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A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Th1-type cytokine profile and to produce interleukin 12.

Leishmania braziliensis causes cutaneous and mucosal leishmaniasis in humans. Most patients with cutaneous leishmaniasis heal spontaneously and may therefore have developed protective immunity. There appears to be a mixed cytokine profile associated with active cutaneous or mucosal disease, and a dominant T helper (Th)1-type response associated with healing. Leishmanial antigens that elicit these potent proliferative and cytokine responses from peripheral blood mononuclear cells (PBMC) are now being identified. Herein, we report on the cloning and expression of a L. braziliensis gene homologous to the eukaryotic ribosomal protein eIF4A (LeIF) and patient PBMC responses to rLeIF. Patients with mucosal and self-healing cutaneous disease had significantly higher proliferative responses than those with cutaneous lesions. Whereas the parasite lysate stimulated patient PBMC to produce a mixed Th1/Th2-type cytokine profile, LeIF stimulated the production of interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor alpha but not IL-4 or IL-10. Recombinant LeIF (rLeIF) downregulated both IL-10 mRNA in the "resting" PBMC of leishmaniasis patients and LPS-induced IL-10 production by patient PBMC. rLeIF also stimulated the production of IL-12 in cultured PBMC from both patients and uninfected individuals. The production of IFN-gamma by patient PBMC stimulated with either rLeIF or parasite lysate was IL-12 dependent, whereas anti-IFN-gamma monoclonal antibody only partially blocked the LeIF-induced production of IL-12. In vitro production of both IFN-gamma and IL-12 was abrogated by exogenous human recombinant IL-10. Therefore, we have identified a recombinant leishmanial antigen that elicits IL-12 production and Th1-type responses in patients as well as IL-12 production in normal human PBMC.

L ishmania are obligate intracellular protozoan parasites of macrophages that cause a spectrum of human diseases, including self-healing skin lesions, diffuse cutaneous and mucosal manifestations, or severe visceral disease (1). Leishmania braziliensis commonly causes localized cutaneous leishmaniasis (CL) 1. Most patients with CL heal spontaneously (2). However, a chronic mucosal leishmaniasis (ML) develops in some infected individuals, with severe and progressive destruction of the nasal, oral, and/or pharyngeal mucous membranes (3,4). Patients with CL generally have positive delayed hypersensitivity and in vitro proliferative responses to leish-1 Abbreviations used in this paler: CL, cutaneous leishmaniasis; LelF, L. braziliensis homologue of the eukaryotic initiation factor 4A; ML, mucosal leishmaniasis; rAg, recombinant antigen; rLelF, recombinant LeIF. manial Ag during both active and cured infection. These responses are often exaggerated in ML patients (5,6).
The induction of Thl effector cells capable of activating macrophages to a microbicidal state is necessary for eliminating these intramacrophage parasites. In experimental leishmaniasis, immunological interventions to direct CD4 § T cell response must be administered before the establishment of Thl or Th2 effector subsets. IL-12, which facilitates the development of Thl responses by stimulating the production of IFN-'y while downregulating the production of IL-4, has been demonstrated to cure susceptible BALB/c mice against L. major infection (7,8), whereas neutralization of IL-12 made resistant mice susceptible to infection (8). Although neutralization of IFN-3' abrogates the natural resistance of C3H/HeN mice to infection with L. major (9), by itself, IFN-7 did not confer protection to susceptible mice (10).
The determination of cytokine profiles in PBMC from leishmaniasis patients with different clinical presentations as well as the identification of defined Ag that induce and elicit cellmediated immune responses is important to our understanding of the nature of immune responsiveness during infection, as well as the potential identification of vaccine molecules. The elucidation of cytokines associated with Thl and/or Th2 responses by defined L. braziliensis Ag in patients with leishmanial infections has not been previously described. Because the course of human infection with L. braziliensis is variable, ranging from self-healing infection to chronic disease, it is an excellent system in which to study immunoregulatory aspects of leishmaniasis, including the effects of parasite Ag on host responses. In the present study, Leishmania-specific PBMC responses in patients representing a spectrum ofL. braziliensis infection were analyzed. A leishmanial Ag, L. braziliensis homologue of the eukaryotic initiation factor 4A (LelF), capable of stimulating strong Thl-type responses in leishmaniasis patients' PBMC as well as IL-12 production in patient and nonpatient cells, was identified. binant antigen (rAg) (LelF.1) was purified, the pBSK(-) phagemid excised, nested deletions generated using Exonuclease III for singlestranded template preparations and sequencing (12). Clone pLelE1 lacked the first 48 amino acid residues (144 nucleotides) of the fulllength sequence. The insert was subsequently used to isolate the full-length genomic sequence.
Expression and Purification of Recombinant LelF (rLelF). kD rag of the LelF.1 genomic clone was purified from 500 ml of isopropyl ~ D-thiogalactoside (IFFG)-induced cultures (12 Immunoblot Analysis. 5-10/~g of parasite or cell extracts or 0.5-1.0 gg of rAg were separated on 12.5% SDS-PAGE and dec. trophoretically transferred to nitrocellulose membranes. Reactivities of the antisera were assessed as previously described (12) using nSI-protein A, followed by autoradiography.
Patients. Peripheral blood was obtained from individuals living in an area endemic for L. braziliensis (Corte de pedra, Bahia, Brazil) in which epidemiological, clinical, and immunological studies of leishmaniasis have been performed for over a decade (3). Diagnosis was made by clinical findings associated with at least one of the following: parasite isolation, leishmanial skin test, or positive serology. An epidemic in the field area without access to glucantime enabled us to detect patients who had healed spontaneously (selfhealing).
PBMC Culture, Proliferation, and Cytokine Assays. Peripheral blood from Leishmania infected or uninfected individuals were collected and PBMC isolated from whole blood by density centrifugation through Ficoll (Pharmacia Biotech Inc., Piscataway, NJ). For in vitro proliferation assays, 2-4 x 10 s cells/well were cultured in complete medium (RPMI 1640 supplemented with gentamycin, 2-ME, t-glutamine, and 10% screened pooled A+ human serum; Trimar, Hollywood, CA) in 96-well flat bottom plates with or without 10 gg/ml of the indicated Ag or 5 gg/ml PHA (Sigma Immunochemicals, St. Louis, MO) for 5 d. The cells were then pulsed with 1/zCi of [3H]thymidine for the final 18 h of culture. Data are represented as mean cpm of triplicate cultures and the stimulation index (SI) defined as mean cpm of cultures with Ag/mean cpm of cultures without Ag. For determination of cytokine production, PBMC were cultured at 1-2 x 106 cells/ml in medium containing endotoxin-free FCS (CeUect Gold; ICN Biomedicals, Inc., Costa Mesa, CA) with or without leishmanial Ag and in the presence or absence of human (Hu) rlL-10 (10 ng/ml, DNAX, Palo Alto, CA), anti-IFN-y (10 /~g/ml; Chemicon, Temucula, CA), or anti-IL-12 (Wistar Institute). Supernatants and cells were harvested and analyzed for secreted cytokine or cytokine mRNAs after 24 (for IL12) or 72 (for ILl0 and fiN-T) h of culture as described below.
Inserts were isolated from plasmids containing the human sequences for Ib2, IFN-3', IL-4, IL-10, and fl-actin and radiolabeled 32p-probes generated as described (17). PCR products were analyzed by dectrophoresis on 1.5% agarose gels, transferred to nylon membranes, and probed with the appropriate szp-labeled DNA insert. Hybridizations were at 55~ overnight. Posthybridization washes were at 55~ for 20 min twice each with 2x and lx SSC containing 0.2% SSC.
Nucleotide Sequence Accession Number. The nucleotide sequence of pLelF has been submitted to the EMBL/GenBank/DDBJ/Data Bank under accession number U19888.

Results
Molecular Cloning and Sequence Characterization of L. braziliensis Ribosomal elF4A. As part of a strategy for identifying leishmanial Ag, we screened a L. braziliensis genomic expression library with sera from a ML patient, purified immunoreactive rAg, and analyzed them in patient T cell assays for their ability to stimulate proliferative responses and preferential Thl cytokine production. This led to the identification of several cDNAs. One of these was identified by GenBank sequence comparison as the L. braziliensis homologue of the eukaryotic initiation factor 4A (LelF).
The open reading frame of the full-length LelF encodes a protein of 403 amino acids and a predicted molecular mass of 45.3 kD (not shown). The lengths (403, 413, 407, and 395 1529 Skeiky et al. amino acids), molecular mass (45.3, 46.8, 46.4, and 44.7 kD), and isoelectric points (5.9, 5.4, 5.5, and 4.9) of 1,elF, and the homologous proteins from tobacco, mouse, and yeast respectively (18)(19)(20) are similar. 1.elF contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases represented by the eukaryotic initiation factor 4A (elF4A). The elF4A is one of the best characterized members of the " D E A D box" family of KNA helicases. These proteins share a series of conserved motifs, with two of them representing specialized versions of the A and B motifs previously described in other ATP-binding proteins (21). The four amino acid sequence Asp-Glu-Ala-Asp (DEAD) is part of the specialized version of the B motif.
Southern analysis of L. braziliensis genomic DNA digested with enzymes that cut both within (PstI and NotI) and outside (BamHI, EcoRI, EcoRV, HindlII, PvulI, and SstI) of LelF and probed with an *0.94-kb fragment (nuc!eotides 143 to 1083) of the coding region of 1.elF revealed at least two copies of LelF (Fig. 1). The same figure also illustrates the cross-species conservation between l.eiF of L. braziliensis and other Leishmania species. Northern analysis revealed that LelF is transcribed as '~2.5 kb mRNA species (not shown).
Expression, Purification, and Immunoblot Analysis of 1.elF Ag. Fig. 2 shows the expression and purification of the m45 kD LelF gene product. Immunoblots of lysates from L. braziliensis as well as other Leishmania species (promastigote and amastigote stages) were performed with the polyclonal rabbit anti-LelF serum which detected one dominant protein species o f -4 5 kD. The immune rabbit sera did not react with E. coli lysate (not shown), demonstrating the lack of E. coli proteins in the purified rLelF.
rLelF Stimulates PBMC from Leishmaniasis Patients to Proliferate and to Express a Predominant Thl Cytokine Profile Purified rLelF was tested for its effectiveness in stimulating T cells from L. braziliensis-infected individuals. Proliferation and cytokine responses of PBMC from patients with active or healed CL, ML, or individuals with self-healing CL were compared using rLelF and L. braziliensis lysates. PBMC from most (>70%) ML and CL patients proliferated to rLelF with stimulation indices ranging from 12 to 233 and 2 to 64, respectively (Table 1). PBMC from all six individuals with selfhealing CL proliferated in response to rLelF with stimulation indices  comparable to those of ML patients. PBMC from two of the self-healing individuals (MS and DJ), proliferated to LelF to a greater degree than to lysate. Cells from normal individuals were only marginally stimulated by LelF. The results demonstrate that rLelF is a potent T cell Ag recognized by a majority of L. braziliensis-infected individuals in different stages or manifestations of infection.
A more detailed analysis of cytokine patterns of PBMC from patients with confirmed cases of L. braziliensis infection was performed using PCR. Fig. 3 A shows the PCR results of cytokine mKNA for three of the six ML patients' PBMC analyzed (JV, SZ, and TE). Cytokine mRNA analyses were performed with freshly isolated PBMC (lanes 0), or PBMC cultured in the absence (lanes -) or presence of rLelF. In three of six patients (TE, Fig. 3 A; NO and EO, not shown), PBMC not cultured in vitro had detectable levels of mKNA for IFN-y and IL-4, and two were positive for IL-2 (patients TE and EO). IL-10 mKNA was not detected in uncultured ("resting") PBMC from any of the ML patients. However, after in vitro culture without Ag, IL-10 mKNA was upregulated in most of the ML patient PBMC analyzed. In addition, the levels of other cytokine mRNAs detected in the resting PBMC of patients TE, NO, and EO, decreased to background levels. In three of six patients, leish-mania1 lysate stimulated the expression of mRNA for the Thl cytokines (IFN-q, and IL-2) as well as for the Th2 cytokine IL-4. Increased IL-10 mRNA was detected in one of the patients' PBMC (SZ, Fig. 3 A) after culture with leishmanial lysate. In contrast, rLelF Ag elicited increased mKNA for IFN-'y and IL-2 from all ML patient PBMC, an exclusive Thl cytokine profile. In fact, the addition ofLelF downregulated the production of IL-10 mRNA in the cultured PBMC of most ML patients (Fig. 3 A, lanes elF).
Similar PCR analyses were performed on PBMC derived from CL patients. The resting PBMC from three (VS, JP, and CA) of the four patients analyzed revealed readily detectable mKNA for both Thl (IFN-3' and IL-2) and Th2 (IL-4 and IL-10) associated cytokines (Fig. 3 B). Little or no mRNA for IFN-% IL-2, IL-IO, or IL-4 was detected in the resting PBMC of the fourth (AS) CL patient. Therefore, in contrast to ML patients, most patients with CL had RNA for IL-10 as well as for IL-4, IL-2, and IFN-3'. For most patients, whereas the mRNA for IL-2 and IFN-y were reduced to barely detectable levels after the in vitro culturing of PBMC in the absence of Ag, mKNA for IL-10 remained either unaffected or increased.
All CL patients tested responded to rLelF or to leishmanial lysate by upregulating the expression of mKNA for IL-2 and IFN-% However, IL-4 mRNA was also enhanced in one of the CL patients (AS) after stimulation with LelF. In three of four patients (JP, VS, and AS), the level of IL-4 mKNA also increased after stimulation with parasite lysate. In the three patients (VS, JP, and CA) with resting levels of IL-IO mKNA, rLelF downregulated their expression of IL-IO mKNA. We also studied PBMC from a diffuse cutaneous leishmaniasis (DCL) patient (L. amazonensis infection). Interestingly, as with PBMC from patients with L. braziliensis infection, LelF downregulated IL-10 mRNA while increasing IFN-3' and IL-2 mRNA in the DCL patient VA (Fig. 3 B). PBMC from patient VA also proliferated in the presence of l.eiF but not in response to parasite tysate (Table 1). In general, the PCK products for IL-2 and IFN-q, of patient PBMC after stimulation with l.eiF or parasite lysate were readily detected on ethidium bromide-stained gels, suggesting relative abundance, whereas IL-4 and IL-IO were detected only by radioactive probing of the resolved PCK products.
The cytokine mRNA profiles of PBMC from self-healing CL were similar to those of ML patients in that (a), except for one individual (HS) with detectable levels of IL-10 mRNA, resting PBMC from three of the four patients (GS, AH, DJ, and HS) analyzed had detectable levels of IL-2, IFN-% and IL-4 but little or no IL-IO mKNA; (b) IL-IO mKNA was upregulated after culture of PBMC without Ag whereas those of IL-2, IFN-y, and IL-4 decreased to background levels and; (c), leishmanial lysate stimulated the expression of a mixed Thl/Th2 cytokine profile whereas LelF elicited increased mRNA expression of only the Thl-type cytokines and downregulated the expression of IL-10 mRNA in the cultured PBMC of most self-healing CL individuals (not shown).
To complement the cytokine mRNA analysis, we assayed PBMC culture supernatants for IFN-% TNF-ol, IL-4, and IL-10. Cells from all three patient groups as well as from  (Fig. 4). The levels of both IFN-3' and TNF-ot in the supernatants of patient PBMC (0.3-26 ng/ml and 0.1-3 ng/ml, respectively) were significantly higher compared to those from uninfected controls. In the absence of rLelF stimulation, only PBMC from ML patients (five of six) produced detectable levels of supernatant TNF-ot (60-190 pg/ml). Little or no IL-4 or IL-10 were detected in any of the supernatants analyzed (not shown), indicating levels below the detection limit of the ELISA assay employed. By comparison, leishmanial lysate also stimulated patient PBMC 1531 Skeiky et al.
to secrete IFN-3' and TNF-c~ and, in some patients, IL-10 was also detected (not shown). Therefore, and in agreement with the PCR cytokine analysis, whereas the cytokine profile of patient PBMC stimulated with LelF reflects an exclusive Thl pattern, parasite lysate stimulates a mixed Thl/Th2 cytokine profile. l_elF Downregulates the LPS-induced IL-IO Production by Patient PBMC. Taken together, the above results demonstrate that rLelF elicits a predominant Thl cytokine profile from the PBMC ofML, CL, or DCL patient while downregulating the expression of IL-10 mRNA. To further demonstrate After hybridization with the respective cytokine probes, the autoradiographs were exposed either at room temperature (11.,2 and IFN-"/) for 30 min to 1 h or at -70~ for 6 h to overnight (11. -10 and 1I-,4).  directly the effect of LelF in downregulating IL-10 production, PBMC from three ML patients were cultured in the presence of LPS with or without I.eiF. LPS stimulated PBMC from all three individuals tested to secrete IL-10 with values ranging from 185 to 424 pg/ml (Fig. 5). However, in the presence of LelF, the production of LPS-induced IL-10 secretion was reduced by 49-65% (83-166 pg/ml). Because IFN-y can inhibit IL-10 production by activated macrophages (22) and I.eiF stimulated patient PBMC to secrete IFN-"/, the secreted IFN-'y may be responsible for partially inhibiting the production of LPS-induced IL-10, LelF Stimulates both Patient and Normal PBMC to Secrete IL-12. IL-12 has been shown to play a pivotal immunoregulatory role in the development of cell-mediated immunity, including the generation of Thl responses and IFN-3' production in intracellular bacterial or parasitic infections (23). IL-12 is a heterodimeric molecule comprised of p40 and p35 subunits which must be coexpressed for the production of biologically active IL-12 p70 (24). The p40 subunit is produced only by IL-12-producing cells and is induced in vitro and in vivo after bacterial and parasitic stimulation (13,25), whereas the p35 subunit is both ubiquitous and constitutively expressed. Therefore, cells producing IL-12 p70 also have a large excess (lO-100-fold) of biologically inactive free 1040 chains (13). Because LelF stimulated a dominant Thl cytokine profile and downregulated Th2 responses, we explored a possible role for IL-12 in PBMC responses to rLelF. Fig. 6 A shows that l_eiF stimulated IL-12 p40 production from patient PBMC with values consistently higher than those observed with parasite lysate, and that IL-10 abrogated the production of IL-12 p40 by patient PBMC after stimulation with either LelF or parasite lysate.
PBMC from uninfected individuals also produced IL-12 p40 when cultured with LelF ( Fig. 6 B), although no p40 was detected in response to parasite lysate. This may suggest a role for IFN-~/in the lysate-induced p40 observed in patient PBMC, which produced 5-100-fold more IFN-'y than normal PBMC after antigen stimulation (Fig. 4 A). To determine whether the IL-12 p40 production observed in Ag-stimulated PBMC cultures reflected biologically active cytokine, IL-12 p70 was also determined in these cultures (Fig. 6, C and D). In general, the p70 production pattern paralleled that of p40, demonstrating that biologically active IL-12 was produced in response to LelF in both patient and normal PBMC. after stimulation with LelF or parasite lysate may be mediated by IL-12. LelF stimulated higher IL-12 p40 in patient PBMC than in normal PBMC indicating that the production of IL-12 may be upregulated by IFN-% To determine the interaction between IL-12, IFN-y, and IL-10 in response to LelF, PBMC from ML patients were stimulated in vitro with LelF in the absence or presence of anti-IL-12, anti-IFN-y mAb, or IL-10, and the cultured supernatants were assayed for IFN-q, and II.-12 p40 secretion. The addition of anti-IFN-3, mAb reduced the I.eIF-induced production of IL-12 by 65 and 80% but abrogated the lysate-induced IL-12 (Fig. 7). Both anti-IL-12 mAb and IL-10 abrogated the production of LelF-induced IFN-7 ( Fig. 8 A) whereas anti-IL-12 mAb only partially decreased the production of IFN-q, after stimulation with leishmanial lysate (Fig. 8 B). Exogenous IL-IO abrogated IL-12 p40 production by patient PBMC in response to either LelF or lysate (Fig. 8). IL-10 has also been shown to inhibit IL-12 production by human PBMC (26) and by human myeloid leukemia cell lines (14). These results indi-cate that the production of IFN-3, by LelF-stimulated patient PBMC is IL-12 dependent, whereas the production of IL-12 is regulated by both IFN-q,-dependent and -independent pathways.

Discussion
We have cloned and characterized rLeIF, a Leishmania Ag, that elicits proliferation and a Thl-type cytokine profile from a leishmaniasis patient PBMC. Although resting and lysate stimulated PBMC from L. braziliensis-infected individuals have a mixture of Thl-and Th2-1ike cytokine profiles, rLelF shifted this pattern towards an exclusive Thl (IFN-3' and IL-2) cytokine profile. In addition, rLelF stimulated both patient and normal PBMC to produce IL-12 whereas lysate did not stimulate the production of IL-12 in uninfected individuals. Finally, both rLelF and parasite lysate stimulated PBMC from ML and self-healing CL patients to secrete TNF-ot. The observation that LelF stimulated T cells from all patients with self-healing CL to proliferate and produce IFN-'y suggests that PBMC responses to LelF may be associated with protective immunity. In contrast, we have identified other L. braziliensis Ag that stimulate patient PBMC to produce a mixed Thl/Th2 or a predominant Th2 cytokine profile or that stimulate PBMC from ML patients but not from self-healing individuals (Skeiky, Y.A.W., et al., manuscript in preparation). Thus, l.eiF appears to have unique immunological properties.
Cytokines appear to be essential mediators of immunity to Leishmania (27). IFN-3, and TNF-o~ may synergize to induce leishmanicidal activity in macrophages (28)(29)(30). In addition, TNF-ol and IL-2 can also synergize with IL-12 to stimulate IFN-y production by NK cells (31)(32)(33). Studies in mice have demonstrated that IL-12 (0.25-1/.tg/d) can induce IL-10 production (34,35). Another study using human monocytes has demonstrated that the addition of TNF-ot (5-10 rig) can also induce IL-10 production (36). In our study, TNF-c~ and IL-12 but not IL-IO were produced by patient PBMC stimulated with l.eiF. This may be due to the relatively lower level of either TNF-ot or IL-12 produced by pa-  II  I III  II III III   I  i  L  t  t  i  i  i  i   0  5  10  15  20  25  30  0  5  10  15  20  25 IFN-gamma (ng/ml) IFN-gamma (ng/ml) tient PBMC, as well as the ability of IFN-'y, produced in high amounts by patient PBMC, to inhibit the production of IL-10 (22). The use of patient PBMC, rather than purified cell populations, may be more relevant to the in vivo situation since the dominant cytokine pattern is dependent on the interplay of modulatory cytokines. The presence of a mixture of Thl and Th2 cytokine mRNAs in the resting PBMC of ML patients is similar to the cytokine profile reported in lesions of ML patients (37,38), although in our study, IL-IO mRNA was not detected in the resting PBMC from any of the ML patients characterized. However, IL-IO mRNA was detected after in vitro culture of ML patient PBMC in the absence of Ag. In contrast, resting PBMC from CL patients had, in addition to mRNA for IL-4 and IFN-% detectable levels of IL-10 mRNA which remained generally unaffected after culture in the absence of Ag. Therefore, the levels of cytokine mRNA in unstimulated controls may reflect an activated state from in vivo exposure to parasite Ag. In this regard, rLelF downregulated the resting/constitutive levels of IL-IO in patient PBMC's as well as in LPS-stimulated PBMC. It therefore appears that certain Leishmania Ag may be able to elicit a dominant Thl cytokine profile as well as inhibit the production of Th2 cytokines (IL-10) by mechanisms that are not fully understood.
Because IL-12 plays a central role in the initiation and maintenance of Thl responses in humans and mice (23,(39)(40)(41)(42)(43) and is a potent inducer of IFN-3, (33) which can inhibit the production of IL-10 (22), we reasoned that rLelF may be mediating a dominant Thl cytokine profile by an IL-12-dependent mechanism. Manetti et al. (41) showed that patients with allergies who have mixed Thl/Th2 cytokine patterns could be shifted towards an experimentally induced Thl phenotype by the addition of IL-12. A significant finding of the present study was the demonstration that rLelF stimulated patient PBMC to produce IL-12 and that the production of IFN-3/was, at least in part, IL-12 dependent. Other studies have demonstrated a role for II,-12 in enhancing IFN-'y production in both mouse and human cells (44,45). Recent studies from several groups have led to the conclusion that the dominant factor of Thl cell development in response to infection may be determined by the ability of the pathogen to stimulate IL-12 production by macrophages. In this regard, several pathogens including L. monocytogenes, Toxoplasma gondii, Mycobacterium tuberculosis, Staphylococcus aureus, and L. major, that induce a Thl response correlate with their capacity to stimulate the production of IL-12 (13,14,24,32,43,44,46,47). However, the components or factors responsible for stimulating IL-12 production remain to be identified.
Our results have demonstrated that a single component of Leishmania is capable of stimulating the production of IL-12. This study represents the first reported identification of a recombinant parasite protein that stimulates the production of IL-12 in both patient and normal PBMC. Therefore, our finding that LelF can stimulate IL-12 production as well as the elicitation of an exclusive Thl cytokine profile in patient PBMC indicates that LelF could confer protective immunity against Leishmania infections and/or act as an adjuvant. In human peripheral blood, monocyte/macrophages are the major source of IL-12 although B cells and other minor populations of HLA-DR + accessory cells also produce IL-12 (13). Our preliminary results indicate the monocytes/macrophages population of patient PBMC as the dominant IL-12producing cell types after stimulation with LelF. In addition, I.eiF stimulated purified monocytes from uninfected individuals to produce IL-12 but not IL-10, thus suggesting the direct ability of 1.elF to function as an adjuvant (Skeiky Y. A. W., M. Kubin, G. Trinchieri, and S. G. Reed, manuscript in preparation). The mechanisms by which l.eiF induce IL-12 production are currently being investigated.
In summary, we have identified a Leishmania Ag that stimulates IL-12 and elicits a dominant Thl-type cytokine profile in human PBMC. An ideal prophylactic and therapeutic vaccine for leishmanial infection would be one that induces the expansion of Ag-specific Thl cells. These cells would produce IFN-y and IL-12 which would promote parasite destruction within the macrophage and downregulate Th2 responses. Because Leishmania I.eiF induced a powerful Thl-type response, including the two cytokines most clearly associated with protection in experimental leishmaniasis, IFN-'y and IL-12, LelF is an antigen that merits further study.
We thank Dr. Dalmo Correia Filho for identifying the self-healing CL patients, Cari Brownell for help with PCR analysis, Jacqueline A. Whittle for performing cytokine ELISA, Lettie Goltry for DNA sequencing, and Karen Kinch for assistance with manuscript preparation.