Inhibition of Human Lymphoproliferative Responses by Mycobacterial Phenolic Glycolipids JEAN-JACQUES FOURNIE,T* ELIZABETH ADAMS, RAYMOND J

INFECTION AND IMMUNITY, Nov. 1989, p. 3653-3659 Vol. 57, No. 11 0019-9567/89/113653-07$02.00/0 Copyright X3 1989, American Society for Microbiology Inhibition of Human Lymphoproliferative Responses by Mycobacterial Phenolic Glycolipids JEAN-JACQUES FOURNIE,t* ELIZABETH ADAMS, RAYMOND J. MULLINS, AND ANTONY BASTEN Clinical Immunology Research Centre, University of Sydney, 2006 Sydney, Australia Received 17 April 1989/Accepted 8 August 1989

The effect of mycobacterial phenolic glycolipids from Mycobacterium leprae, M. bovis BCG, and M. kansasii on in vitro proliferative responses by human blood mononuclear cells from healthy BCG vaccinees was investigated. All three phenolic glycolipids inhibited proliferation in a concentration-dependent manner. Inhibition was independent of the stimulus used and involved neither antigen-presenting cells nor antigen- specific CD8+ suppressor T cells. It was concluded that the phenomenon may be a general property of mycobacterial phenolic glycolipids, perhaps analogous to the growth-modulating properties of gangliosides. Despite the lack of specificity of inhibition in vitro, de facto specificity may occur in vivo by virtue of the localization of glycolipid in the leprosy lesions.

The apparent lack of protective immunity in patients with surface of the antigen-presenting cell (APC) for several days leprosy is associated with the persistence of viable myco- (18), thereby interfering with the subsequent proliferative bacteria within host tissues and a lack of degradation by responses of murine lymphocytes to a variety of stimuli (8). macrophages. Because the microbicidal function of macro- Thus, the studies with both these antigens emphasize the phages is largely dependent on activation by antigen-specific importance of the potential interaction between mycobacte- T lymphocytes, the occurrence of infection could be second- rial glycolipids and macrophages in interfering with T-cell ary to interference in the activation process by mycobacte- activation. rial antigens. Among these antigens, the abundant mycobac- The most widely studied glycolipid from M. leprae is Phe terial cell wall glycolipids constitute the active interface GL-I, largely on account of its capacity to elicit a strong between the pathogen and its environment and therefore antibody response in leprosy patients, particularly those at have the potential to modulate the host response. The the lepromatous end of the spectrum (2). However, this Phe glycolipids are of different types: the phosphatidylinositol GL-I also interacts with the cellular arm of the immune mannosides, the phosphorylated lipoarabinomannam (LAM) system at the level of both macrophage function and lym- found in most mycobacteria, the glycopeptidolipids from the phocyte proliferation. For example, pretreatment of mono- Mycobacterium avium-M. intracellulare-M. scrofulaceum cytes with Phe GL-I from M. leprae results in inhibition of complex, the trehalose-containing lipooligosaccharides from the oxidative burst in response to bacterial antigens (30), M. kansasii and M. xenopi, and the phenolic glycolipids (Phe whereas the ability of macrophages to kill Phe GL-I-coated GL) found in M. bovis BCG, M. leprae, M. kansasii, M. Staphylococcus aureus cells has been shown to be grossly marinum, and M. gastri (reviewed in reference 5). impaired (26). In addition, there is some evidence that the Data about the effects of some but not all of these Phe GLs may act as virulence factors by scavenging oxygen glycolipids on the in vitro response by human lymphoid cells radicals released by activated macrophages (10). are available. LAM is a potent inhibitor of in vitro prolifer- Phe GL-I was initially reported to cause inhibition of ation of peripheral blood mononuclear cells (PBM) from concanavalin A (ConA)-induced proliferation of PBM from healthy subjects or patients with tuberculosis when stimu- patients with lepromatous disease but not from those with lated by mycobacterial or other antigens, the effect being tuberculoid disease or from healthy controls (23). The mech- dependent on the presence of monocytes in culture (14). anism of this effect was thought to be due to triggering of M. Further studies have revealed that this molecule can also leprae-specific T suppressor cells by the specific oligosac- inhibit purified protein derivative (PPD)- or M. Ieprae- charide epitope on Phe GL-I (24). On the other hand, induced responses by PBM from leprosy and patients attempts to induce M. leprae-specific T lymphocytes in mice healthy individuals (20) as well as influenza virus-specific proliferation of CD4+ T-cell clones (25). Since proliferation with Phe GL-I have yielded little evidence of a specific cellular response to the glycolipid (6, 21). induced by macrophage-independent stimuli such as inter- More recently, the effect of Phe GL-I on proliferative leukin-2 (IL-2) and anti-CD3 antibody was not inhibited by responses to ConA in leprosy patients across the clinical LAM, the findings indicate that LAM exerted its effect at the level of antigen processing by macrophages. spectrum as well as in healthy contacts has been studied. Similar results have been found with glycopeptidolipids, The results indicated that Phe GL-I usually exerts a nonspe- (the serovar-specific antigens of M. avium, M. intracellulare cific inhibitory effect on proliferation, which occurs regardless of the type or even the presence of leprosy, although in and M. scrofulaceum). This type of glycolipid is resistant to a minority of patients and healthy contacts the mitogen degradation by macrophages, so that it remains intact on the response was actually enhanced (27). These findings suggested that Phe GL-I was unlikely to determine the type of * Corresponding author. leprosy or to induce a specific T-suppressor cell response. t Present address: Centre de Recherches de Biochimie et de The conflicting results in the literature prompted the G6ndtique Cellulaires du Centre National de la Recherche Scienti- current study, which was designed to test whether higher fique, 118 route de Narbonne, 31062 Toulouse, France. purified mycobacterial glycolipids could in fact influence the 3653 3654 FOURNIE ET AL. INFECT. IMMUN. in vitro proliferative response by T cells and, if so, by what 30 ng/ml. Leu-O-Me was freshly diluted in PBS and used at mechanism. A variety of antigens and mitogens were used to a final concentration of 5 nM as previously described (29). control for specificity, and cells from healthy BCG vaccinees The IL-2 source was the culture supernatant from the gibbon and continuous cell lines served as targets to avoid the cell line MLA 144 used at a final concentration of 20%. potentially confounding effects of chronic infection and Monoclonal antibodies. The anti-CD3, CD4, CD8, and treatment associated with the use of PBM from leprosy CD25 (Tac) monoclonal antibodies were purchased from patients. Initial experiments were performed with the Phe Becton Dickinson and Co., Mountain View, Calif., and the GLs from M. leprae (Phe GL-I), M. kansasii (Phe GLK-I), secondary anti-mouse antibodies fluorescein isothiocyanate and M. bovis BCG (Phe GLB-I) (which differ only in their and phycoerythrin conjugate were obtained from Silenius oligosaccharide portion), and Phe GLK-I was used in the Laboratories, Hawthorn, Australia, and Amersham Interna- remainder because of its fully elucidated structure and ready tional Plc, Amersham, United Kingdom, respectively. availability in pure form (15, 16). Preparation of cells. Samples of blood from healthy BCG Inhibition of proliferation was found to be independent of vaccinees were collected in preservative-free heparin at 10 the Phe GL studied, of the stimulus to proliferation, and of IU/mi (Weddel Pharmaceuticals, Thomleigh, New South the presence of APC or CD8' T cells. The mechanism Wales, Australia). PBM were separated on Ficoll-Hypaque appeared to be a nontoxic functional effect on proliferating gradients. T lymphocytes and APC were further separated CD4+ lymphocytes. The degree of inhibition was correlated from PBM by AET-treated sheep erythrocyte rosetting, as in most cases with the concentration of glycolipid in culture, described previously (28), into E+ and E- cell-enriched although there was a large degree of individual variation. populations, respectively. Fluorescence-activated cell sorter Despite the apparent lack of specificity of the effect, it was (FACS) profiles of the E+ population revealed consistently concluded that inhibition may be a general property of less than 3% of remaining monocytic cells. CD8+-depleted glycolipids, with the potential for de facto specificity owing and CD8+-enriched T-cell populations were obtained from to their high concentration within the lesions of leprosy. E+ cells by using an anti-CD4 monoclonal antibody panning procedure (32). The nonadherent (CD8+-enriched) popula- MATERIALS AND METHODS tion was 52% CD8+, and the adherent (CD8+-depleted) population was less than 4% CD8+, as assessed by FACS Preparation of purified Phe GLs. Phe GLK-I, the major analysis. Monocyte-macrophage-selective depletion of the tetrasaccharidic Phe GL from M. kansasii, was extracted lymphocyte population was performed by treatment of E+ from cultures with chloroform-methanol and purified by cells with 5 mM Leu-O-Me in serum-free medium (5 x 106 liquid chromatography as already described (15). Purified M. cells per ml) for 40 min at room temperature. The remaining bovis BCG Phe GL B-I and M. leprae Phe GL-I were kind population was found by FACS analysis to contain less than gifts from, respectively, A. Vercelonne and C. Asselineau, 0.5% monocytes or macrophages, as described previously Centre de Recherches de Biochimie et de Genetique Cellu- (29), and consequently could not be stimulated to proliferate laires du Centre National de la Recherche Scientifique, by lectins (data not shown). Toulouse, France. The glycolipids were stored in sterile The antigen-specific helper T-cell line EABCG reactive to filtered ethanol stock solutions at 4°C and a concentration of M. bovis BCG sonic extract is part of a larger collection of 100 ,ug/ml. BCG-specific T-cell lines and was generated from PBM of a Reagents. Tuberculin PPD was purchased from Statens BCG-vaccinated subject stimulated for 7 days with the Seruminstitut, Copenhagen, Denmark, and reconstituted in antigen, followed by a 1-week expansion phase in the sterile 0.15 M phosphate-buffered saline (pH 7.2) (PBS). The presence of IL-2-containing medium and two successive M. bovis BCG sonic extract was prepared as described 1-week expansions in the presence of antigen, irradiated earlier (7). Streptokinase-streptodornase was purchased autologous APC, and IL-2 (E. Adams, unpublished results). from Lederle Laboratories, Sydney, Australia. After recon- The CD4+ phenotype of this cell line was confirmed by stitution in sterile water, the solution was dialyzed in 25-mm FACS analysis (data not shown). dialysis tubing (Union Carbide Corp., Chicago, Ill.) for 48 h Jurkat and MOLT-4 T-cell leukemia lines were obtained against cold PBS to remove preservatives. The protein from Karen Walker, Clinical Immunology Research Centre, concentrations of antigens were adjusted to 1 mg/ml with University of Sydney. The phenotype of these cells by PBS before being subjected to sterile filtration through a FACS analysis was found to be negative for the markers membrane filter (pore size, 0.22 pRm; Millipore Corp., Bed- Tac, CD3, CD4, and CD8. Cell lines were grown in RPMI ford, Mass.) and stored in aliquots at either 4°C (PPD) or 1640 with 10% fetal calf serum and maintained in log-phase -70°C (BCG and streptokinase-streptodornase). growth before being seeded into tissue culture wells in the ConA was purchased from Calbiochem-Behring, La Jolla, presence or absence of glycolipid, pulsed with tritiated Calif., and the purified leucoagglutinin fraction of phyto- thymidine, and harvested the next day. haemagglutinin was purchased from Pharmacia Laborato- Proliferation assays. The phenolic glycolipid stock solu- ries, Sydney, Australia. Both mitogens were freshly sus- tions in ethanol (or ethanol alone in control experiments) pended in PBS and sterile filtered before being used in cell were added to the wells of culture trays prior to cell addition. cultures at the concentrations indicated in the text. The Briefly, 50 ,ul of either sterile filtered ethanol or 100-,ug/ml calcium ionophore ionomycin was kindly donated by N. phenolic glycolipid solutions in sterile ethanol were added Hunt, Department of Pathology, University of Sydney, and dropwise and dried under sterile conditions. On the basis of was used at a final concentration of 300 ng/ml. Indometh- a 2,124-dalton molecular mass for the major Phe GLK-I acin, L-leucine-O-methyl ester (Leu-O-Me), 2-aminoethyl- homolog (15), the glycolipid concentration in culture wells isothiouronium bromide hydrochloride (AET), and phorbol was thus 11.7 ,uM, unless otherwise specified. The antigen or myristate acetate were purchased from Sigma Chemical Co., mitogen solutions were added to the wells at the final St. Louis, Mo. Phorbol myristate acetate was stored as a concentrations specified. Cells were then added in a 200-,ul stock solution in ethanol at -20°C and freshly diluted in PBS final volume per well of complete RPMI 1640 culture me- before being used in cell cultures at a final concentration of dium obtained from Flow Laboratories, Sydney, Australia, VOL. 57, 1989 PROLIFERATION INHIBITION BY MYCOBACTERIAL GLYCOLIPIDS 3655 supplemented with 0.85 g of NaHCO3 per liter, 10 mM TABLE 1. Inhibition of the proliferative response to PPD N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid by Phe GLK-I (HEPES), 50 mg ofpenicillin per liter, 100 mg of streptomycin [3H]thymidine incorporation (103 DPM) by: Inhibition per liter, 50 mg of gentamicin per liter, and 0.2 mM L- Subject glutamine and containing 10% human heat-inactivated A' Medium Phe GLK-I PPD PPD + Phe GLK-I(1 ) serum. 1 3.1 3.8 38.3 23.2 39 Cell concentrations were 106/ml for PBM and 2. 105/ml for 2 4.6 18.4 65.9 32.7 51 T-cell lines or T-ceil subsets, which were cultured in the 3 5.2 5.0 20.3 5.8 72 presence of 5.105 irradiated (2,500 rads) autologous histo- 4 2.7 NTa 91.5 62.1 33 compatible APC per ml, unless otherwise specified. The 5 8.3 7.0 83.8 57.1 31 cultures were incubated at 37°C in a humidified atmosphere 6 7.8 2.7 78.7 35.7 54 of 5% CO2 in air. The total duration of cultures with antigens 7 3.5 7.7 59.5 63.4 -6b or mitogens was 6 or 3 days, respectively, and these were a NT, Not tested. pulsed with 0.5 ,uCi of [methyl-3H]thymidine (Amersham b Indicates an enhanced reponse to PPD of 6%. International) per well in sterile PBS 24 h before being harvested unless otherwise specified. Cultures were performed in triplicate, and results are expressed as 103 mean inhibition was due to the presence of Phe GLK-I per se in the disintegrations per minute ± standard deviations. When culture. Furthermore, it was not caused by toxicity of the indicated, results were expressed as the difference from glycolipid, as assessed by FACS analysis of cells stained background proliferations in unstimulated control culture: with propidium iodide (data not shown). dDPM. The inhibition of proliferation was calculated as To determine whether there was an optimal concentration follows: D (%) = 1 - [(DPM of cultures with glycolipid)/ of Phe GLK-I for inhibiting proliferation, PBM from two of (DPM of similar cultures without glycolipid)]. the above healthy subjects were stimulated with PPD (10 FACS analysis. Cells cultured as indicated above were ,ug/ml) and cultured in the presence of various concentra- analyzed for their surface expression of CD3 (T-cell antigen tions of Phe GLK-I. Although individual variability was receptor marker), CD4 (T-helper cell marker), CD8 (sup- again observed, maximal inhibition occurred with 11 ,uM Phe pressor or cytotoxic T-cell marker), and CD25 (Tac antigen, GLK-I, and this concentration was therefore used in all the p55 component of IL-2 receptor). Triplicate cultures subsequent experiments (Fig. 1). were pooled and staining was performed as previously The inhibitory effect of Phe GLs is unrelated to structural described (1). Stained cells were fixed in 1% paraformalde- carbohydrate specificities. The previous experiments demon- hyde in PBS and stored at 4°C in the dark before being strated a clear-cut reduction in the proliferative response to analyzed. For each sample, 20,000 cells were analyzed on a PPD in the presence of Phe GLK-I, but it was uncertain FACS 440 (Becton Dickinson). whether this was specific to Phe GLK-I or was a general CD3-fluorescein isothiocyanate and CD25-phycoerythrin property of Phe GLs as a whole. Parallel cultures were double-labeled cells were excited by the 488-nm line of an therefore set up with Phe GLs from M. leprae, M. bovis argon ion laser, and the fluorescent emissions were selec- BCG, and M. kansasii. The inhibitory effects exerted by all tively collected by using 535 + 15 nm and 575 ± 26 nm band three glycolipids on the PPD response were comparable pass filters, respectively. (Table 2), suggesting that the mechanism responsible was The proportion of Tac+ CD3+ double-labeled cells in independent of the carbohydrate epitopes on the glycolipids cultures was calculated from the FACS analysis of 20,000 involved. In view of its ready availability in pure form, gated lymphocytes by the ratio %Tac+ CD3+/CD3+ = subsequent experiments were performed with Phe GLK-I. [(number of CD3-fluorescein isothiocyanate and CD25-phy- Inhibition of proliferation by glycolipids is independent of coerythrin fluorescent cells in stained pooled triplicate cul- the stimulus and acts throughout the response. It was impor- tures) - (number of nonspecifically fluorescent cells in tant to determine whether the inhibition observed was re- unstained pooled triplicate control cultures)/[(number of stricted to the response to mycobacterial antigens as PPD. CD3-fluorescein isothiocyanate fluorescent cells detected in To answer this question, PBM from healthy subjects were cultures) - (number of nonspecifically fluorescent cells in unstained control cultures)].

RESULTS 100 Effect of Phe GLK-I on the PPD-induced proliferative response of PBM from healthy BCG vaccinees. In preliminary experiments, PBM from seven healthy BCG vaccinees were a6a stimulated to proliferate with PPD (10 ,xg/ml) in the presence or absence of Phe GLK-I (11 ,uM, equivalent to 25 jig/ml), to determine whether the glycolipid could influence the cellular 20. response to antigen. The presence of Phe GLK-I in culture resulted in a decrease in proliferation in six of the subjects, with a mean inhibition of 39.1 + 24.2%, whereas an en- 0 10-8 i0 7 10-6 1iO5 hanced response was found in the seventh subject (Table 1). When one subject was retested a total of 12 times, the effect PheGLKI MOLAR CONCENTRATION on the PPD response was reproducible with a mean inhibi- FIG. 1. Sensitivity to Phe GLK-I of stimulated PBM from two tion of 51% + 18%. The decreased response to PPD oc- subjects. Symbols: El, subject 1, stimulated cells; 0, subject 1, curred despite the use of different batches of PPD, RPMI unstimulated cells (control); *, subject 2, stimulated cells; 0, 1640, human A sera, and culture plates, suggesting that the subject 2, unstimulated cells (control). 3656 FOURNIE ET AL. INFECT. IMMUN.

TABLE 2. Inhibition of the proliferative response to PPD by mycobacterial glycolipids Mean [3H]thymidine incorporation Glycolipid (103 DPM) ± SD by: Inhibition Medium PPD None 4.9 ± 2.1 92.9 ± 10.3

Phe GLK-I 3.8 ± 1.3 31.6 ± 7.7 66 20 Phe GLB-I 5.2 ± 3.6 45.4 ± 11.5 51 ± ± Phe GL-I 3.8 3.1 23.6 5.1 75 4 12 24 48 ( hours) Time of cell transfer on PheGL KI cultured with various concentrations of the mycobacterial antigen BCG and the unrelated antigen streptokinase- after stimulation by Con A streptodornase, as well as the nonspecific mitogens ConA FIG. 2. Suppression of lymphoproliferative response to ConA by and PHA. In each case, the proliferative response was transfer of stimulated cultures into wells containing Phe GLK-I (LI) significantly reduced when compared with that in the pres- or into control wells without Phe GLK-I (U). ence of antigen or mitogen alone (Table 3). Furthermore, time course experiments suggested that the effect on both mitogen- and antigen-induced proliferation was not due to a was required, prostaglandin E2 was not involved. Second, delay in the kinetics of the response in the presence of various numbers of irradiated E- cells containing APC were glycolipid (data not shown). cultured with a fixed number of E+ T lymphocytes and then These results, however, did not indicate whether inhibi- stimulated with PPD. However, there was no relationship tion with glycolipid occurred during the induction phase of between the numbers of APC present and the degree of the proliferative response, i.e., at the level of antigen pre- inhibition seen (Table 4). Finally, unseparated PBM treated sentation to T cells, or acted later on replicating cells. In an with Leu-O-Me to kill macrophages were cultured in the attempt to distinguish between these two possibilities, a presence of a combination of ionomycin and PMA, which two-stage culture system was used in which PBM were are known to stimulate lymphocytes to proliferate indepen- initially stimulated with 10 ,ug of ConA per ml. After dently of APC. Once again, significant inhibition of prolifer- exposure to ConA alone for a period ranging from 4 to 24 h, ation by Phe GLK-I was observed, providing the strongest the cells were transferred to new wells either containing no evidence against a role for APC in mediating this phenome- glycolipid or containing 11 ,uM Phe GLK-I. Regardless ofthe non (Fig. 3). time of transfer, proliferation was inhibited to a similar Phe GLs inhibit the proliferation of CD4+ cells without extent, suggesting that the inhibitory effect was active involvement of CD8+ suppressor cells. The lack of a require- throughout the response and not just during the induction ment for APC in mediating inhibition raised the possibility phase (Fig. 2). that the glycolipid was exerting its effect on proliferating T APC are not involved in the glycolipid-mediated inhibition. cells either by switching off CD4+ cells directly or by The fact that the inhibitory effect operated throughout the preferentially activating CD8+ suppressor T cells. Lympho- proliferative response rather than just during induction im- cytes were therefore enriched either for CD4+ or CD8+ cells plied that APC were not involved. Several experiments were by monoclonal antibody panning after initial enrichment for therefore performed to confirm that the APC had no role in CD3+ cells by E cell rosetting. Inhibition of the response by mediating this phenomenon. First, addition of indomethacin CD4+ lymphocytes (>98% purity) cultured with autologous to the culture medium failed to reverse the inhibitory effect irradiated E- cells and stimulated with 10 jig of ConA per ml of Phe GLK-I (data not shown), suggesting that even if APC was unaffected by depletion of CD8+ cells (Table 5). Con- versely, enrichment for CD8+ lymphocytes did not result in enhanced inhibition, thereby arguing against a significant TABLE 3. Inhibition of the proliferative response to mitogens role for CD8+ cells and suggesting that CD4+ lymphocytes and antigens by Phe GLK-I might be the primary target of the inhibitory effect. To confirm this, a BCG-specific T-cell line, EABCG, of CD4+ Mean [3H]thymidine incorporation phenotype was stimulated with 10 ,ug of BCG sonic extract Mitogen (103 DPM) + SD by: Inhibition per ml in the presence of autologous irradiated E- cells for Medium Phe GLK-I 3 days. On addition of Phe GLK-I, comparable inhibition None 0.4 + 0.1 0.32 ± 0 ConA TABLE 4. Effect of the number of APC on Phe GLK-I- 10,ug/ml 66.0 ± 0.8 26.4 ± 6.3 60 induced inhibition of the proliferative response to PPD 1 ,Ug/ml 7.0 ± 0.8 4.8 ± 0.6 31 PHA Mean [3H]thymidine incorporation 10,g/ml 190.3 ± 20.3 131.7 ± 33.4 31 No. of APC (103 dDPM) ± SD by: Inhibition 1 ,Ug/ml 215.8 ± 15.3 141.3 ± 37.6 35 added to i05 % purified T cells PPD PPD + Phe SKSDa GLK-I 10 ,Ug/ml 47.3 ± 17.2 24.8 ± 9.6 47 1 ,Ug/ml 31.5 ± 7.3 15.4 ± 2.7 51 2 x 103 93.2 ± 1.3 51.7 ± 10.4 44 BCG S x 103 85.6 ± 5.6 48.8 ± 5.0 43 10 ,Ug/ml 94.3 ± 8.8 62.5 ± 1.3 34 1 X 104 82.7 ± 36.5 47.9 ± 4.9 42 1 ,Ug/ml 49.6 ± 6.4 30.9 ± 15.2 37 5 x 104 100.6 ± 40.5 40.6 ± 1.4 59 1 x 105 76.0 ± 35.7 31.9 ± 2.8 58 a SKSD, Streptokinase-streptodornase. VOL. 57, 1989 PROLIFERATION INHIBITION BY MYCOBACTERIAL GLYCOLIPIDS 3657

100 I UNTREATED 100_ CELLS

50- 50

0 0 Jurkat MOLT-4 100 Leu -0-Me TREATED CELLS FIG. 4. Suppression of spontaneous proliferation of T leukemia cell lines Jurkat and MOLT-4 by Phe GLK-I. Spontaneous proliferation with (1) or without (E) glycolipid is shown. 50 Jurkat and MOLT-4 were cultured in the presence and absence of Phe GLK-I. Spontaneous proliferation of both I cell lines was reproducibly inhibited to a degree comparable to that observed previously with PBM (Fig. 4). In other 0 words, the inhibitory effect was not due to down-regulation PMA Both of IL-2 receptor per se, although it did act on proliferating lonomycin APC. FIG. 3. Suppression of ionomycin- and PMA-triggered prolifer- cells independently of ation of whole PBM and macrophage-depleted PBM. Whole PBM (untreated cells) or macrophage-depleted PBM (Leu-O-Me-treated DISCUSSION cells) stimulated by either ionomycin alone, PMA alone, or both, were cultured for 3 days with (L) or without (U) Phe GLK-I. Mycobacterial antigens are widely distributed throughout the tissues of patients infected with M. leprae. M. Ieprae bacteremia has been found in patients with lepromatous was obtained to that with PBM (Table 4), which effectively leprosy (13), and acid-fast bacilli are frequently identifiable excluded active suppression by CD8+ cells. in bone marrow, kidneys, and liver, as well as the main sites Phe GLK-I decreases the expression of IL-2 receptor on the of infection in the skin and nerves (3). Furthermore, antigens T-cell surface. Given that the addition of glycolipids to such as Phe GL-I have been isolated in serum and urine and culture appeared to interfere with the expansion of prolifer- have been purified from skin lesions of patients with leprosy ating T cells (the latter being dependent on the interaction (11, 19, 31). In view of their abundance and location in the between free IL-2 and cell-bound IL-2 receptor) (9), the cell wall, the glycolipids such as Phe GL-I are recognized by question arose of whether glycolipid was inhibiting the the immune system and thus are likely to play a significant expression of IL-2 receptor. PBM were therefore cultured role in the interaction between the pathogen and its environ- for 48 h in the presence of 10 ,ug of ConA per ml and double ment. stained for CD3 and Tac. The proportion of Tac-positive, The current experiments were designed to explore the role CD3-positive cells from freshly isolated PBM was 5.4%, of Phe GLs in the immune response to mycobacteria. In whereas culture for 48 hours yielded values of 0% for particular, attention was focused on whether they are inhib- unstimulated cells, 10.6% for cells stimulated with ConA, itory rather than stimulatory and whether their effects are and 0.2% for cells grown with ConA and Phe GLK-I. That specific or nonspecific, given the controversy in the litera- this was a selective effect for Tac was shown by the fact that ture on this subject. no detectable change occurred in the cell surface markers for Phe GLK-I from M. kansasii was used as the prototype CD3, CD4, and CD8 during culture with glycolipid (data not glycolipid since it was readily available in pure form and in shown). fact exhibited similar inhibitory effects on in vitro prolifera- To determine whether the apparent decrease in Tac tive responses to the glycolipids obtained from both M. expression was the cause of the reduction in proliferation or leprae and M. bovis BCG (Table 2). This inhibition was a merely indicative of a nonspecific modulatory effect on the general property of Phe GLs and could not be attributed to T-cell membrane, cells from the Tac-negative T-cell lines the carbohydrate moieties which express specific B-cell epitopes (12). The lack of specificity was confirmed by showing (i) that responses to mitogens as well as mycobac- TABLE 5. Effect of Phe GLK-I on proliferation of purified T-cell terial antigens were inhibited to a comparable degree (Table populations to ConA and of the CD4+ cell line 3) and (ii) that the inhibitory effect operated throughout the EABCG to BCG sonic extract proliferative response rather than being confined to the Mean [3H]thymidine induction phase of antigen presentation to specific T cells Cell type per Phenotype incorporation Inhibition (Fig. 2). culture (% (103 dDPM) ± SD by: (% The target of glycolipid-induced inhibition of PBM ap- Medium Phe GLK-I peared to be the proliferating CD4+ T cell, in contrast to that of LAM, which acts at the level of antigen presentation by CD4+ enriched CD4+ (>98%) 14.5 ± 2.7 6.0 ± 3.4 57 macrophages (4, 25). Evidence in favor of this conclusion CD8+ enriched CD8+ (52%) 29.0 ± 1.3 18.9 ± 1.6 34 ± ± came from two sets of experiments. In the first, a role for EABCG cell CD4+ (>95%) 20.9 24.4 5.7 0.2 60 APC was effectively excluded. For example, addition of the line prostaglandin inhibitor indomethacin to the culture did not 3658 FOURNIE ET AL. INFECT. IMMUN. abrogate the inhibitory effect. Furthermore, the APC-inde- Institute of Pathology, Washington, D.C. pendent proliferative response to ionomycin and PMA was 4. Bloom, B. R., and V. Mehra. 1984. Immunological unrespon- inhibited to a similar degree to that obtained with macro- siveness in leprosy. Immunol. Rev. 80:5-28. phage-containing suspensions of PBM (Fig. 3), as was spon- 5. Brennan, P. J. 1988. Mycobacterium and other actinomycetes, taneous proliferation of the p. 203-298. In C. Ratledge and S. G. Wilkinson (ed.), Microbial Jurkat and MOLT-4 cell lines. lipids, vol. 1. Academic Press, Inc. (London), Ltd., London. Second, the effect was not dependent on the presence of Editors, London. CD8+ suppressor T cells and was in fact observed when 6. Brett, S. J., C. Lowe, S. N. Payne, and P. Draper. 1984. Phenolic purified CD4+ T cells from PBM or a CD4+ BCG-specific glycolipid 1 of Mycobacterium leprae causes nonspecific inflam- cell line were used in the culture system (Table 4). mation but has no effect on cell-mediated responses in mice. The mechanism of inhibition of lymphocyte proliferation Infect. Immun. 46:802-808. remains unclear. Although glycolipid leads to down-regula- 7. Britton, W. J., L. Hellqvist, A. Basten, and R. L. Raison. 1985. tion of IL-2 receptors on the T-cell surface, proliferation of Mycobacterium leprae antigens involved in human immune the Tac-negative Jurkat and MOLT-4 cell lines was reduced responses. J. Immunol. 135:4171-4177. as well. 8. Brownback, P. E., and W. W. Barrow. 1988. Modified lympho- Expression of CD3 and CD4 cell surface markers on cyte response to mitogens after intraperitoneal injection of PBM was unaffected by exposure to glycolipid, thereby glycopeptidolipid antigens from Mycobacterium avium com- excluding a role for these markers in mediating the effect. plex. Infect. Immun. 56:1044-1050. Furthermore, the inhibitory influence of glycolipid on the 9. Cantrell, D. A., and K. A. Smith. 1983. Transient expression of CD3- CD4- CD8- Jurkat and MOLT-4 cell lines suggests interleukin 2 receptors. Consequences for T cell growth. J. Exp. that the glycolipids are exerting a nonspecific modulatory Med. 158:1895-1911. effect on the membranes of proliferating cells, perhaps 10. Chan, J., T. Fujiwara, P. Brennan, M. McNeil, S. J. Turco, J. C. analogous to the effects of glycolipids such as gangliosides Sibille, M. Snapper, P. Aisen, and B. R. Bloom. 1989. Microbial on growth and differentiation (17, 22). glycolipids: possible virulence factors that scavenge oxygen Differences in radicals. Proc. Natl. Acad. Sci. USA 86:2453-2457. the concentration of glycolipid and its 11. Cho, S. N., S. W. Hunter, R. H. Gelber, T. H. Rea, and P. J. presentation in culture may account for the conflicting data Brennan. 1986. Quantitation of the phenolic glycolipid of My- in the literature. Mehra et al. reported a reduction in cobacterium leprae and relevance to glycolipid antigenaemia in ConA-induced proliferation of PBM from lepromatous pa- leprosy. J. Infect. Dis. 153:560-569. tients when Phe GL-I encapsulated in liposomes was used 12. Cho, S. N., D. L. Yanagihara, S. W. Hunter, R. H. Gelber, and (23). The effect was maximal at 0.5 jig/ml but disappeared P. J. Brennan. 1983. Serological specificity of phenolic gly- when the terminal trisaccharide was removed and was not colipid I from Mycobacterium leprae and use in serodiagnosis of observed during analogous experiments with Phe GLK-I and leprosy. Infect. Immun. 41:1077-1083. Phe GLB-I (23). By contrast, Prasad et al. (27) demonstrated 13. Drutz, D. J., T. S. N. Chen, and L. Wen-Hsiang. 1972. The the continuous bacteraemia of lepromatous leprosy. N. Eng. J. inhibition of a ConA-induced response by PBM from Med. 287:159-164. healthy contacts and leprosy patients of all clinical types 14. Ellner, J. J., and T. M. Daniel. 1979. Immunosuppression by with Phe GL-I in liposomes but at a concentration of 1 mycobacterial arabinomannan. Clin. Exp. Immunol. 35:250- mg/ml. In our experiments, an intermediate concentration of 257. 25 jig/ml (or 11.7 ,uM) was used, but this was dried onto the 15. Fournie, J. J., M. Riviere, and G. Puzo. 1987. Structural base of the wells instead of being suspended in particulate elucidation of the major phenolic glycolipid from Mycobacte- form in the culture medium. rium kansasii. J. Biol. Chem. 262:3174-3179. Clearly, further work is required to resolve the contro- 16. Fournie, J. J., M. Riviere, and G. Puzo. 1987. Absolute config- versy surrounding the mechanism of inhibitory effects of uration of the unique 2,6-dideoxy-4-O-methyl-arabinohexopyra- glycolipids. Nevertheless, it is unnecessary to postulate nose of the major phenolic glycolipid antigen from Mycobacte- rium kansasii. Eur. J. Biochem. 167:181-183. antigen-specific suppression to explain the biological phe- 17. Hakomori, S. I. 1987. Ganglioside mediated modulation of nomenon seen. Despite the lack of specificity observed in growth factor receptor function and cell adhesion. NATO ASI vitro, de facto specificity occurs in vivo owing to the Ser. H7:465-479. presence of large amounts of glycolipids within the lesions of 18. Hooper, L. C., M. M. Johnson, V. R. Khera, and W. W. Barrow. leprosy, which could compromise effective clearance of 1986. Macrophage uptake and retention of radiolabeled glyco- mycobacteria. peptidolipid antigens associated with the superficial Li layer of Mycobacterium intracellulare serovar 20. Infect. Immun. 54: ACKNOWLEDGMENTS 133-141. 19. Kaldany, R. D. J., K. Maasho, R. Ohman, D. Reitz-Vick, S. This work was supported by grants from Leprosy Eradication and Britton, and M. Lefford. 1987. Methods for the detection of a Education Program of Lions International, the Centre National de la specific Mycobacterium leprae antigen in the urine of leprosy Recherche Scientifique, France, (to J.J.F.), and the National Health patients. Scand. J. Immunol. 25:37-43. and Medical Research Council of Australia (to R.J.M.). 20. Kaplan, G., R. R. Gandhi, D. E. Weinstein, W. R. Levis, M. E. We are grateful to Karen Walker for providing useful T-cell lines Patarroyo, P. J. Brennan, and Z. A. Cohn. 1987. Mycobacterium and to Joelle Maurel for secretarial assistance. We thank G. Puzo leprae antigen-induced immunosuppression of T cell prolifera- and J. P. Zalta for special encouragement. tion in vitro. J. Immunol. 138:3028-3034. 21. Koster, F. T., C. Teuscher, P. Matzner, E. Umland, D. Yanagi- LITERATURE CITED hara, P. J. Brennan, and K. S. K. Tung. 1986. Strain variation in 1. Adams, E., J. Wotherspoon, L. Hellqvist, and A. Basten. 1987. the murine cellular immune response to the phenolic glycolipid High dose suppression of human anti influenza A virus re- I antigen of Mycobacterium leprae. Infect. Immun. 51:495-500. sponses using T cell clones. Immunol. Cell. Biol. 65:25-33. 22. Laine, R. A., and S. I. Hakomori. 1973. Incorporation of 2. Baumgart, K., W. Britton, A. Basten, and A. Bagshawe. 1987. exogenous glycosphingolipids in plasma and concurrent change Use of phenolic glycolipid I for serodiagnosis in a high preva- of growth behavior. Biochem. Biophys. Res. 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