In an earlier study, we generated a large number of Mycobacterium leprae-responsive and M. leprae-nonresponsive T cell clones (TCC) from the lesional skin of immunologic unstable borderline leprosy patients. In that study, we divided TCC into type 1- and type 2-like on the basis of their IFN-γ and IL-4 expression. To explore whether other cytokines are coproduced along with IFN-γ and IL-4, we investigated the secretion of a panel of other cytokines (TNF-α, IL-5, IL-6, IL-10, and IL-13) by a large number of these TCC. Upon analysis of 139 M. leprae-responsive TCC, we observed a positive correlation in the coproduction of IFN-γ/TNF-α (r = 0.81), and in that of IL-4/IL-5 (r = 0.83), IL-4/IL-13 (r = 0.80), and IL-5/IL-13 (r = 0.82). Polarized type 1-like TCC produced dominantly IFN-γ/TNF-α, and polarized type 2-like TCC predominantly IL-4/IL-5/IL-13. Most type 0-like TCC produced both sets of cytokines. In contrast, type 1- and type 2-like subsets of M. leprae-nonresponsive TCC (n = 58) did not show the same coexpression of these cytokines. Furthermore, when the differential expression of a broad panel of cytokines by individual M. leprae-responsive TCC is considered, it appeared that additional phenotypes could be recognized. These results suggested that distinct isotypes of type 1- and type 2-like T cells, based on the secretion of a panel of cytokines, may reflect M. leprae-specific characteristics.

Discrete T cell subsets, through their release of cytokines, are recognized either to regulate cell-mediated immunity with the expression of delayed-type hypersensitivity (CMI-DTH)3 or humoral immune responses, and are associated with the tissue damage in human diseases (1, 2). Th1 or type 1 T cells producing IL-2, IFN-γ, and lymphotoxin are known to sustain CMI responses against intracellular pathogen and DTH reactions, whereas Th2 or type 2 T cells secrete IL-4 and IL-5, favoring humoral immune responses. On the other hand, the majority of T cells known as Th0 or type 0 T cells produce all lymphokines that are associated with both the Th1 and Th2 subsets.

Leprosy is a dynamic disease model in which distinct Mycobacterium leprae-responsive T cell subsets appear to control the clinical and immunologic spectrum (3, 4, 5, 6). Type 1-like T cells are associated with tuberculoid leprosy patients that are characterized by strong M. leprae-specific CMI-DTH responses restricting the growth of bacilli in the lesions (3, 4, 5). On the other hand, type 2-like T cells are observed predominantly in lepromatous leprosy patients that are characterized by strong humoral immune responses in association with the absence of M. leprae-specific T cell responses paralleling the dissemination of infection (3, 4). Between the two polar forms of leprosy, the majority of the patients are the immunologically unstable borderline patients that are classified as borderline tuberculoid (BT), mid-borderline (BB), or borderline lepromatous (BL). As a consequence, these patients often experience acute immunologic changes in the form of reactions during the course of the disease. To elucidate the mechanism of the pathology of reactions, we undertook studies on the contribution of the distinct T cell subsets in the lesional skin of these patients in the form of a follow-up study (6). We observed that when borderline patients experienced acute episodes of increased CMI-DTH responses during treatment, known as reversal reaction (RR) (7, 8), the IFN-γ/IL-4 secretion profile of the skin-derived M. leprae-responsive T cells shifted to a polarized type 1-like phenotype. In the same study, we demonstrated, in concordance with the results reported in recent literature, that irrespective of the clinical status of the patient, a major subset of M. leprae-responsive T cells have a type 0-like phenotype, producing both IFN-γ and IL-4 (6, 9).

It is now increasingly appreciated that besides IFN-γ and IL-4, other cytokines produced by T cells (TNF-α, IL-6, IL-10, and IL-13) also regulate CMI-DTH and humoral immune responses (10, 11). Several lines of evidence suggest that coproduction of various cytokines by type 1- and/or type 2-like human T cell subsets is associated with the type of disease, from which the T cells originate, and/or their Ag specificities. In this respect, type 1-like Mycobacterium tuberculosis-reactive TCC were found to coproduce predominantly TNF-α and IL-10, and occasionally IL-5 (12), whereas type 1-like TCC generated from psoriasis lesional skin produced little or no TNF-α and IL-10 (13). Coproduction of IL-4 and IL-6 was found in T cells generated from synovial fluid of patients with rheumatoid arthritis (14). Allergen-specific type 1- and type 2-like TCC did not differ in respect to their TNF-α or IL-6 production (15), whereas other investigators showed positive correlation of IL-10 production with allergen-specific type 2-like T cells (16).

To date, simultaneous production of cytokines other than IFN-γ and IL-4 by M. leprae-responsive T cells from lesional skin was studied with only limited number of M. leprae-reactive TCC (4, 17), and to our knowledge no evaluations were conducted to determine in parallel the extent of coproduction of a large panel of cytokines. In particular, studies regarding the production of IL-13 by skin-derived M. leprae-specific TCC have never been reported. Since a large panel of M. leprae-responsive and M. leprae-nonresponsive TCC was produced from the lesional skin of borderline leprosy patients with distinct CMI-DTH status and was classified on the basis of their IFN-γ/IL-4 ratio (6), we extended our studies by examining the simultaneous production of a broad panel of cytokines (TNF-α, IL-5, IL-6, IL-10, and IL-13) by these TCC. The cytokine secretion profile of both M. leprae-responsive and M. leprae-nonresponsive TCC was compared to elucidate the paradigm concerning the classification of T cell subsets as well as to evaluate the extent of association of the coproduction of these cytokines by TCC with the M. leprae responsiveness.

Both M. leprae-responsive and M. leprae-nonresponsive TCC used in this study were generated from lesional skin biopsies of three untreated borderline leprosy patients (one BT patient with RR (P6), one BB patient with RR (P7), and one BL patient (P4)), and again from the lesions of these patients when they (re)experienced an RR during treatment in the course of the disease. The details of the methods of generation and phenotyping (CD4+, CD8+, or TCR-γ/δ+) of these TCC were described previously (6). Briefly, primary T cell lines (TCL), spontaneously migrated from lesional skin biopsy specimen, were expanded by mitogenic stimulation with 0.05% PHA (Difco, Detroit, MI) in the presence of 3000-rad-irradiated allogenic feeder cells comprised of PBMC from two unrelated donors, EBV-transformed B cells (JY) and rIL-2 (a gift from Eurocetus, Amsterdam, The Netherlands). After expansion, these TCL were analyzed for their responsiveness to M. leprae using an Ag-induced stimulation assay (as described earlier, 6). When the TCL were found M. leprae responsive, rIL-2 was added to identical cultures for further promoting the expansion of the M. leprae-responsive T cells. These expanded T cells were then cloned by limiting dilution using a protocol, as described previously (18). The individual TCC were further expanded by mitogenic stimulation in the presence of an irradiated allogenic feeder cell mixture, as described above, and screened for their M. leprae responsiveness. In parallel, all TCC (both M. leprae responsive and nonresponsive) were stimulated for cytokine production, as described below.

As described previously (6), the IFN-γ/IL-4 secretion ratios of the M. leprae-responsive TCC upon stimulation with either M. leprae Ag or PMA/anti-CD3 did not differ, whereas the absolute secretion levels of both cytokines by these TCC stimulated by mitogen were higher. A similar trend of differences in the secretion of other cytokines between mitogenic stimulation and Ag-specific stimulation in the presence of autologous PBMC as APC was found (data not shown). Moreover, some cytokines that are presently investigated (particularly TNF-α and IL-6) were also produced by irradiated PBMC in the presence of M. leprae Ags (data not shown). Such basal secretion levels of cytokines by irradiated PBMC may mask the actual secretion level of Ag produced by T cells. Most importantly, we were interested in establishing the coexpression profile of several cytokines by M. leprae-responsive as compared with those M. leprae-nonresponsive TCC generated from the same lesions. Therefore, the supernatants of TCC stimulated by PMA/anti-CD3, according to the methods described elsewhere (6, 19), have been used for measuring the cytokine secretion profiles. Briefly, 10 days after the last mitogenic stimulation with PHA (0.05%) and allogenic feeder cells, T cells (105 cells/well) were incubated with immobilized CD3 mAb (OKT3, 1 mg/ml) and PMA (1 ng/ml) in a total volume of 200 μl/well in a flat-bottom 96-well plate. After 24 h, 100 μl of cell-free supernatants were harvested from each well and stored at −80°C until tested. The remaining cultures were subsequently cultured for an additional 16 h in the presence of 0.3 μCi/well [3H]thymidine to confirm stimulation.

All cytokines were measured with specific sandwich ELISA, testing the serial dilutions of supernatants starting at a 1/10 dilution.

IFN-γ.

The ELISA was performed as described before (20). Briefly, flat-bottom EIA/RIA ELISA microtiter plates (Costar, Cambridge, MA) were coated for 3 h with a mouse anti-human capture mAb MD2 (Innogenetics, Gent, Belgium) at 10 μg/ml in a 0.1 M carbonate buffer (pH 9.6, 50 μl/well) in a 37°C humidified atmosphere containing 5% CO2. All subsequent incubation steps were in 50 μl volumes and were followed by a washing step with PBS supplemented with 0.05% (v/v) Tween-20. The plates were washed twice and incubated for 1 h with PBS/3% BSA (37°C, 5% CO2) as a blocking step. After washing, freshly thawed IFN-γ-containing samples diluted in PBS/3% BSA were then added and incubated overnight at 4°C. Plates were washed, and biotinylated mAb MD1 was added for 2 h (37°C, 5% CO2). Thereafter, the plates were washed and incubated with horseradish peroxidase-coupled streptavidin (Genzyme Corp., Cambridge, MA), 1/5000 diluted (according to the manufacturer’s instructions) in PBS/3% BSA for 1 h (37°C, 5% CO2), and washed, and the enzymatic activity was determined with the substrate 1,2-phenylene-diamine (Fluka, Buchs, Switzerland) with H2O2 (30%) in substrate buffer, pH 5.4. The reaction was stopped by adding an equal volume of 1 M H2SO4 to the wells. Plates were read at dual wavelength of 490 and 405 nm in a micro plate reader (Bio-Rad, Richmond, CA). Human IFN-γ was used as a standard; the detection limit for IFN-γ was found to be at the detection level of 50 pg/ml.

IL-4 ELISA.

The assay was performed identical to the IFN-γ ELISA, and has been described before (21). For coating mAb CLB-IL-4/5 was used at 1 μg/ml, and for detection biotinylated mAb CLB-IL-4/1 was used at 1 μg/ml. Human IL-4 was used as a standard, and the detection limit was 40 pg/ml.

IL-5 ELISA.

The assay was performed identically to the IFN-γ ELISA, with the following exceptions (22): the IL-5-containing samples were incubated for 1 h at 37°C, the incubation time of the biotinylated Ab was 1 h, and the horseradish peroxidase-coupled streptavidin was diluted in PBS/3% BSA in the presence of 2% (v/v) milk and incubated for 0.5 h. For coating mAb TRFK5 (PharMingen, San Diego, CA) was used at 2 μg/ml, and for detection biotinylated JES1-5A10 (PharMingen) at 0.1 μg/ml. Human rlL-5 (Glaxo Institute for Molecular Biology, Geneva, Switzerland) was used as a standard; the detection limit was 20 pg/ml.

TNF-α ELISA.

We used the Pelikan compact human TNF-α ELISA kit (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB), Amsterdam, the Netherlands). The detection limit was 8 pg/ml.

IL-13 ELISA.

The ELISA was performed as described before (23). Flat-bottom microtiter plates (Nunc, Maxisorb) were coated overnight at 4°C with mouse anti-human capture mAb CLB-IL-13/3 at 1 μg/ml in 0.1 M carbonate buffer (pH 9.6, 100 μl/well). All subsequent incubations were in 100 μl volumes at room temperature. The plates were washed with PBS, 0.02% (v/v) Tween-20 and incubated for 30 min with PBS, containing 2% (v/v) milk as blocking step. After washing, biotinylated mAb CLB-IL-13/2 at 0.5 μg/ml was added together with the IL-13-containing samples diluted in high performance ELISA buffer (CLB) for 2 h. Thereafter, the plates were washed and incubated with polystreptavidin-horseradish peroxidase (PolyHRP; CLB), 1/10,000 diluted (according to the manufacturer’s instruction) in PBS containing 2% (v/v) milk for 0.5 h, washed, and developed with a solution of 100 μg/ml of 3,5,3′,5′-tetramethybenzidine (Merck, Darmstadt, Germany) with 0.003% (v/v) H2O2 in 0.11 M sodium acetate, pH 5.5 (100 μl/well). The reaction was stopped by adding an equal volume of 1 M H2SO4 to the wells. Plates were read at 450 nm in a Titertek Multiskan reader Labsystems Multiskan (Multisoft, Helsinki, Finland). Background absorbance at 540 nm was subtracted. Human rIL-13 (Pretotech, London, U.K.) was used as a standard. The detection limit was 8 pg/ml.

IL-6 ELISA.

Procedures were identical to the IL-13 ELISA, except that the blocking step was eliminated (24). For coating mAb CLB-IL-6/16 was used at 1 μg/ml. Affinity-purified polyclonal sheep anti-IL-6 was used for detection at 0.25 μg/ml. PBMC-derived IL-6, calibrated to human rIL-6 (25), was used as a standard. The detection limit was 2 pg/ml.

IL-10 ELISA.

The assay was performed identical to the IL-13 ELISA, except that the blocking step was omitted (26). For coating mAb B-N10 was used at 0.5 μg/ml in PBS, and for detection biotinylated mAb anti-IL-10 (B-T10) was used at 0.125 μg/ml. Human rIL-10 was used as a standard. The detection limit was 15 pg/ml.

Spearman correlation coefficients (r) of coproduction of pairs of cytokines were calculated. Comparison of the production level of a cytokine between subsets of TCC was performed using the Mann-Whitney test. p values < 0.05 were considered significant.

Table I shows the origin of 139 M. leprae-responsive TCC and their phenotypes according to their IFN-γ and IL-4 production levels upon stimulation with PMA plus anti-CD3, as previously described (6). The absolute values of IFN-γ and IL-4 production by these TCC are illustrated in Figure 1. One hundred twenty-eight of these TCC were CD4+, seven were CD8+ and/or TCR-γ/δ+, and four were not phenotyped. The TCC with a type 0-like phenotype (IFN-γ/IL-4 ratio ranging from 0.4 to 20) were generated from all lesions, irrespective of the immune status of the patient. Type 1-like TCC (IFN-γ/IL-4 ratio > 20) predominantly originated from the lesional skin of patients, characterized by high levels of CMI-DTH against M. leprae, and were most prominently seen in the lesions from those patients (re)experiencing an RR during treatment. Type 2-like TCC (IFN-γ/IL-4 ratio < 0.4) dominated in the lesion of an untreated patient (P4) with strong humoral immune responses in the absence of CMI-DTH responses against M. leprae. As described previously (6), it appeared that the polarized cytokine profile of these M. leprae-responsive TCC reflected the local immunopathologic status of the borderline leprosy patient.

Table I.

Type 1-, type 0-, and type 2-like distribution of M. leprae-responsive CD4+ TCC that were generated from the lesional skin of borderline leprosy patients with distinct immunologic status

Clinical StatusT Cell Subset Distributiona
Patient no./ClassUntr./Tr.Episode of RRImmune status HI/CMI-DTHTCCType 1Type 0Type 2
P6/BT Untr. 1st CMI-DTH 32 8b 19b 
 Tr. 2nd CMI-DTH 12 7c 
P7/BB Untr. 1st CMI-DTH 26 24 
 Tr. 2nd CMI-DTH 20 17 
P4/BL Untr. — HI 14 1d 3d 10d 
 Tr. 1st CMI-DTH 34 25e 9d 
Clinical StatusT Cell Subset Distributiona
Patient no./ClassUntr./Tr.Episode of RRImmune status HI/CMI-DTHTCCType 1Type 0Type 2
P6/BT Untr. 1st CMI-DTH 32 8b 19b 
 Tr. 2nd CMI-DTH 12 7c 
P7/BB Untr. 1st CMI-DTH 26 24 
 Tr. 2nd CMI-DTH 20 17 
P4/BL Untr. — HI 14 1d 3d 10d 
 Tr. 1st CMI-DTH 34 25e 9d 
a

The distribution of TCC within distinct T cell subsets was based on the following criteria: TCC that produced IFN-γ but no IL-4 (<40 pg/ml) or with an IFN-γ/IL-4 ratio of >20.0 were defined as type 1-like; TCC with an IFN-γ/IL-4 ratio between 20.0 and 0.4 were defined as type 0-like; TCC with an IFN-γ/IL-4 ratio <0.4 were defined as type 2-like.

Distinct phenotypes: b2 TCC with unidentified phenotype; c1 CD8+/TCRγ/δ+ TCC; d1 CD8+ TCC, e2 CD4/CD8/TCRγ/δ+ TCC.

HI, humoral immunity; CMI-DTH, cell-mediated immunity-delayed type hypersensitivity type IV; RR, reversal reaction; Untr., untreated; Tr., treated.

FIGURE 1.

The IFN-γ and IL-4 secretion profile of M. leprae-responsive TCC generated from the lesional skin of borderline leprosy patients. Ten-day rested TCC cells (105) were stimulated with PMA (1 ng/ml) and immobilized anti-CD3 mAb (OKT3, 1 mg/ml). Twenty-four-hour supernatants were collected and analyzed for their IFN-γ and IL-4 production by ELISA. The distribution of TCC within distinct T cell subsets was based on the criteria described in Table I. The absolute IFN-γ and IL-4 levels (ng/ml) by type 1-like (•), type 0-like (○), and type 2-like (▴) TCC are presented. Two TCC produced exceptional high amounts of IFN-γ, and those data were transformed to fit the figure axis; the absolute production of IFN-γ/IL-4 was for a, 77,050/1,890 pg/ml, and for b, 184,300/ 19,475 pg/ml.

FIGURE 1.

The IFN-γ and IL-4 secretion profile of M. leprae-responsive TCC generated from the lesional skin of borderline leprosy patients. Ten-day rested TCC cells (105) were stimulated with PMA (1 ng/ml) and immobilized anti-CD3 mAb (OKT3, 1 mg/ml). Twenty-four-hour supernatants were collected and analyzed for their IFN-γ and IL-4 production by ELISA. The distribution of TCC within distinct T cell subsets was based on the criteria described in Table I. The absolute IFN-γ and IL-4 levels (ng/ml) by type 1-like (•), type 0-like (○), and type 2-like (▴) TCC are presented. Two TCC produced exceptional high amounts of IFN-γ, and those data were transformed to fit the figure axis; the absolute production of IFN-γ/IL-4 was for a, 77,050/1,890 pg/ml, and for b, 184,300/ 19,475 pg/ml.

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The production of TNF-α, IL-5, IL-6, IL-10, and IL-13 by all M. leprae-responsive TCC was measured by ELISA in the same supernatants that were used for IFN-γ and IL-4 detection. Most TCC did not produce IL-6 above detection level (<15 pg/ml), and coproduction of IL-6 with other cytokines was therefore not further analyzed. Spearman correlation coefficients (r) of coproduction of cytokines are presented in Figure 2 and summarized in Table II. The production of IFN-γ and that of TNF-α was strongly correlated (r = 0.81, Fig. 2,A), whereas the production of both of these cytokines did not correlate with the production of either IL-4 (r = 0.10, r = 0.15, respectively) or IL-5 (r = 0.09, r = 0.17, respectively), and correlated weakly with IL-13 (r = 0.34, r = 0.41, respectively). On the other hand, the production of IL-4 strongly correlated with the production of IL-5 (r = 0.83, Fig. 2,B) and IL-13 (r = 0.80, Fig. 2,C). In addition, the production of IL-5 and that of IL-13 strongly correlated (r = 0.82, Fig. 2,D). The production of IL-10 weakly correlated with the production of all other cytokines: e.g., IFN-γ (r = 0.40), TNF-α (r = 0.61), IL-4 (r = 0.55), IL-5 (r = 0.61), and IL-13 (r = 0, 63). Figure 2, E and F, illustrates two representative examples of the production of IL-10 in conjunction with IFN-γ and IL-4, respectively.

FIGURE 2.

Coproduction of cytokines by M. leprae-responsive TCC generated from the lesional skin of borderline leprosy patients. Ten-day rested TCC cells (105) were stimulated with PMA (1 ng/ml) and immobilized anti-CD3 mAb (OKT3, 1 mg/ml). Twenty-four-hour supernatants were collected and analyzed for their IFN-γ, IL-4, TNF-α, IL-5, IL-10, and IL-13 production by ELISA. The production of IFN-γ/TNF-α (A), IL-4/IL-5 (B), IL-4/IL-13 (C), IL-5/IL-13 (D), IFN-γ/IL-10 (E), and IL-4/IL-10 (F) by 139 TCC is presented. r, Spearman correlation coefficients.

FIGURE 2.

Coproduction of cytokines by M. leprae-responsive TCC generated from the lesional skin of borderline leprosy patients. Ten-day rested TCC cells (105) were stimulated with PMA (1 ng/ml) and immobilized anti-CD3 mAb (OKT3, 1 mg/ml). Twenty-four-hour supernatants were collected and analyzed for their IFN-γ, IL-4, TNF-α, IL-5, IL-10, and IL-13 production by ELISA. The production of IFN-γ/TNF-α (A), IL-4/IL-5 (B), IL-4/IL-13 (C), IL-5/IL-13 (D), IFN-γ/IL-10 (E), and IL-4/IL-10 (F) by 139 TCC is presented. r, Spearman correlation coefficients.

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Table II.

Spearman correlation coefficients (r) between IFN-γ, IL-4, TNF-α, IL-5, IL-10, and IL-13 production of M. leprae-responsive TCC (n=139)

Spearman correlation coefficient (r)a
IFN-γIL-13IL-10IL-5TNF-α
IL-4 0.10 (p = 0.25) 0.80 (p = <0.001) 0.55 (p = <0.001) 0.83 (p = <0.001) 0.15 (p = 0.07) 
TNF-α 0.81 (p = <0.001) 0.41 (p = <0.001) 0.61 (p = <0.001) 0.17 (p = 0.05)  
IL-5 0.09 (p = 0.32) 0.82 (p = <0.001) 0.61 (p = <0.001)   
IL-10 0.40 (p = < 0.001) 0.63 (p = <0.001)    
IL-13 0.34 (p = <0.001)     
Spearman correlation coefficient (r)a
IFN-γIL-13IL-10IL-5TNF-α
IL-4 0.10 (p = 0.25) 0.80 (p = <0.001) 0.55 (p = <0.001) 0.83 (p = <0.001) 0.15 (p = 0.07) 
TNF-α 0.81 (p = <0.001) 0.41 (p = <0.001) 0.61 (p = <0.001) 0.17 (p = 0.05)  
IL-5 0.09 (p = 0.32) 0.82 (p = <0.001) 0.61 (p = <0.001)   
IL-10 0.40 (p = < 0.001) 0.63 (p = <0.001)    
IL-13 0.34 (p = <0.001)     
a

Ten-day rested TCC cells (105) were stimulated with PMA (1 ng/ml) and immobilized anti-CD3 mAb (OKT3, 1 mg/ml). Twenty-four-hour supernatants were collected and analyzed for their IFN-γ, IL-4, TNF-α, IL-5, IL-10, and IL-13 production by ELISA. Bold-face type indicates the significant correlation coefficients.

Since the production of IFN-γ/TNF-α, and the production of IL-4/IL-5, IL-4/IL-13, and IL-5/IL-13 by M. leprae-responsive TCC were positively correlated, we investigated whether these cytokines were differentially produced by the predesignated type 1- and type 2-like T cell subsets. Indeed, such differences in the coproduction of cytokines with either IFN-γ or IL-4 can be further seen when the mean cytokine secretions by these type 1- and type 2-like TCC were calculated (Table III). Additionally, the absolute values of TNF-α (p < 0.004), IL-5 (p = 0.001), and IL-13 (p < 0.001) are significantly different between type 1-like (n = 57) and type 2-like (n = 17) TCC. In spite of the fact that the production of IL-10 was found to correlate weakly with the production levels of either IFN-γ or IL-4, type 2-like T cells produce significantly more IL-10 (p = 0.008).

Table III.

Mean secretion values of IFN-γ, IL-4, TNF-α, IL-5, IL-10, and IL-13 production by type 1- and type 2-like M. leprae-responsive TCC

PhenotypeMean Cytokine Secretion (ng/ml) ± SDa
IFN-γIL-4TNF-αIL-5IL-6IL-10IL-13
Type 1 (n = 57) 8.9 ± 12.6 0.1 ± 0.3 4.8 ± 6.7 0.3 ± 0.8 0.03 ± 0.09 3.9 ± 9.7 4.0 ± 5.7 
Type 2 (n = 17) 1.2 ± 1.2 9.0 ± 8.5 1.2 ± 1.2 11.2 ± 10.5 0.05 ± 0.08 10.7 ± 13.5 18.4 ± 11.6 
pb <0.001 <0.001 0.004 <0.001 0.74 0.008 <0.001 
PhenotypeMean Cytokine Secretion (ng/ml) ± SDa
IFN-γIL-4TNF-αIL-5IL-6IL-10IL-13
Type 1 (n = 57) 8.9 ± 12.6 0.1 ± 0.3 4.8 ± 6.7 0.3 ± 0.8 0.03 ± 0.09 3.9 ± 9.7 4.0 ± 5.7 
Type 2 (n = 17) 1.2 ± 1.2 9.0 ± 8.5 1.2 ± 1.2 11.2 ± 10.5 0.05 ± 0.08 10.7 ± 13.5 18.4 ± 11.6 
pb <0.001 <0.001 0.004 <0.001 0.74 0.008 <0.001 
a

The experimental conditions were identical to those described in Table II.

b

To test whether the secretion of a cytokine differed significantly between the distinct subsets of TCC, the measured values were analyzed in the Mann-Whitney test. Statistic significance was defined as p < 0.05.

Differences in coproduction of cytokines can be distinctly recognized when the most polarized M. leprae-responsive TCC within this panel are taken into account (Table IV). It can be seen that polarized type 1-like TCC (IFN-γ/IL-4 ratio ≥ 187, n = 9) dominantly coproduced high levels of IFN-γ and TNF-α, but with only low amounts of IL-13. Only one TCC (78.13) produced high amounts of IL-13. On the other hand, polarized type 2-like TCC (IFN-γ/IL-4 ratio ≤ 0.25, n = 9) dominantly coproduced high levels of IL-4, IL-5, and IL-13. In contrast with the significantly different production level of IL-10 between type 1- and type 2-like T cell subsets, as presented in Table III, the absolute values of IL-10 did not differ significantly (p = 0.19) between these two polarized subsets of TCC. As presented in Table IV, polarized type 1-like TCC produced either significant amounts of IL-10 or below detection level. The latter results may explain the weak correlation of IL-10 production with either IFN-γ or IL-4, as was presented in Table II.

Table IV.

Cytokine secretion profiles of polar type 1- and type 2-like M. leprae-responsive TCC

Clone No.bPhenotypeIFN-γ/IL-4 RatioSecretion of Cytokines (ng/ml)a
IFN-γIL-4TNF-αIL-5IL-6IL-10IL-13
Type 1-like          
78.8 CD4 580 34.8 0.06 10.7 0.22 0.03 8.0 7.1 
78.46 CD4 448 17.9 0.04 11.3 <0.02 <0.02 37.3 0.2 
78.15 CD4 299 26.9 0.09 11.1 0.05 <0.02 2.5 1.5 
78.13 CD4 260 18.2 0.07 8.9 0.19 <0.02 20.0 14.3 
18.17 CD4 255 10.2 <0.04 2.0 0.06 <0.02 <0.08 1.6 
18.15 CD4 238 9.5 <0.04 1.1 0.05 <0.02 <0.08 3.1 
18.46 CD4 228 9.1 <0.04 2.0 <0.02 <0.02 <0.08 0.2 
78.52 CD4 213 12.8 0.06 6.1 0.19 <0.02 <0.08 4.9 
78.38 CD4 187 20.6 0.11 13.9 0.63 <0.02 7.9 2.4 
Type 2-like          
74.33 CD4 0.25 1.6 6.4 0.8 15.8 0.04 33.4 19.3 
13.16 CD4 0.18 0.9 4.9 0.9 22.4 <0.02 1.9 11.5 
74.32 CD4 0.16 0.9 5.5 0.8 8.9 0.03 16.7 18.0 
74.3 CD4 0.10 2.1 20.4 3.6 32.7 <0.02 32.4 26.2 
74.5 CD8 0.10 0.5 5.2 0.2 3.4 <0.02 6.5 14.0 
74.10 CD4 0.07 1.3 19.6 3.1 30.9 <0.02 6.4 40.0 
74.6 CD4 0.05 0.4 7.7 0.2 2.7 <0.02 5.4 12.3 
74.25 CD4 0.02 0.7 31.2 1.1 16.3 <0.02 27.5 11.2 
74.21 CD4 0.02 0.1 5.1 0.6 4.6 <0.02 3.8 7.6 
pc     0.002 <0.001  0.19 0.002 
Clone No.bPhenotypeIFN-γ/IL-4 RatioSecretion of Cytokines (ng/ml)a
IFN-γIL-4TNF-αIL-5IL-6IL-10IL-13
Type 1-like          
78.8 CD4 580 34.8 0.06 10.7 0.22 0.03 8.0 7.1 
78.46 CD4 448 17.9 0.04 11.3 <0.02 <0.02 37.3 0.2 
78.15 CD4 299 26.9 0.09 11.1 0.05 <0.02 2.5 1.5 
78.13 CD4 260 18.2 0.07 8.9 0.19 <0.02 20.0 14.3 
18.17 CD4 255 10.2 <0.04 2.0 0.06 <0.02 <0.08 1.6 
18.15 CD4 238 9.5 <0.04 1.1 0.05 <0.02 <0.08 3.1 
18.46 CD4 228 9.1 <0.04 2.0 <0.02 <0.02 <0.08 0.2 
78.52 CD4 213 12.8 0.06 6.1 0.19 <0.02 <0.08 4.9 
78.38 CD4 187 20.6 0.11 13.9 0.63 <0.02 7.9 2.4 
Type 2-like          
74.33 CD4 0.25 1.6 6.4 0.8 15.8 0.04 33.4 19.3 
13.16 CD4 0.18 0.9 4.9 0.9 22.4 <0.02 1.9 11.5 
74.32 CD4 0.16 0.9 5.5 0.8 8.9 0.03 16.7 18.0 
74.3 CD4 0.10 2.1 20.4 3.6 32.7 <0.02 32.4 26.2 
74.5 CD8 0.10 0.5 5.2 0.2 3.4 <0.02 6.5 14.0 
74.10 CD4 0.07 1.3 19.6 3.1 30.9 <0.02 6.4 40.0 
74.6 CD4 0.05 0.4 7.7 0.2 2.7 <0.02 5.4 12.3 
74.25 CD4 0.02 0.7 31.2 1.1 16.3 <0.02 27.5 11.2 
74.21 CD4 0.02 0.1 5.1 0.6 4.6 <0.02 3.8 7.6 
pc     0.002 <0.001  0.19 0.002 
a

The experimental conditions were identical to those described in Table II.

b

TCC with no. 18 were generated from the untreated lesion of P6, with no. 13 from the untreated lesion of P7, and with no. 74 or 78 from the untreated and treated lesions of P4, respectively.

c

To test whether the secretion of a cytokine differed significantly between the distinct subsets of TCC, the measured values were analyzed in the Mann-Whitney test. Statistical significance was defined as p < 0.05.

We found that two main sets of cytokines are coproduced by M. leprae-responsive TCC: IFN-γ/TNF-α prominently by type 1-like T cells and IL-4/IL-5/IL-13 prominently by type 2-like T cells. TCC with the type 0-like phenotype (n = 65) were found to produce both sets of cytokines (data not shown). However, when analyzing quantitatively the cytokine secretion profile of individual TCC, a correlated production of IL-4/IL-5/IL-13 by type 1- and type 0-like TCC was not always observed. We considered TCC to be low producers of IL-4, IL-5, or IL-13 when the production of IL-4 or IL-5 was below 1 ng/ml, and the production of IL-13 below 10 ng/ml, respectively. Following such criteria, a subset of type 1- and type 0-like TCC producing either high levels of IL-4 and IL-5, IL-5 and IL-13, IL-4 and IL-13, or high levels of any of those cytokines alone was identified (Table V). Another exception can also be seen in that one type 2-like TCC (74.21) produced high amounts of IL-4 and IL-5, but low levels of IL-13 (see Table IV).

Table V.

Type 1- and type 0-like T cells with indeterminant cytokine secretion profile in respect to their IL-4, IL-5, and IL-13 production

Clone (no.)bTypeSecretion of Cytokines (ng/ml)a
IFN-γIL-4TNF-αIL-5IL-13
High IL-4/High IL-5/Low IL-13       
74.34 3.8 3.1 3.9 1.3 9.6 
Low IL-4/High IL-5/High IL-13       
78.36 9.8 0.2 5.7 2.0 10.5 
43.16 23.1 0.3 5.2 5.9 32.9 
13.5 2.5 0.2 1.9 14.5 16.0 
13.39 4.8 0.4 5.7 15.1 16.5 
13.68 1.6 0.6 2.2 11.0 11.5 
13.72 1.2 0.6 1.9 15.2 18.5 
13.33 3.3 0.9 5.5 26.9 29.5 
High IL-4/Low IL-5/High IL-13       
43.22 77.1 1.9 45.6 0.1 13.1 
18.23 0.5 1.2 4.8 0.1 18.1 
High IL-4/Low IL-5/Low IL-13       
18.47 2.0 1.4 1.2 0.7 4.8 
18.10 1.0 2.0 1.0 0.7 4.7 
18.53 1.7 2.0 1.4 0.5 4.2 
Low IL-4/High IL-5/Low IL-13       
43.19 1.9 0.3 0.9 8.9 9.1 
18.48 0.7 0.6 0.7 2.0 8.4 
13.61 1.2 0.5 1.2 4.2 8.5 
Low IL-4/High IL-5/Low IL-13       
78.13 18.2 0.1 8.9 0.2 14.3 
78.4 6.9 0.1 5.7 0.3 16.3 
13.12 6.1 0.2 6.0 0.6 12.5 
18.50 8.9 0.3 2.1 0.1 13.8 
78.24 4.5 0.3 6.7 0.8 31.9 
18.45 3.6 0.5 2.6 0.1 11.3 
18.36 1.5 0.7 0.9 0.6 12.0 
Clone (no.)bTypeSecretion of Cytokines (ng/ml)a
IFN-γIL-4TNF-αIL-5IL-13
High IL-4/High IL-5/Low IL-13       
74.34 3.8 3.1 3.9 1.3 9.6 
Low IL-4/High IL-5/High IL-13       
78.36 9.8 0.2 5.7 2.0 10.5 
43.16 23.1 0.3 5.2 5.9 32.9 
13.5 2.5 0.2 1.9 14.5 16.0 
13.39 4.8 0.4 5.7 15.1 16.5 
13.68 1.6 0.6 2.2 11.0 11.5 
13.72 1.2 0.6 1.9 15.2 18.5 
13.33 3.3 0.9 5.5 26.9 29.5 
High IL-4/Low IL-5/High IL-13       
43.22 77.1 1.9 45.6 0.1 13.1 
18.23 0.5 1.2 4.8 0.1 18.1 
High IL-4/Low IL-5/Low IL-13       
18.47 2.0 1.4 1.2 0.7 4.8 
18.10 1.0 2.0 1.0 0.7 4.7 
18.53 1.7 2.0 1.4 0.5 4.2 
Low IL-4/High IL-5/Low IL-13       
43.19 1.9 0.3 0.9 8.9 9.1 
18.48 0.7 0.6 0.7 2.0 8.4 
13.61 1.2 0.5 1.2 4.2 8.5 
Low IL-4/High IL-5/Low IL-13       
78.13 18.2 0.1 8.9 0.2 14.3 
78.4 6.9 0.1 5.7 0.3 16.3 
13.12 6.1 0.2 6.0 0.6 12.5 
18.50 8.9 0.3 2.1 0.1 13.8 
78.24 4.5 0.3 6.7 0.8 31.9 
18.45 3.6 0.5 2.6 0.1 11.3 
18.36 1.5 0.7 0.9 0.6 12.0 
a

The experimental conditions were identical to those described in Table II.

b

TCC with no. 18 and 43 were generated from the untreated and treated lesion of P6, respectively, with no. 13 from the untreated lesion of P7, and with no. 74 and 78 from the untreated and treated lesions of P4, respectively.

To further substantiate the specific characteristic of M. leprae-responsive T cell subsets, we also investigated in parallel the coproduction of the same panel of cytokines for a number of M. leprae-nonresponsive TCC (n = 58) that were generated from the same lesions as an internal control. According to their IFN-γ and IL-4 production levels, type 1-like (n = 16), type 0-like (n = 25), and type 2-like (n = 17) TCC were included. Within this panel, a subset of TCC was found to produce IL-6. In contrast with the M. leprae-responsive TCC, strongly correlated production of IFN-γ/TNF-α, IL-4/IL-5, and IL-4/IL-13 by these TCC was not observed (data not shown). Only the IL-5 and IL-13 expression were found to be related (r = 0.80). The absence in coproduction of IFN-γ/TNF-α, and IL-4/IL-13 can be further seen when the mean cytokine secretions by the predesignated type 1- and type 2-like M. leprae-nonresponsive TCC were calculated (Table VI). The absolute production levels of TNF-α (p = 0.22) and IL-13 (p = 0.09) did not significantly differ between those two subsets of TCC. However, in spite of weak correlated production of IL-5, IL-6, and IL-10 with the production of IL-4 (r = 0.65, r = 0.39, and r = 0.53, respectively), these cytokines were found to be significantly more produced by type 2-like T cells.

Table VI.

Mean secretion values of IFN-γ, IL-4, TNF-α, IL-5, IL-6, IL-10, and IL-13 production by type 1- and type 2-like M. leprae-nonresponsive TCC

PhenotypeMean Cytokine Secretion (ng/ml) ± SDa
IFN-γIL-4TNF-αIL-5IL-6IL-10IL-13
Type 1 (n = 16) 16.4 ± 15.9 0.3 ± 0.3 7.2 ± 7.6 1.0 ± 2.6 0.07 ± 0.13 0.2 ± 0.1 9.1 ± 11.7 
Type 2 (n = 17) 0.6 ± 0.7 4.9 ± 4.1 3.8 ± 3.8 10.6 ± 11.7 0.22 ± 0.3 3.0 ± 3.6 16.8 ± 16.1 
pb <0.001 <0.001 0.22 <0.001 <0.001 0.002 0.09 
PhenotypeMean Cytokine Secretion (ng/ml) ± SDa
IFN-γIL-4TNF-αIL-5IL-6IL-10IL-13
Type 1 (n = 16) 16.4 ± 15.9 0.3 ± 0.3 7.2 ± 7.6 1.0 ± 2.6 0.07 ± 0.13 0.2 ± 0.1 9.1 ± 11.7 
Type 2 (n = 17) 0.6 ± 0.7 4.9 ± 4.1 3.8 ± 3.8 10.6 ± 11.7 0.22 ± 0.3 3.0 ± 3.6 16.8 ± 16.1 
pb <0.001 <0.001 0.22 <0.001 <0.001 0.002 0.09 
a

The experimental conditions were identical to those described in Table II.

b

To test whether the secretion of a cytokine differed significantly between the distinct subsets of TCC, the measured values were analyzed in the Mann-Whitney test. Statistic significance was defined as p < 0.05.

The classification of human T cell subsets on the basis of their cytokine profile either in relation to diseases or Ag specificities is a subject of intense studies. In this respect, the immunopathology of leprosy spectrum is closely studied with the T cell repertoire and their cytokine network (4, 5, 6, 9). To date, the current classification of M. leprae-responsive type 1- or type 2-like T cells was based merely on the exclusive or predominant production of IFN-γ or IL-4, respectively. This restricted cytokine secretion profile, however, may not reflect the complex regulatory role of T cells in vivo. Therefore, we undertook to investigate the secretion profile of a broad panel of cytokines (IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-10, and IL-13) by a large panel of TCC with specificity toward M. leprae. These TCC were derived from the lesional skin of borderline leprosy patients with changing immune status that were investigated in a follow-up study (6). As previously described, based on their IFN-γ/IL-4 secretion profile, the polarized type 1- or type 2-like phenotype of the M. leprae-responsive TCC reflected the local immune status of the patient from which they were generated. In the same study, a large number of type 1-, type 0-, or type 2-like M. leprae-nonresponsive TCC was also generated in parallel from the same lesions and offered the opportunity to compare the cytokine secretion profiles of these TCC with those of M. leprae-responsive TCC under the same experimental conditions. By doing so, the specific characteristics of the M. leprae-responsive T cell subsets can be better understood.

Despite the similarities in functional properties of TNF-α with IFN-γ (27), and of IL-6, IL-10, and IL-13 with IL-4 (28, 29, 30, 31, 32), the production of these cytokines is not found to be restricted predominantly to either the type 1- or type 2-like human T cell subsets, respectively (2, 33, 34). In the present study, we showed that the production of IFN-γ/TNF-α, and IL-4/IL-5/IL-13 by M. leprae-responsive TCC was positively correlated. Consequently, the production levels of TNF-α, IL-5, and IL-13 were found to differ significantly between the type 1- and type 2-like T cell subset. Since type 1- and type 2-like M. leprae-nonresponsive TCC did not differ in their production levels of TNF-α and IL-13, our findings suggest that coproduction of IFN-γ/TNF-α vs IL-4/IL-13 may be characteristic for T cells with reactivity to M. leprae. Coproduction of IL-5 and IL-4 has already been shown for human T cells, particularly with the type 2-like phenotype, with varying Ag specificities (1, 2). Since TNF-α is also produced by M. tuberculosis-responsive type 1-like TCC (12), coproduction of these proinflammatory cytokines may be characteristic for T cells in mycobacterial infections in general. Indeed, type 1-like TCC from psoriasis lesional skin produced little or no TNF-α (13), whereas allergen-specific type 1- as well as type 2-like TCC were found to produce similar amounts of TNF-α (15). To date, other studies failed to show that production of IL-13 is associated with a type 2-like phenotype of human T cells. In this respect, correlated production of IL-4 and IL-13 was found to be absent in TCC with specificity to allergen (34). Additionally, similar production levels of IL-13 were found for type 1-, type 0-, and type 2-like TCC with varying Ag specificities (35) (Dr. Eddy A. Wierenga, personal communication).

It should be noted that TNF-α was produced predominantly by the type 1-like M. leprae-responsive T cells. These T cells were generated predominantly from the lesional skin of borderline leprosy patients undergoing RR characterized by CMI-DTH responses against M. leprae. (6). Furthermore, such type 1-like Ag-responsive TCC were also generated from BT patients with high Ag-specific CMI-DTH responses (6). In the context of the role of the T cell cytokines in leprosy, it is suggestive that TNF-α may contribute to the elimination of bacilli (27), as well as the granuloma formation (36) and tissue damage in the lesions. Interestingly, TNF-α has also been described as an important cytokine in mediating myelin and oligodendrocyte damage in vitro (37). Since most polarized type 1-like TCC originated from the skin lesions of patients with RR (as presented in Table I), it is suggestive that this cytokine is crucial to cause nerve damage in those patients. Such assumption is compatible with the observation by other investigators that showed increased levels of TNF-α in the lesions with RR (38, 39). On the other hand, IL-13 was coproduced dominantly by type 2-like M. leprae-responsive TCC that were generated predominantly from a borderline lepromatous leprosy (BL) patient characterized by the lack of CMI-DTH responses to M. leprae, but with significant humoral immunity (6). Since IL-13 shares with IL-4 the functional properties of inhibiting CMI responses by the inactivation of monocyte functions (31, 40), as well as favoring humoral immunity by the activation of B cells (40), our findings implied that IL-13 may contribute to high Ab serum levels and unrestricted replication of bacilli in the lesion of such patients. Further studies are required to more fully understand the role of T cell-derived TNF-α and IL-13 in the immunopathologic spectrum of leprosy.

An interesting finding of the present study is that type 1- and type 0-like TCC did not always show coproduction of IL-4/IL-5/IL-13. These data may support the hypothesis that cytokine-producing T cells display a spectrum of cytokine profiles, of which the polarized type 1- and type 2-like T cells represent the two possible extremes (41). A preliminary analysis of our data indicates that most of these TCC showing the variable spectrum of cytokine secretion profiles could be generated from the lesions of borderline leprosy patients irrespective of their clinical status. However, these indeterminate TCC, depending on their coexpressions of particular cytokine profiles, might be pivotal in the pathophysiology of the disease process by interacting with other type 1- or type 2-like T cell subsets in the microenvironment of the granuloma. Such assumption, nevertheless, should be taken with caution until a detailed analysis of the cytokine secretion profile of these TCC in relation to the disease activities of the individual patient has been evaluated. At present it should be emphasized that the significance of these indeterminate subsets of TCC in respect to the pathology of leprosy could not be drawn from this study.

In this study, we also showed that IL-10 was produced predominantly by type 2-like M. leprae-responsive T cells. However, a weak correlation between the production of IL-10 and other type 2-like cytokines, as determined in the whole panel of TCC, was found. This may be explained by the fact that a subset of type 1-like M. leprae-responsive TCC produced IL-10 with varying secretion levels. It is interesting to note that the type 1-like M. leprae-responsive TCC, but not the type 1-like M. leprae-nonresponsive TCC, could be divided into a subset of IFN-γ+/TNF-α+/IL-10 type 1-like TCC (n = 35; IL-10 production below detection level 0.2 ng/ml) and a subset of IFN-γ+/TNF-α+/IL-10+ type 1-like TCC (n = 35; varying production of IL-10 between 0.2 and 56.3 ng/ml). These data correlate well with other studies that showed that human type 1-, type 0-, and type 2-like T cells have the capacity to produce IL-10 upon activation (12, 33, 42), whereas highest mean levels of IL-10 were synthesized by Th0- and Th2-like TCC (42). Our data may support the idea that IL-10, by down-regulating through monocytes the Ag-induced proliferation and cytokine production of Th1 as well as Th2 cells (33), may be involved in dampening both ongoing type 1- and type 2-like Ag-driven immune responses. Recent evidence for such a regulatory role of IL-10 in mycobacterial infection came from a study that shows that within the antimycobacterial response of transgenic mice that secrete IL-10 only from the T cell compartment, IL-10 overrides the antimycobacterial effects of IFN-γ on macrophages infected with Calmette-Guerin bacillus (Mycobacterium bovis) (43). M. leprae-responsive TCC may be characterized further by minimal IL-6 production, irrespective of the secretion profile of other cytokines. This finding is compatible with other studies showing that M. leprae-responsive TCL (44) and TCC (4) generated from leprosy patients do not produce IL-6. Although production of IL-6 by T cells is generally regarded to be minimal, the production of this cytokine by T cells has been reported in rheumatoid arthritis (14), and was found to be produced predominantly by the type 2-like M. leprae-nonresponsive TCC investigated in the present study. Since additionally M. tuberculosis-specific T cells lacked the expression of IL-6 mRNA (45), it is suggestive that the absence of IL-6 production is another characteristic for Mycobacterium-responsive T cells in general. The implications of the absence of IL-6 production by M. leprae-responsive T cells cannot be drawn from this study.

In conclusion, it appears that the classification of T cell subsets on the basis of cytokine profiles may be regarded as pathogen specific. In respect to the pathology of leprosy, the balanced production of IFN-γ/TNF-α vs IL-4/IL-5/IL-13 by type 1- and type 2-like M. leprae-responsive T cells, respectively, may play a dominant role in the T cell-mediated immune responses.

We thank Dr. E. A. Wierenga from the Cell Biology and Histology Department (Academic Medical Center, Amsterdam, The Netherlands) for IL-5 ELISA. We thank Dr. J. Oosting for his assistance in statistic analysis.

1

This work was supported by grants from the Netherlands Leprosy Relief Association (The Netherlands) and Q.M. Gastmann Wichers Foundation (The Netherlands), and was conducted under the research programs of ODP/DE1 and ODP/PA2 of Van Loghem Immunology Institute of the Faculty of Medicine, Academic Medical Center, University of Amsterdam. C.E.V. and A.A.M.B. are recipients of Netherlands Leprosy Relief Association maintenance grants.

3

Abbreviations used in this paper: CMI, cell-mediated immunity; BL, borderline lepromatous; BT, borderline tuberculoid; BB, mid-borderline; DTH, delayed-type hypersensitivity; RR, reversal reaction; TCC, T cell clone; TCL, T cell line.

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