IL-12 Is Required to Maintain a Th1 Response During Leishmania major Infection

Audrey Y. Park, Brian D. Hondowicz and Phillip Scott
J Immunol July 15, 2000, 165 (2) 896-902; DOI: https://doi.org/10.4049/jimmunol.165.2.896

Abstract

IL-12 initiates Th1 cell development and cell-mediated immunity, but whether IL-12 contributes to the maintenance of a Th1 response is unclear. To address this question, we infected IL-12 p40−/− C57BL/6 mice with Leishmania major, an intracellular protozoan parasite controlled by a cell-mediated immune response, and simultaneously administered IL-12. Whereas untreated p40−/− mice developed an uncontrolled infection, p40−/− mice treated with IL-12 for the first 2 or 4 wk of infection developed a Th1 response and resolved their lesions. However, the induction of this protective Th1 cell response by IL-12 treatment was not associated with long term immunity. We observed that on rechallenge in the absence of IL-12, the mice exhibited a susceptible phenotype. In addition, without rechallenge, lesions in the IL-12-treated p40−/− mice developed several weeks after cessation of IL-12 treatment. In both cases, disease was associated with the loss of a Th1 response and the development of a Th2 response. Our observations are not limited to the C57BL/6 strain, because IL-12 treatment was also unable to provide lasting protection to p40−/− BALB/c mice. Finally, we found that although Th1 cells from healed wild-type C57BL/6 mice adoptively transferred protection to L. major-infected RAG−/− mice, they were unable to protect p40−/− mice. In conclusion, these studies provide the first demonstration that IL-12 is required not only to initiate Th1 cell development but also throughout infection to maintain a Th1 cell response and resistance to L. major.

Interleukin 12 is a heterodimeric cytokine formed by two chains, p35 and p40 (1, 2, 3, 4). It is mainly produced by activated APC, including macrophages and dendritic cells, and is central in the development of both the early innate and subsequent adaptive immune responses (5). IL-12 induces cytokine production, in particular IFN-γ, from T and NK cells (1, 6), as well as enhancing the cytotoxic activity of these cells (1, 2). However, its most critical role may be to initiate the development of cell-mediated immunity by promoting the differentiation of Th1 cells from naive T cells (7, 8, 9, 10, 11). On the other hand, once a Th1 response is established, the role of IL-12 has been less clear.

Infection of mice with the intracellular protozoan parasite, Leishmania major, has provided a valuable model for the study of Th cell responses, because resistant strains of mice develop a Th1 response and other strains develop nonhealing lesions associated with a Th2 response (12, 13). As predicted, IL-12 is required in the initiation of Th1 cell development in leishmaniasis. In the absence of IL-12, normally resistant strains of mice infected with L. major fail to develop a Th1 response and consequently are unable to control their infections (14, 15, 16, 17). In contrast, susceptible BALB/c mice treated with IL-12 during the first 2 wk of infection (18, 19) or vaccinated with IL-12 and soluble leishmanial Ag (SLA)4 acquire a Th1 phenotype and exhibit resistance to L. major (20).

Once a Th1 response is initiated during an infection, it is believed that IL-12 is not required to sustain the response. For example, administration of anti-IL-12 Abs to mice that had healed from a primary L. major infection did not alter the resistance of these mice on reinfection (21). Similarly, in other infectious diseases, such as Toxoplasma gondii (22) and Listeria monocytogenes (23), maintenance of a protective Th1 response appeared to be IL-12 independent. A major limitation in the interpretation of these experiments is that IL-12 may not have been completely blocked by the neutralizing mAb, and even a small amount of IL-12 could be enough to sustain a Th1 response. Therefore, to conclusively address this issue, L. major-infected IL-12 p40−/− mice (referred to as p40−/− mice in this article) were transiently treated with IL-12 and monitored for maintenance of resistance after cessation of IL-12 treatment. Surprisingly, we observed that despite the development of a Th1 response in p40−/− mice treated with IL-12, the animals were unable to sustain a Th1 response in the absence of IL-12. Thus, in contrast to previous findings, our results indicate that IL-12 is important for both initiation and maintenance of cell-mediated immunity to L. major infection.

Materials and Methods

Animals

Female C57BL/6, BALB/cByJ, and RAG 1−/− C57BL/6 mice, 4–6 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME). IL-12 p40−/− C57BL/6 mice used in the initial studies were generously provided by Dr. Jeanne Magram (Hoffmann-LaRoche, Nutley, NJ). All subsequent studies as shown in this paper used IL-12 p40−/− mice on the C57BL/6 and BALB/cByJ background, which were purchased from The Jackson Laboratory. Animals were maintained in a specific pathogen-free environment and tested negative to the presence of murine pathogens.

Parasites and Ag

L. major parasites (MHOM/IL/80/Friedlin) were grown in Grace’s insect culture medium (Life Technologies, Gaithersburg, MD) with 20% heat-inactivated FBS (HyClone, Logan, UT), 2 mM glutamine, 100 U/ml penicillin 6-potassium, and 100 μg/ml streptomycin sulfate. Metacyclic promastigote parasites were isolated by negative selection with Arachis hypogae agglutinin as previously described (24). Mice were injected in the hind footpad with 2 × 106 metacyclic parasites. In certain instances, 8 wk after primary infection, mice were inoculated in the contralateral hind footpad with 2 million metacyclic promastigotes. SLA was prepared as previously described (25). To quantitate the number of parasites in the footpad, single-cell suspensions of the lesions were plated in 10-fold serial dilutions in Grace’s insect culture medium starting with a 1:100 dilution. Each sample was plated in triplicate, and the mean of the negative log parasite titer was calculated after 5 days.

In vivo IL-12 treatment

Mice were treated intralesionally with 0.2 μg IL-12 per footpad (Genetics Institute, Cambridge, MA) three times a wk during the first 2 or 4 wk of infection. The course of infection for IL-12-treated and untreated mice was monitored by measuring the footpad sizes with a dial caliper (Starrett, Athol, MA). The lesion size was determined by subtracting the size of the normal hind footpad from the size of the infected hind footpad. In secondary infections, the lesion size was determined by subtracting the size of the hind footpad before challenge from the size of the challenged hind footpad.

In vitro recall responses

Popliteal lymph nodes were harvested, and single-cell suspensions were prepared. Cells were resuspended at 4 × 106/ml in DMEM, 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin 6-potassium, 100 mg/ml streptomycin sulfate, 25 mM HEPES, and 5 × 10−5 M 2-ME and plated at 200 μl/well in 96-well tissue culture plates. Cells were stimulated with or without 50 μg/ml SLA Ag. In addition, 5 μg/ml anti-IL-4R mAb (M1; a generous gift from Dr. Fred Finkleman, University of Cincinnati, Cincinnati, OH) was added to block consumption of IL-4. After 3 days, supernatants were collected and assayed for IFN-γ and IL-4 production by ELISA as previously described (26).

Intracellular cytokine staining

Pooled lymphocytes from the popliteal lymph nodes were resuspended at 5 × 106/ml and plated at 1 ml in 24-well tissue cultures plates with or without 50 μg/ml SLA. Intracellular cytokine staining was performed as previously described with minor modifications (27). Briefly, after 3 days, the cells were stimulated with 50 ng/ml PMA, 500 ng/ml ionomycin, and 10 μg/ml brefeldin A for 5 h. The cells were washed and incubated with 10 μg anti-FCγIII/IIR Ab (2.4G2) and 10 μg rat IgG (Sigma, St. Louis, MO) to block nonspecific binding of Abs and then stained with anti-CD4- or CD8-FITC (PharMingen, San Diego, CA) and fixed overnight with 1% paraformaldehyde. Cells were then permeabilized with 0.1% saponin in FACS buffer (PBS/BSA/Na3) and stained with anti-IFN-γ APC and anti-IL-4 PE mAbs or the appropriate isotype controls (PharMingen). Cells were washed and run on a FACSCalibur cytometer (Becton Dickinson, San Jose, CA). Analysis was performed with CellQuest (Becton Dickinson) software.

Adoptive transfer

Draining lymph nodes from C57BL/6 mice that had healed from a L. major infection (6–8 wk postinfection) were collected, and single-cell suspensions were prepared. Cells were incubated with 5 μg/ml anti-CD8 (H35) for 30 min on ice and washed, and rabbit complement (Pel-Freez, Brown Deer, WI) was added at a 1:10 dilution for 1 h at 37°C. The remaining cells were then panned with anti-Ig Abs (Jackson Immunoresearch, West Grove, PA) for 30 min at 37°C to remove B cells. The CD4+-enriched cell population (80–85%) was adoptively transferred by i.v. injection (10 × 106/mouse) into RAG−/−, p40−/−, or wild-type C57BL/6 mice. One day later, the mice were injected with L. major.

Results

Administration of IL-12 to p40−/− C57BL/6 mice promotes resistance to L. major and a Th1 response

IL-12 p40−/− C57BL/6 and BALB/c mice were infected with L. major, and some mice were treated intralesionally with IL-12 six times during a 2-wk period. As expected, untreated p40−/− C57BL/6 and BALB/c mice developed progressive lesions, although the kinetics of lesion growth was slightly slower in the p40−/− C57BL/6 mice than in the BALB/c mice (Fig. 1⇓). In contrast, both the IL-12-treated BALB/c and p40−/− C57BL/6 mice controlled their lesions for at least the first 7 wk of infection (Fig. 1⇓). The ability to control lesion size correlated with lower parasite counts in the footpad (Table I⇓). At 2 wk postinfection, lesions from IL-12-treated p40−/− mice contained significantly lower parasite levels compared with those in untreated p40−/− mice (Table I⇓). We also observed that at 2 wk postinfection, popliteal lymph node cells from IL-12-treated p40−/− mice produced significantly higher levels of IFN-γ and no detectable IL-4, unlike cells from control p40−/− mice which exhibited a Th2 phenotype (Fig. 2⇓A). At this early time point, control C57BL/6 mice do not have a pronounced Th1 response in agreement with previous findings (28).

FIGURE 1.
FIGURE 1.

p40−/− C57BL/6 mice treated with IL-12 exhibit resistance to L. major infection. Mice were inoculated with 2 million metacyclic promastigotes into the left hind footpad. Some mice were treated intralesionally with IL-12 six times over the first 2 wk of infection. Results are expressed as the mean ± SD of four to five mice per group. Data are representative of four independent experiments.

FIGURE 2.
FIGURE 2.

Early after IL-12 treatment, p40−/− C57BL/6 mice develop a Th1 response. Two weeks after primary infection, popliteal lymph node cells were harvested and stimulated with SLA. A, After 3 days, supernatants were collected and assayed by ELISA for IFN-γ and IL-4. Results are expressed as the mean ± SD of five individual mice per group. B, After 3 days, cells were restimulated with PMA, ionomycin, and brefeldin A for 5 h. Cells were then stained for CD4, IFN-γ, and IL-4. Data are representative of four independent experiments.

View this table:
Table I.

Parasite quantitation from footpads of L. major-infected B6 p40−/−, IL-12-treated p40−/−, and B6 micea

Because we observed that the IL-12 treatment was capable of inducing a Th1 response in the p40−/− mice, we were interested in determining which cells were responsible for the IFN-γ production. To examine this, popliteal lymph nodes from 2-wk IL-12-treated p40−/−, control p40−/−, and C57BL/6 mice were harvested. Lymphocytes were stimulated with SLA for 3 days, and then PMA, ionomycin, and brefeldin A were added to the cultures for 5 h. Cells were then stained for either CD4 or CD8, as well as intracellular cytokine expression. Consistent with the ELISA data, IL-12-treated p40−/− mice displayed higher IFN-γ and reduced IL-4 levels than did the untreated p40−/− mice (Fig. 2⇑B). Interestingly, the percentage of CD4+ T cells in the IL-12-treated p40−/− mice that produced IFN-γ was similar to those in C57BL/6 mice (Fig. 2⇑B). However, only a small percentage (0.5%) of CD8+ T cells of both IL-12-treated p40−/− and C57BL/6 mice exhibited IFN-γ expression (data not shown). We also observed that the IL-12 treatment did not change the cellular composition of lymphocytes, which was tested by staining for CD4, CD8, B220, γδ TCR, and Mac-1 (data not shown). Thus, IL-12 treatment in the p40−/− mice promotes differentiation of CD4+ T cells to the Th1 phenotype. Together, these data indicate that IL-12 treatment was effective in providing resistance to L. major and initiating a Th1 response in p40−/− C57BL/6 mice.

Administration of IL-12 to p40−/− C57BL/6 mice fails to induce long term protection

To determine whether the protective immune response exhibited in IL-12-treated p40−/− mice would be maintained in the absence of IL-12, we challenged the IL-12-treated p40−/− mice 8 wk after the primary infection in the contralateral footpad without administering IL-12. Surprisingly, IL-12-treated p40−/− mice that had developed a Th1 response and controlled a primary L. major infection were unable to control a secondary infection without exogenous IL-12 administration (Fig. 3⇓). In addition, on challenge of these IL-12-treated p40−/− mice, the lesions at the primary site reemerged (data not shown). In contrast, the resistance induced by IL-12 treatment in BALB/c mice was maintained on secondary infection (Fig. 3⇓). Eleven weeks after the secondary infection, the increased lesion size in the IL-12-treated p40−/− mice correlated with a strongly polarized Th2 response, where little IFN-γ was detected (Fig. 4⇓). However, challenged IL-12-treated BALB/c mice produced high IFN-γ and low IL-4 levels, which were comparable with that of the healed C57BL/6 mice (Fig. 4⇓). These data demonstrate that although exogenous IL-12 treatment is sufficient in protecting BALB/c mice from a primary, as well as a secondary L. major infection, it is insufficient in providing long term protection to p40−/− mice.

FIGURE 3.
FIGURE 3.

p40−/− C57BL/6 mice treated with IL-12 for 2 wk fail to maintain resistance to secondary infection with L. major. Eight weeks after primary infection, the mice shown in Fig. 1⇑ were inoculated with 2 million metacyclic promastigotes into the contralateral footpad and were not further treated with IL-12. Results are expressed as the mean ± SD of four to five mice per group. Data are representative of four independent experiments.

FIGURE 4.
FIGURE 4.

On secondary infection, p40−/− C57BL/6 mice treated with IL-12 for 2 wk during a primary infection develop a Th2 response. Eleven weeks after secondary infection, popliteal lymph node cells were stimulated with SLA. Supernatants were collected after 3 days and assayed by ELISA for IL-4 and IFN-γ. Results are expressed as the mean ± SD of four to five individual mice per group. Data are representative of two independent experiments.

Longer administration of IL-12 is unable to induce long term protection in p40−/− C57BL/6 mice

To determine whether longer treatment with IL-12 could induce a protective response that could be maintained in the absence of IL-12, we administered IL-12 during the first 4 wk of infection. As before, IL-12-treated p40−/− mice were initially able to control the primary infection, unlike the untreated p40−/− group (Fig. 5⇓A). Nevertheless, when challenged at 8 wk, the p40−/− mice treated with IL-12 for the first 4 wk of infection were still susceptible to infection (Fig. 5⇓B), similar to the 2-wk IL-12-treated p40−/− mice (Fig. 3⇑). As expected, the increased lesion size was associated with a Th2 response in the 4-wk IL-12-treated p40−/− mice (data not shown). Thus, these results suggest that IL-12 needs to be present throughout the course of infection to maintain resistance to L. major.

FIGURE 5.
FIGURE 5.

Longer treatment with IL-12 is unable to protect p40−/− C57BL/6 mice from a secondary infection with L. major. A, Mice were inoculated with 2 million metacyclic promastigotes into the left hind footpad and treated intralesionally with IL-12 12 times during the first 4 wk of infection. B, Eight weeks after primary infection, mice in A were inoculated with 2 million metacyclic promastigotes in the contralateral footpad and were not further treated with IL-12. Results are expressed as the mean ± SD of four to five mice per group. Data are representative of three independent experiments.

We then wanted to determine whether secondary challenge was a requirement to reveal the inability of the IL-12 treatment to fully protect p40−/− mice, because it is known that resistance to L. major is not associated with sterile immunity in that low parasite levels persist in clinically resistant mice (29, 30, 31, 32). Therefore, we extended the course of primary infection of 2-wk IL-12-treated p40−/− mice. We observed that lesions spontaneously reappeared at the site of primary infection and steadily increased in size (Fig. 6⇓). Thus, taken together with the studies where mice were rechallenged at 8 wk of infection, these experiments suggest that exogenous IL-12 treatment given to p40−/− mice induces short term protection but fails to induce long term, IL-12-independent resistance.

FIGURE 6.
FIGURE 6.

IL-12 treatment cannot provide long term protection to p40−/− mice from a primary L. major infection. Mice were inoculated with 2 million metacyclic promastigotes and treated with IL-12 for 2 wk as in Fig. 1⇑. Results are expressed as the mean ± SD of three to five mice per group. Data are representative of two independent experiments. All p40−/− mice that were treated with IL-12 developed lesions between weeks 5 and 13 postinfection, although at variable rates. This variability in lesion development is reflected in the SD observed in the figure.

IL-12 treatment does not protect p40−/− BALB/c mice from L. major infection

Because IL-12 treatment can protect BALB/c mice, but not p40−/− C57BL/6 mice, we wanted to determine whether endogenous IL-12 could account for the resistance in BALB/c mice. To address this question, we treated p40−/− mice on the BALB/c background with IL-12 during the first 2 wk of L. major infection and monitored their course of infection. Similar to the p40−/− C57BL/6 mice, IL-12 treatment delayed lesion growth but ultimately did not protect p40−/− BALB/c mice (Fig. 7⇓). Because IL-12 treatment of wild-type BALB/c, but not p40−/− BALB/c mice, leads to long term protection, it appears that endogenous IL-12 is involved in perpetuating the Th1 response in IL-12-treated BALB/c mice. This fact also extends our results to the BALB/c genetic background, indicating that the requirement for IL-12 to maintain a Th1 response is not unique to C57BL/6 mice.

FIGURE 7.
FIGURE 7.

IL-12 treatment fails to protect p40−/− BALB/c mice from L. major infection. Mice were inoculated with 2 million metacyclic promastigotes and treated with IL-12 for 2 wk as in Fig. 1⇑. Results are expressed as the mean ± SD of three to five mice per group. Data are representative of two independent experiments.

Adoptive transfer of Th1 cells from healed C57BL/6 mice protects RAG−/− but not p40−/− mice

To further investigate the role of IL-12 in maintaining a Th1 response, we asked whether Th1 cells from L. major-infected C57BL/6 mice would be able to adoptively transfer protection to p40−/− mice. We transferred CD4+-enriched T cells from healed wild-type C57BL/6 mice into RAG−/−, p40−/−, and wild-type C57BL/6 mice. As expected, the CD4+-enriched T cells exhibited a Th1-type response to SLA in a recall assay, producing high levels of IFN-γ and little IL-4 (data not shown). C57BL/6 recipients of Th1 cells exhibited enhanced resistance to L. major, as indicated by a significant decrease in the number of parasites within their lesions compared with control C57BL/6 mice (Table II⇓). In addition, after adoptive transfer of the Th1 cell population, RAG−/− recipients were resistant to L. major infection, whereas control RAG−/− mice were susceptible to infection (Fig. 8⇓). As expected, protection from lesion development was associated with lower parasite counts. Adoptively transferred RAG−/− and control RAG−/− mice contained 103.4 and >109 parasites within their lesions, respectively (Table II⇓). However, when CD4+ T cells from healed wild-type mice were transferred to p40−/− recipients, they failed to induce any protection to L. major infection (Fig. 8⇓). Furthermore, the parasite levels in the footpads were very high in both p40−/− recipients of Th1 cells and control p40−/− mice (>109) (Table II⇓). Thus, adoptive transfer of Th1 cells enhanced parasite clearance in C57BL/6 recipients and protected RAG−/− mice but failed to protect p40−/− mice, further demonstrating that IL-12 is required for established Th1-committed cells to provide resistance to L. major.

FIGURE 8.
FIGURE 8.

Adoptive transfer of Th1 cells from healed C57BL/6 mice protects RAG 1−/− but not p40−/− mice. RAG 1−/−, p40−/−, and wild-type C57BL/6 mice received Th1 cells from healed C57BL/6 mice by i.v. injection. One day after transfer, mice were inoculated with 2 million metacyclic promastigotes. Results are expressed as the mean ± SD of three to five mice per group. Data are representative of two independent experiments.

View this table:
Table II.

Parasite quantitation from footpads of L. major-infected control and adoptively transferred p40−/−, RAG−/−, and B6 micea

Discussion

IL-12 is critical in initiating a Th1 response that is centrally involved in protection against many pathogens (18, 19, 20, 33, 34, 35), as well as exacerbation of certain autoimmune diseases (36, 37, 38). However, the role of IL-12 in maintaining a Th1 response has been unclear. To address this issue, we treated p40−/− mice with IL-12 during the early stages of L. major infection and then monitored them for maintenance of resistance after cessation of IL-12 treatment. Surprisingly, we found that once IL-12 treatment was terminated, p40−/− mice did not maintain resistance to L. major infection, despite mounting an early leishmanial-specific Th1 response and controlling parasite replication. However, the IL-12 treatment was sufficient to protect BALB/c mice from both a primary and a secondary infection, which we believe was due to the ability of BALB/c mice to produce endogenous IL-12. Because IL-12 treatment was unable to provide long-lasting protection to the p40−/− mice, one could argue that longer administration (>4 wk) may have been necessary to develop fully committed Th1 cells. However, even Th1 cells from healed resistant mice were incapable of controlling parasite growth and lesion progression in L. major-infected p40−/− recipients but could protect RAG−/− recipients. Thus, in the absence of IL-12, a leishmanial-specific Th1 immune response could not be maintained, which argues that IL-12 is important beyond the initiation of cell-mediated immunity.

Our findings differ from earlier reports which concluded that IL-12 is not required for continued protection in several infectious diseases but is essential in the maintenance of autoimmune diseases (39). We had previously shown that healed C3H mice reinfected with L. major maintained resistance after administration of anti-IL-12 Abs (21). Similarly, others observed that anti-IL-12-treated mice chronically infected with T. gondii or Listeria monocytogenes survive and produce IFN-γ in a manner comparable with that of untreated controls (22, 23). However, a major caveat in these studies is that the neutralization of IL-12 may have been incomplete and low amounts of IL-12 could be sufficient in maintaining Th1 immune responses. To overcome the technical limitation of these studies, we examined L. major-infected p40−/− mice after cessation of IL-12 treatment and clearly found the requirement for IL-12 in the maintenance of Th1 responses. Thus, the necessity of IL-12 for the maintenance of a leishmanial-specific Th1 response is similar to that seen in murine autoimmune models. For example, administration of blocking Abs to IL-12 given to mice with experimental autoimmune encephalomyelitis decreased disease severity (36, 37). Furthermore, in a model of autoimmune uveitis, wild-type cells could transfer disease to wild-type recipients, but not to p40−/− mice unless IL-12 was also administered (40). Because the IL-12 dependence of a Th1 response seen here in experimental leishmaniasis may apply to other infectious diseases previously thought to be IL-12 independent, it is possible that no qualitative differences exist for the role of IL-12 in the maintenance of a Th1 response in infection vs autoimmunity.

There are several possible explanations as to why IL-12 may be necessary to maintain an established Th1 response. For example, IL-12 may be required for optimal proliferation of Th cells or for optimal IFN-γ production (41, 42). Proliferation and IFN-γ production of murine Th1 cell clones are significantly enhanced in the presence of IL-12 (41). However, several studies using Th1 clones have shown that in the absence of IL-12, Th1 clones are still capable of producing IFN-γ (21, 43). Alternatively, IL-12 could be acting to ensure Th1 cell survival. IL-12 prevents Fas-mediated apoptosis of Ag-specific T cells (44), and because Th1 cells have been characterized to be more vulnerable to Fas-mediated death (45), IL-12 may be especially important for maintaining Th1 cell survival. Similarly, IL-12 may function as a tonic survival signal for Th1 cells in the manner that IL-15 has been shown to maintain CD8+ T cells in vivo (46). Another intriguing possibility is that IL-12 may be required to prevent Th2 cell development and replenish the Th1 cell pool from naive or uncommitted Th0 cells. Despite the ability of p40−/− mice to initially make a polarized Th1 response after IL-12 treatment, these mice developed a Th2 response several weeks after cessation of treatment. Our observations suggest that IFN-γ alone is insufficient to suppress Th2 development and fails to sustain a Th1 response, as seen in other studies (47, 48). Thus, in the absence of IL-12, leishmanial-specific naive cells or uncommitted Th0 cells may become Th2 cells. In fact, Mocci and Coffman (49, 50) have observed that within a polarized Th1 population from L. major-infected mice, Mel 14high T cells can differentiate into either Th1 or Th2 cells. Therefore, IL-12 may be important in the continuous renewal of Th1 cells from these undifferentiated precursors. Overall, any one or combination of these possibilities for how IL-12 is involved in sustaining Th1 responses may apply, and additional studies will be required to resolve this issue.

Our finding that IL-12 is an absolute requirement to maintain a Th1 response suggests that the efficacy of vaccines depends on induction and continuous IL-12 production. It may also explain why plasmid IL-12 DNA when used as an adjuvant in an experimental leishmanial vaccine provides longer term protection against L. major than does soluble IL-12 (51). Our data underscore the need to elicit continuous IL-12 production to provide lasting protection against infections requiring cellular immunity.

Acknowledgments

We thank Drs. Joseph Sypek and Stan Wolf (Genetics Institute) for generously donating IL-12; Dr. Fred Finkelman (University of Cincinnati) for donating anti-IL-4R mAb; Dr. Jeanne Magram (Hoffmann-La Roche) for donating IL-12 p40−/− C57BL/6 mice used in initial studies; Drs. Jay Farrell, Chris Hunter, M. Merle Elloso, and Steve Reiner (University of Pennsylvania) for helpful discussion; and Nadine Blanchard for technical assistance.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grant AI35914.

  • ↵2 Current address: Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Queensland, Australia 4029.

  • ↵3 Address correspondence and reprint requests to Dr. Phillip Scott, Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, 216 Rosenthal Building, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: pscott{at}phl.vet.upenn.edu

  • ↵4 Abbreviation used in this paper: SLA, soluble leishmanial Ag.

  • Received March 7, 2000.
  • Accepted April 26, 2000.

References

  1. ↵
    Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170: 827
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Stern, A. S., F. J. Podlaski, J. D. Hulmes, Y. C. Pan, P. M. Quinn, A. G. Wolitzky, P. C. Familletti, D. L. Stremlo, T. Truitt, R. Chizzonite, et al 1990. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 87: 6808
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Gubler, U., A. O. Chua, D. S. Schoenhaut, C. M. Dwyer, W. McComas, R. Motyka, N. Nabavi, A. G. Wolitzky, P. M. Quinn, P. C. Familletti, M. K. Gately. 1991. Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc. Natl. Acad. Sci. USA 88: 4143
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Wolf, S. F., P. A. Temple, M. Kobayashi, D. Young, M. Dicig, L. Lowe, R. Dzialo, L. Fitz, C. Ferenz, R. M. Hewick, et al 1991. Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. J. Immunol. 146: 3074
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Trinchieri, G.. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. 13: 251
    OpenUrlCrossRefPubMed
  6. ↵
    Chan, S. H., B. Perussia, J. W. Gupta, M. Kobayashi, M. Pospisil, H. A. Young, S. F. Wolf, D. Young, S. C. Clark, G. Trinchieri. 1991. Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173: 869
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, K. M. Murphy. 1993. Development of Th1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260: 547
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Manetti, R., P. Parronchi, M. G. Giudizi, M. P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani. 1993. Natural killer cell stimulatory factor (interleukin 12 (IL-12)) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177: 1199
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Manetti, R., F. Gerosa, M. G. Giudizi, R. Biagiotti, P. Parronchi, M. P. Piccinni, S. Sampognaro, E. Maggi, S. Romagnani, G. Trinchieri, et al 1994. Interleukin 12 induces stable priming for interferon γ (IFN-γ) production during differentiation of human T helper (Th) cells and transient IFN-γ production in established Th2 cell clones. J. Exp. Med. 179: 1273
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Seder, R. A., R. Gazzinelli, A. Sher, W. E. Paul. 1993. Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon γ production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90: 10188
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Wu, C.-Y., C. Deumeure, M. Kiniwa, M. Gately, G. Delespesse. 1993. IL-12 induces the production of IFN-γ by neonatal human CD4 T cells. J. Immunol. 151: 1938
    OpenUrlAbstract
  12. ↵
    Reiner, S. L., R. M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13: 151
    OpenUrlCrossRefPubMed
  13. ↵
    Scott, P., J. P. Farrell. 1998. Experimental cutaneous leishmaniasis: induction and regulation of T cells following infection of mice with Leishmania major. Chem. Immunol. 70: 60
    OpenUrlCrossRefPubMed
  14. ↵
    Hondowicz, B. D., T. M. Scharton-Kersten, D. E. Jones, P. Scott.. 1997. Leishmania major-infected C3H mice treated with anti-IL-12 mAb develop but do not maintain a Th2 response. J. Immunol. 159: 5024
    OpenUrlAbstract
  15. ↵
    Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN-γ production and type 1 cytokine responses. Immunity 4: 471
    OpenUrlCrossRefPubMed
  16. ↵
    Mattner, F., J. Magram, J. Ferrante, P. Launois, K. Di Padova, R. Behin, M. K. Gately, J. A. Louis, G. Alber. 1996. Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 26: 1553
    OpenUrlCrossRefPubMed
  17. ↵
    Scharton-Kersten, T., L. C. Afonso, M. Wysocka, G. Trinchieri, P. Scott. 1995. IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J. Immunol. 154: 5320
    OpenUrlAbstract
  18. ↵
    Sypek, J. P., C. L. Chung, S. E. Mayor, J. M. Subramanyam, S. J. Goldman, D. S. Sieburth, S. F. Wolf, R. G. Schaub. 1993. Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response. J. Exp. Med. 177: 1797
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Heinzel, F. P., D. S. Schoenhaut, R. M. Rerko, L. E. Rosser, M. K. Gately. 1993. Recombinant interleukin 12 cures mice infected with Leishmania major. J. Exp. Med. 177: 1505
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Afonso, L. C., T. M. Scharton, L. Q. Vieira, M. Wysocka, G. Trinchieri, P. Scott. 1994. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 263: 235
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Constantinescu, C. S., B. D. Hondowicz, M. M. Elloso, M. Wysocka, G. Trinchieri, P. Scott. 1998. The role of IL-12 in the maintenance of an established Th1 immune response in experimental leishmaniasis. Eur. J. Immunol. 28: 2227
    OpenUrlCrossRefPubMed
  22. ↵
    Gazzinelli, R. T., M. Wysocka, S. Hayashi, E. Y. Denkers, S. Hieny, P. Caspar, G. Trinchieri, A. Sher. 1994. Parasite-induced IL-12 stimulates early IFN-γ synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153: 2533
    OpenUrlAbstract
  23. ↵
    Tripp, C. S., O. Kanagawa, E. R. Unanue. 1995. Secondary response to Listeria infection requires IFN-γ but is partially independent of IL-12. J. Immunol. 155: 3427
    OpenUrlAbstract
  24. ↵
    Sacks, D. L., P. V. Perkins. 1984. Identification of an infective stage of Leishmania promastigotes. Science 223: 1417
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Scott, P., E. Pearce, P. Natovitz, A. Sher. 1987. Vaccination against cutaneous leishmaniasis in a murine model. II. Immunologic properties of protective and nonprotective subfractions of soluble promastigote extract. J. Immunol. 139: 3118
    OpenUrlAbstract
  26. ↵
    Mosmann, T. R., T. A. T. Fong. 1989. Specific assays for cytokine production by T cells. J. Immunol. Methods 116: 151
    OpenUrlCrossRefPubMed
  27. ↵
    Openshaw, P., E. E. Murphy, N. A. Hosken, V. Maino, K. Davis, K. Murphy, A. O’Garra. 1995. Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J. Exp. Med. 182: 1357
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Scott, P., A. Eaton, W. C. Gause, X. di Zhou, B. Hondowicz. 1996. Early IL-4 production does not predict susceptibility to Leishmania major. Exp. Parasitol. 84: 178
    OpenUrlCrossRefPubMed
  29. ↵
    Titus, R. G., M. Marchand, T. Boon, J. A. Louis. 1985. A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol. 7: 545
    OpenUrlCrossRefPubMed
  30. ↵
    Aebischer, T., S. F. Moody, E. Handman. 1993. Persistence of virulent Leishmania major in murine cutaneous leishmaniasis: a possible hazard for the host. Infect. Immun. 61: 220
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Moll, H., S. Flohe, C. Blank. 1995. Dendritic cells seclude Leishmania parasites that persist in cured mice: a role in the maintenance of T-cell memory?. Adv. Exp. Med. Biol. 378: 507
    OpenUrlCrossRefPubMed
  32. ↵
    Stenger, S., N. Donhauser, H. Thuring, M. Rollinghoff, C. Bogdan. 1996. Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase. J. Exp. Med. 183: 1501
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Decken, K., G. Kohler, K. Palmer-Lehmann, A. Wunderlin, F. Mattner, J. Magram, M. K. Gately, G. Alber. 1998. Interleukin-12 is essential for a protective Th1 response in mice infected with Cryptococcus neoformans. Infect. Immun. 66: 4994
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Zhou, P., M. C. Sieve, J. Bennett, K. J. Kwon-Chung, R. P. Tewari, R. T. Gazzinelli, A. Sher, R. A. Seder. 1995. IL-12 prevents mortality in mice infected with Histoplasma capsulatum through induction of IFN-γ. J. Immunol. 155: 785
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Gazzinelli, R. T., S. Hieny, T. A. Wynn, S. Wolf, A. Sher. 1993. Interleukin 12 is required for the T-lymphocyte-independent induction of interferon γ by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90: 6115
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Constantinescu, C. S., B. Hilliard, M. Wysocka, E. S. Ventura, M. K. Bhopale, G. Trinchieri, A. M. Rostami. 1999. IL-12 reverses the suppressive effect of the CD40 ligand blockade on experimental autoimmune encephalomyelitis (EAE). J. Neurol. Sci. 171: 60
    OpenUrlCrossRefPubMed
  37. ↵
    Leonard, J. P., K. E. Waldburger, S. J. Goldman. 1995. Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12. J. Exp. Med. 181: 381
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Trembleau, S., G. Penna, E. Bosi, A. Mortara, M. K. Gately, L. Adorini. 1995. Interleukin 12 administration induces T helper type 1 cells and accelerates autoimmune diabetes in NOD mice. J. Exp. Med. 181: 817
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Seder, R. A., B. L. Kelsall, D. Jankovic. 1996. Differential roles for IL-12 in the maintenance of immune responses in infectious versus autoimmune disease. J. Immunol. 157: 2745
    OpenUrlAbstract
  40. ↵
    Tarrant, T. K., P. B. Silver, C. C. Chan, B. Wiggert, R. R. Caspi. 1998. Endogenous IL-12 is required for induction and expression of experimental autoimmune uveitis. J. Immunol. 161: 122
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Murphy, E. E., G. Terres, S. E. Macatonia, C. S. Hsieh, J. Mattson, L. Lanier, M. Wysocka, G. Trinchieri, K. Murphy, A. O’Garra. 1994. B7 and interleukin 12 cooperate for proliferation and interferon γ production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180: 223
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Bertagnolli, M. M., B.-Y. Lin, D. Young, S. H. Herrmann. 1992. IL-12 augments antigen-dependent proliferation of activated T lymphocytes. J. Immunol. 149: 3778
    OpenUrlAbstract
  43. ↵
    Dubey, C., M. Croft, S. L. Swain. 1996. Naive and effector CD4 T cells differ in their requirements for T cell receptor versus costimulatory signals. J. Immunol. 157: 3280
    OpenUrlAbstract
  44. ↵
    Marth, T., M. Zeitz, B. R. Ludviksson, W. Strober, B. L. Kelsall. 1999. Extinction of IL-12 signaling promotes Fas-mediated apoptosis of antigen-specific T cells. J. Immunol. 162: 7233
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Zhang, X., T. Brunner, L. Carter, R. W. Dutton, P. Rogers, L. Bradley, T. Sato, J. C. Reed, D. Green, S. L. Swain. 1997. Unequal death in T helper cell (Th) 1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185: 1837
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Zhang, X., S. Sun, I. Hwang, D. F. Tough, J. Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity 8: 591
    OpenUrlCrossRefPubMed
  47. ↵
    Gajewski, T. F., F. W. Fitch. 1988. Anti-proliferative effect of IFN-gamma in immune regulation. I. IFN-γ inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol. 140: 4245
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Wenner, C. A., M. L. Guler, S. E. Macatonia, A. O’Garra, K. M. Murphy. 1996. Roles of IFN-γ and IFN-α in IL-12-induced T helper cell-1 development. J. Immunol. 156: 1442
    OpenUrlAbstract
  49. ↵
    Mocci, S., R. L. Coffman. 1995. Induction of a Th2 population from a polarized Leishmania-specific Th1 population by in vitro culture with IL-4. J. Immunol. 154: 3779
    OpenUrlAbstract
  50. ↵
    Mocci, S., R. L. Coffman. 1997. The mechanism of in vitro T helper cell type 1 to T helper cell type 2 switching in highly polarized Leishmania major-specific T cell populations. J. Immunol. 158: 1559
    OpenUrlAbstract
  51. ↵
    Gurunathan, S., C. Prussin, D. L. Sacks, R. A. Seder. 1998. Vaccine requirements for sustained cellular immunity to an intracellular parasitic infection. Nat. Med. 4: 1409
    OpenUrlCrossRefPubMed
Back to top

In this issue

The Journal of Immunology: 165 (2)
The Journal of Immunology
Vol. 165, Issue 2
15 Jul 2000
Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section