower proliferative reactivity was pronounced in the case of the 38-G peptide, which has previously been reported to cause relative anergy in tuberculosis patients. 16 Characterization of functional T-cell subsets against isolated mycobacterial antigens was based on the enumeration of IL-4- and IFN-y-producing cells following stimulation with synthetic peptides derived from the 38,000 MW and 19,000 MW antigens, which have been shown previously to be immunogenic in human.'5 17 Interestingly, cytokine production was marked, even in cases where PBMC proliferation to a given antigen was not detectable. This result underlines the importance of measuring several parameters when studying T-cell activation and is thus in accordance with comparable analyses of responses directed to malaria antigens.22 When studying the T-cell reactions in the course of experimental M. tuberculosis infection, T-cell proliferation was an insufficient indicator of antigen recognition, and elevated levels of IFN-y were secreted from apparently non-proliferating T cells.23 According to our results, the numbers of IFN-y-secreting cells did not differ significantly between patients and controls. Thus, the results are not in accordance with the suggestion that deficient IFN-y production is involved with active tuberculosis.' 3In fact, increased secretion of IFN-y was found slightly more frequently in patients than in controls after 3 days of antigen stimulation. Conventionally, IFN-y is measured in 5-7-days culture supernatants, and IFN-y levels are found to be at least marginally decreased in TB patients.' 3 These results may reflect different kinetics of IFN-y production between patients and controls. In vivo-activated T cells in the TB patients may result in faster detection of cytokine production in vitro. Earlier studies based on the characterization of PPDstimulated human T-cell clones claimed that IL-4 production is not involved in immune reactivity against M. tuberculosis infection.2426 We have demonstrated here that PBMC from TB patients responded to MTSE with IL-4 reactivity more frequently than PBMC from healthy subjects. This result was confirmed using the 38,000 MW and 19,000 MW antigens. Even though low, the increase in the number of IL-4-producing cells after antigen stimulation was convincing and comparable to our experience using tetanus toxoid as T-cell stimulating antigen (El Ghagali et al. manuscript in preparation). Whether this is a reflection of antigen-specific IL-4-producing cells being few, or that they are down-regulated by other factors or that they are located somewhere else is not known. An interesting result obtained in this study was that PBMC from different individuals responded to several synthetic peptides but without preference for either IFN-y or IL-4. It could be

argued that this result is due to differences in the HLA-DR genotype among donors, as shown by Pfeiffer et al.,27 who reported that the MHC genotype controlled the selective activation of Thl - and Th2-type cells in response to the collagen IV peptide. We think this explanation for our data to be unlikely; although HLA-DR typing was carried out in the present work, mainly in the healthy subjects, there was no evident association of any of the HLA-DR haplotypes with either IFN-y or IL-4 production. Our results are thus in agreement with those of Haanen et al.28 who showed that human T-cell clones secreted either IFN-y or IL-4, irrespective of HLA haplotype. A corresponding result is represented by our finding that IL-4 and IFN-y production to individual peptides was often but not always mutually exclusive. It remains to be established whether double producers (IFN-y and IL-4) comparable to the ThO phenotype described for the mouse8 are present in the PBMC of tuberculosis patients. Higher IL-4 production in tuberculosis patients can explain the increased antibody levels that have been found previously in active tuberculosis.'4 Moreover, IL-4 together with other cytokines may also act as an influential down-regulator of Th 1-type responsiveness in infections,29 especially if it is secreted at an early stage of the immune response.30 3' Thus it is tempting to speculate that IL-4 production may be involved in the loss of protective host response and linked to the pathogenesis of tuberculosis. ACKNOWLEDGMENTS We are grateful to Dr Peter Klouda from the U.K. Transplant Support Service, Bristol, U.K. and Mr Paul Brookes from the Department of Immunology, RPMS, London, U.K. for HLA typing. We also thank Mr A. Hills, MRC Tuberculosis and Related Infections Unit, London, U.K., for preparing the synthetic peptides.
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