INTRODUCTION

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Interleukin-12 (IL-12) stimulates both NK and T cells and is particularly potent in its ability to induce gamma interferon (IFN-) production (reviewed in references 6, 40, 41, and 45). These biological activities led to the suggestion that IL-12 may play a critical role in the development and determination of effector cell functions. Indeed, IL-12 induces Th1-cell differentiation while inhibiting the development of Th2 cells (16, 19). A variety of models have used recombinant murine IL-12 (rmIL-12) as an adjuvant in prophylactic vaccination protocols (2, 20, 22, 25, 29, 46, 47). Vaccine-induced immunity to Schistosoma mansoni is enhanced by rmIL-12, although only partial protection against challenge infection was achieved (47). Striking results were obtained when an rmIL-12-based vaccine strategy prevented Th2-mediated pathologic changes upon challenge with S. mansoni larvae (46). Other researchers have reported that rmIL-12 promoted Th1 development and, ultimately, protection against leishmaniasis in BALB/c mice vaccinated with leishmanial antigens in combination with rmIL-12 (2, 13). A recent study has demonstrated that rmIL-12 conferred protection against Listeria monocytogenes when delivered with an otherwise nonimmunogenic peptide (22). The effects of rmIL-12 administration have also been studied in combination with immunogens such as keyhole limpet hemocyanin (KLH), hen egg white lysozyme, phospholipase A₂, and alloantigen (4, 7, 10-12, 21). These models have provided evidence that rmIL-12 can induce strong Th1-cell-type responses to soluble protein antigens.

Studies with several different infectious disease models in mice and humans have shown that an existing Th2 response influences the character of the response to challenge with novel antigens. This phenomenon may have an important impact on the use of IL-12 as a vaccine adjuvant in individuals who have an infection in which a Th2 response dominates. It has been demonstrated that individuals infected with S. mansoni produce higher levels of Th2 cytokines in response to mitogen or parasite antigen stimulation (3, 44). Thus, along with parasite antigens, these individuals have a propensity to make strong Th2 responses to other stimuli. Another report showed specifically that S. mansoni-infected persons developed a predominant Th2 response to tetanus toxoid whereas uninfected individuals mounted a Th1 or Th0 response (31). Mouse models of helminthic infection, with S. mansoni or Brugia malayi, have also been used to evaluate the immunological consequence of immunization in the presence of an ongoing Th2 response (1, 27, 28). These models showed that the Th1 response, normally induced to particular immunogens, was diminished in the presence of these helminthic infections. We hypothesized that rmIL-12, a potent inducer of Th1-associated responses, could still enhance Th1 responses to an immunogen during a chronic Th2-associated infection. To test the ability of rmIL-12 to promote a Th1 response in the presence of an ongoing systemic Th2 response, we used two well-defined model systems.

We used a hapten-protein conjugate model system, in which we previously showed that exogenous rmIL-12 administration can promote primary Th1-associated responses in mice primed with 2,4,6-trinitrophenyl (TNP)-KLH (4, 21). The advantage of using a hapten-carrier system is that T cells react to the carrier determinants (KLH) and B cells respond to the hapten (TNP). Therefore, these responses can be examined independently. We also chose a murine model of leishmaniasis to establish a chronic Th2-associated response. Using this infection model, we established a strong Th2 response in mice and then primed them with TNP-KLH alone or in combination with exogenous rmIL-12 to examine the efficacy of rmIL-12 in promoting a Th1 response to TNP-KLH in the presence of the ongoing Th2 response. The parasite-specific responses were also evaluated to confirm the Th2 dominant response and to determine whether this response was altered in conjunction with TNP-KLH and/or rmIL-12 delivery.

	MATERIALS AND METHODS

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Mice. Female BALB/c mice, 8 to 12 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, Maine) and housed and cared for under American Association for the Accreditation of Laboratory Animal Care-approved conditions.

Leishmania spp. Leishmania major, National Institutes of Health Seidman strain (WHOM/SN/74 Seidman), was used to establish experimental infections (24). Amastigotes were propagated in mice by serial infection as described previously (26). Amastigotes were harvested from infected footpad tissue and cultured in complete medium, consisting of RPMI 1640 supplemented with 10% fetal calf serum (FCS) that had been heat inactivated for 30 min at 57°C (HyClone Sterile Systems, Inc., Logan, Utah), 50 µg of gentamicin (Sigma, St. Louis, Mo.) per ml, 10 mM HEPES buffer (Sigma), and 2 mM L-glutamine (GIBCO, Long Island, N.Y.), in 75-ml flasks containing rabbit blood agar (18). Mice were infected subcutaneously in the right hind footpad with 2 × 10⁵ stationary-phase promastigotes (32). Soluble leishmanial antigen (SLA) preparations were generated by freeze-thawing culture-derived promastigotes, resuspended in phosphate-buffered saline (PBS; pH 7.0), four times. Then the preparation was sonicated for 1 h, sterile-filtered, aliquoted, and stored at 20°C (34). A protein assay (Bio-Rad Laboratories, Inc., Hercules, Calif.) based on the Bradford method was performed to determine the concentration of the preparation.

Reagents and immunizations. rmIL-12 (lot MRB02894-1), produced at Genetics Institute, Inc., was diluted in sterile physiologic saline (0.9% NaCl) at 5 µg/ml. Mice were given rmIL-12 intraperitoneally in a 0.2-ml volume on days 25, 26, and 27 for a total of three consecutive doses at 1 µg/mouse/day. KLH was conjugated with TNP as previously described (8); both reagents were purchased from Calbiochem (La Jolla, Calif.). The mice were primed subcutaneously in the left hind footpad with 100 µg of TNP-KLH in PBS on day 26 postinfection.

Cell cultures. Single-cell suspensions were prepared from lymph nodes (popliteal, inguinal, brachial, and mesenteric) by routine methods (9, 35, 42, 43). Cell populations were plated at 2.2 × 10⁶ cells per well in 24-well plates (Costar, Cambridge, Mass.) and cultured in the presence of medium alone, concanavalin A (Sigma) at a final concentration of 2.5 µg/ml, KLH at a final concentration of 50 µg/ml, or live promastigotes at 4.4 × 10⁵ per well. All cell cultures were incubated at 37°C in an atmosphere of 5% CO₂ in air. Cell-free supernatant fluids were harvested from these cell cocultures at 72 h, and cytokine concentrations were determined by cytokine-specific enzyme-linked immunosorbent assays (ELISAs) or enzyme-linked immunospot (Elispot) assays.

Cytokine assays. Cytokine levels in culture supernatant fluids were assessed by ELISAs. Commercially available kits were used to assay for IL-4 (R&D Systems, Minneapolis, Minn.). Reagents for the IFN- ELISAs were capture antibody R46A2 (HB170; American Type Culture Collection, Rockville, Md.) used at 3 µg/ml and the biotinylated detector antibody XMG 1.2 (HB10648; American Type Culture Collection) used at 1 µg/ml. Costar plates (enzyme immunoassay high-binding/flat bottom plates) were used; washes were done with Tris high salt-0.05% Tween 20, and blocking was done with Tris high salt gelatin (50 mM Tris, 0.5 M NaCl, 0.1 mM glycine, 5% gelatin). After the detector step, avidin D-HRP (Vector Laboratories) was added for 1 h at 37°C. The plates were developed with 2,2'-azino-di[3-ethylbenzthiazoline sulfonate (6)] (ABTS) (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) for 9 min at room temperature in the dark after a final wash, and the reactions were then stopped with 1% sodium dodecyl sulfate and read at an optical density of 405 nm (OD₄₀₅) (4).

Antibody isotype ELISAs. Sera from BALB/c mice were evaluated in TNP- and SLA-specific antibody isotype ELISAs. Enzyme immunoassay high binding/flat bottom or Immulon-4 plates were coated with purified TNP-BSA at 50 µg/ml or SLA at 4 µg/ml, respectively (4). The plates were then washed four times with Tris high salt-0.05% Tween 20 or PBS-0.05% Tween 20, blocked with Tris high salt gelatin or PBS-2% bovine serum albumin (Sigma) at 37°C, and washed again. Serum samples were diluted 1/100 (TNP ELISA) or 1/5 (SLA ELISA) and then serially diluted log₄. The ELISA plates were then incubated with horseradish peroxidase (HRP)-conjugated rat anti-mouse immunoglobulin G1 (IgG1) or IgG2a (Southern Biotechnology, Birmingham, Ala.) for the TNP ELISAs or HRP-conjugated rabbit anti-mouse IgG1 or IgG2a (Zymed, San Francisco, Calif.) for the SLA ELISAs. The plates were again washed four times, the substrate for the HRP conjugates, ABTS or o-phenylenediamine dihydrochloride (Sigma), was added, and the assays was developed for 10 min at 25°C, protected from light, after a final wash. The reactions were then stopped with 1% sodium dodecyl sulfate in the TNP-specific ELISA and read at OD₄₀₅. The SLA-specific ELISAs were stopped with 1 M HCl, and the reactions were read at OD₄₉₀ with an automated plate reader (Molecular Devices, Menlo Park, Calif.).

	RESULTS

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Establishment of a progressive infection. Groups of BALB/c mice were infected with L. major to establish a dominant Th2 response (5, 14, 36). Chronic infection was evident on day 21 by the presence of parasites in lymph node cells cultured from a subset of animals (data not shown). Also, infected lymph node cell cultures had a 3.5-fold-larger number of IL-4-secreting cells in response to mitogen stimulation than did naive cell cultures and there was no evidence of a parasite-specific IFN- response (cutoff value of 1 ng/ml). On day 26 postinfection, mice were immunized with TNP-KLH in the presence or absence of exogenous rmIL-12 (Fig. 1). We examined the lymph node cell responses on day 30 by using five animals from each group. The lymph node cells were selected because the draining lymph nodes contain the cell population which is most actively involved in resolving disease at the site of infection, the footpad. The remaining animals from each experimental group were bled on day 39 for antibody isotype analysis, 14 days after the last injection of TNP-KLH. Additionally, serum samples were harvested on day 1, to serve as a naive control, and day 20, to serve as a postinfection, pre-priming with TNP-KLH control. We wanted to provide evidence of class-switching events directed by the cytokine response to the circulating antigens. Therefore, we determined the cytokine production potential before the isotype analysis.

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Experimental protocol. The six experimental groups of BALB/c mice (15 to 18 mice per group) were L. major infected, and primed with TNP-KLH, and given IL-12 (group 1), infected and TNP-KLH primed (group 2), infected and treated with PBS (group 3), TNP-KLH primed and given IL-12 (group 4), TNP-KLH primed only (group 5), and PBS treated only (group 6). Infected groups received 2 × 105 stationary-phase promastigotes (WHOM/SN74 Seidman strain) subcutaneously in the right hind footpad (day 0). IL-12 was administered at 1 µg/mouse/day (days 25, 26, and 27). TNP-KLH (100 µg) was given subcutaneously in the left hind footpad (day 26). On day 21, lymph node cells were harvested from naive and infected test groups (two mice per group) and cultured for promastigote growth to ensure progressive infection. IL-4 Elispots and IFN- ELISAs were also performed. On days 1, 20, and 39, sera was collected from all experimental groups for antibody isotype ELISAs. On days 30 and 33, lymph node cells were harvested from all experimental groups (five mice per group) and stimulated for 72 h with medium, KLH, promastigotes and concanavalin A. Culture supernatant fluids were assayed for IFN- and IL-4 production. Details of all assays are given in Materials and Methods. Numbers in the figure indicate days.

KLH-specific cytokine production. Lymph nodes cells derived from all experimental groups on day 30 were cultured in the presence of medium alone, concanavalin A, KLH, or L. major promastigotes. Supernatant fluids from these cultures were assayed for IFN- production by ELISAs. The response to antigen is shown in Fig. 2A. Control uninfected mice primed with TNP-KLH in the presence of rmIL-12 produced IFN- in response to KLH stimulation in vitro (22.0 ± 0.8 ng/ml; mean ± standard deviation [SD] of triplicate determinations), whereas in the absence of rmIL-12 no IFN- was detected (cutoff value of 1 ng/ml) (4). Infected mice primed with TNP-KLH in the presence of rmIL-12 were also capable of secreting IFN- in response to KLH stimulation (20.8 ± 0.7 ng/ml). In addition, no parasite-specific IFN- was detectable in any culture supernatant fluids.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Cytokine responses from lymph node cell cultures. Cells were derived from all experimental groups (five mice per group) on day 30, and the levels of cytokine in the 72-h culture supernatant fluids were determined by ELISAs (see Materials and Methods for details). (A) IFN-; (B) IL-4. Each bar represents the mean and SD of the values obtained from triplicate cultures. Medium alone (open bars), promastigote-stimulated cultures (hatched bars), and the KLH-stimulated cultures (solid bars) are shown. The limit of detection for the IFN- ELISA was 1 ng/ml, and that for the IL-4 ELISA was 20 pg/ml.

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Parasite-specific IL-4 response from lymph node cell cultures. Cell populations were collected from mice (n = 5/group) on day 30. The number of IL-4-secreting cells per 5 × 105 lymph node cells was determined in an Elispot assay after 24 h of stimulation in the presence of medium alone or live promastigotes. Each bar represents the mean and SD of the values obtained from triplicate cell cultures.

Antibody response. To determine if the chronic Th2-associated disease state affected the B-cell response, subsets of mice from all the experimental groups were bled on days 1, 20, and 39 and the TNP-specific IgG2a and IgG1 antibody isotype titers were determined (Fig. 4). As we have shown previously, BALB/c mice primed with TNP-KLH in the presence of rmIL-12 develop substantially greater titers of TNP-specific IgG2a than do mice primed with TNP-KLH in the absence of rmIL-12 (4). Mice with a chronic Th2 parasitic infection also generated elevated titers of TNP-specific IgG2a when rmIL-12 was administered at the time of priming. The TNP-specific IgG1 titers remained fairly constant in all the groups primed with TNP-KLH (4). Thus, neither infection nor rmIL-12 administration affected the IgG1 response to the hapten in these experiments. Moreover, the Th2 cytokine milieu established in the lymphoid organs in response to the leishmanial infection did not suppress the development of an IgG2a response.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   TNP-specific IgG2a and IgG1 titers in serum. TNP-specific antibody isotypes were assessed from serum samples by ELISAs. Plates were coated with 50 µg of purified TNP-bovine serum albumin, and the titers given are half-maximal values of log4 serial dilutions performed in duplicate for each serum sample on individual mice (n = 5). Results are representative of two independent experiments.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   SLA-specific IgG2a and IgG1 titers in serum. Plates were coated with 4 µg of SLA per ml, and a dilution series of pooled serum samples (n = 5) was incubated in duplicate for 2 h. Next, HRP-conjugated anti-mouse IgG1 or IgG2a was added, and the mixture was incubated for 1 h. Then the substrate (o-phenylenediamine hydrochloride) was added for 10 min, followed by 1 M HCl to stop the reactions. The plates were read at 490 nm. The results are expressed as log cut point titers.

	DISCUSSION

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The studies here have addressed the question whether a Th2 disease state predisposes the immune response to a novel antigen. We also addressed whether rmIL-12, a strong potentiator of a Th1 response, could induce a Th1-type antibody and cellular response to stimulation with a novel antigen in the presence of an ongoing Th2 response. A Th2 disease state was established by infecting BALB/c mice with L. major. The mice were subsequently primed with TNP-KLH on day 26 in the presence or absence of rmIL-12 administration on days 25, 26, and 27.

On day 30, parasite-specific IL-4-secreting cells were detected in lymph node cell Elispot cultures derived from all the infected experimental groups (Fig. 3). These results confirmed that leishmanial infection in the BALB/c mouse strain promotes a Th2 cytokine response. In addition, priming with the immunogen TNP-KLH did not alter parasite-specific IL-4 secretion. Consistent with our previous findings regarding exogenous rmIL-12 given late in the course of leishmanial infection (37), the numbers of parasite-specific IL-4 secretors in the lymph node cell cultures were not suppressed. These results are consistent with the idea that the parasite-specific cells from animals with progressive disease are committed to a Th2-cell phenotype and are no longer responsive to IL-12 due to IL-12 receptor ₂-chain down-regulation (15, 17, 38, 39).

The results presented in Fig. 2A show that KLH-specific IFN- can be induced in vitro from the lymph node cells derived from mice on day 30 postinfection. No KLH-specific IFN- was detected in any cell cultures derived from mice that did not receive rmIL-12 during TNP-KLH immunization. Furthermore, no parasite-specific IFN- was detected. Taken together, these results suggest that rmIL-12 can generate a Th1 cytokine response to a heterologous immunogen in the presence of an ongoing Th2 parasitic infection without provoking a parasite-specific IFN- response. Consistent with these results, a corresponding elevated Th1-mediated antibody isotype response to TNP-KLH was detected (Fig. 4). Sera from both infected and uninfected mice primed with TNP-KLH in the presence of rmIL-12 had a higher titer of TNP-specific IgG2a than did mice that did not receive exogenous rmIL-12. rmIL-12-induced production of KLH-specific IFN- is probably signaling this antibody class-switching event (4, 7, 23), which can still occur despite the presence of an existing Th2 disease state. In contrast, rmIL-12 administration with TNP-KLH did not induce parasite-specific IFN production in BALB/c mice or enhance parasite-specific IgG2a class switching.

KLH-specific IL-4 production was detected in the lymph node cell cultures on day 30 (Fig. 2B). More specifically, both infected and uninfected mice primed with TNP-KLH in the absence of rmIL-12 were able to secrete IL-4 in response to KLH stimulation. On the other hand, no IL-4 was detectable (cutoff value, 20 pg/ml) in the cell cultures derived from mice receiving rmIL-12 at the time of TNP-KLH priming. These results suggest that exogenous rmIL-12 was capable of suppressing KLH-specific IL-4 production during an ongoing systemic parasitic infection. Curiously, no parasite-specific IL-4 was detectable in any of the culture supernatant fluids derived from the infected groups. However, we were able to detect parasite-specific IL-4-secreting cells in an Elispot assay. One explanation is that IL-4 may have been rapidly consumed by Th2 cells and therefore was undetectable in the culture supernatant fluids whereas in the Elispot assay the IL-4 was captured before being taken up by surrounding cells.

Despite the absence of a demonstrable KLH-specific IL-4 response in mice primed with TNP-KLH in the presence of rmIL-12, TNP-specific IgG1 was detectable (Fig. 4). In fact, the IgG1 titers present were similar to that observed in mice primed with TNP-KLH in the absence of rmIL-12 that had a demonstrable IL-4 in vitro response. One possible explanation is that IgG1 production was occurring by an IL-4-independent mechanism. A more likely explanation is that enough immunogen-specific IL-4 was being made in the rmIL-12-treated groups to allow IgG1 class switching but that it was below the limit of detection in our ELISA. For example, although we were unable to detect parasite-specific IL-4 in the culture supernatant fluids by ELISA, we were able to detect parasite-specific IL-4-secreting cells in an Elispot assay. Previous work with the Leishmania model prompted us to perform the Elispot assay to detect parasite-specific IL-4; however, we did not include KLH stimulation in this assay. Nevertheless, we can conclude from our ELISA results that rmIL-12 administration did down-regulate the KLH-specific IL-4 response in vitro (Fig. 2B). However, the IL-4 response may not have been completely suppressed in vivo, given the TNP-specific IgG1 response (Fig. 4).

Overall, our findings with the TNP-KLH immunization model during an ongoing leishmanial infection are in agreement with work done by Sadick et al., who reported that mice with an established Th2 response to a Nippostrongylus brasiliensis infection were still able to elicit a protective Th1 response to a subsequent L. major infection (33). However, in those studies, exogenous rmIL-12 was not required to promote a Th1 response to leishmanial infection. One explanation for this apparent difference in the requirement for exogenous rmIL-12 may be related to the genetic background of the mouse strain used. The studies by Sadick et al. were done with C57BL/6 mice, which are normally resistant to L. major and generate a Th1-cell response, while the present studies were done with BALB/c mice, which typically require exogenous rmIL-12 to promote a Th1 response to both parasite antigen and other immunogens such as TNP-KLH (4, 37). In support of this hypothesis, studies by Rousseau et al. have recently shown that BALB/c mice with an ongoing Th2 response to a helminth infection generated a Th response of a mixed phenotype to a subsequent leishmanial infection in the absence of exogenous rmIL-12 (30). Thus, it appears that BALB/c mice may be more likely to produce IL-4 upon subsequent challenge. Nevertheless, our findings clearly demonstrate that in a dominant Th2 setting, rmIL-12 administration was able to skew the Th response to that of a Th1-cell-type response. This result is perhaps even more compelling since the mouse strain used typically generates a strong Th2 response to both immunogens studied.

In summary, our results show that rmIL-12 administration on days 25 through 27 after leishmanial infection did not alter the parasite-specific responses. These results were predicted based on our previous findings demonstrating that exogenous rmIL-12 given past the first week of infection will not prevent disease dissemination in BALB/c mice (37). More importantly, our results suggest that rmIL-12 is capable of promoting a Th1-cell response to an immunogen even in the presence of an ongoing Th2-promoting infection.