The induction of optimal systemic antitumor immunity involves the priming of both CD4+ and CD8+ T cells specific for tumor-associated antigens. The role of CD4+ T helper cells (Th) in this response has been largely attributed to providing regulatory signals required for the priming of major histocompatibility complex class I restricted CD8+ cytolytic T lymphocytes, which are thought to serve as the dominant effector cell mediating tumor killing. However, analysis of the effector phase of tumor rejection induced by vaccination with irradiated tumor cells transduced to secrete granulocyte/macrophage colony-stimulating factor indicates a far broader role for CD4+ T cells in orchestrating the host response to tumor. This form of immunization leads to the simultaneous induction of Th1 and Th2 responses, both of which are required for maximal systemic antitumor immunity. Cytokines produced by these CD4+ T cells activate eosinophils as well as macrophages that produce both superoxide and nitric oxide. Both of these cell types then collaborate within the site of tumor challenge to cause its destruction.

The exquisite specificity of antigen recognition by the T cell arm of the immune response provides an important basis for cancer immunotherapy. The ability to discriminate tumor cells from normal tissues is critical for enabling effective tumor destruction while minimizing toxicity. Indeed, the isolation of tumor-specific T cells from cancer patients has fueled the search for tumor-associated antigens. The molecular identification of several such antigens, particularly in human melanoma, has enabled the pursuit of vaccine strategies that specifically target defined tumor antigens (13).

In the setting where relevant tumor rejection antigens have yet to be defined, vaccination with modified whole tumor cells as the antigen source has been explored as a means to prime systemic antitumor immunity. The host response to this form of vaccination has been shown to be significantly enhanced when the immunizing tumor cells are transduced with genes encoding cytokines or other molecules involved in the regulation of immune responses (4). For both tumor cell–based and defined antigen vaccine strategies, T cell subset depletion studies have usually demonstrated the requirement for both CD4+ and CD8+ T cells for systemic tumor rejection to occur.

Given that most nonhematopoietic tumors express MHC class I molecules, which serve as the restricting element for CD8+ T cell recognition, but do not express MHC class II molecules, which are required for CD4+ T cell recognition, it has been assumed that the predominant tumoricidal effector mechanism is killing by CD8+ CTL. The requirement for CD4+ T cells in these responses has been attributed to providing help during priming to achieve full activation and effector function of tumor-specific CTL. Indeed, MHC class I restricted CD8+ T cells that specifically lyse tumor cells in vitro are frequently measured to document vaccine efficacy and to serve as a surrogate end point in clinical tumor vaccine trials.

However, several lines of evidence suggest a broader role for CD4+ T cells in mediating other significant antitumor effector functions. First, studies in which mice were vaccinated several weeks before a live tumor challenge demonstrated that although immunologically intact mice were able to reject tumor, the depletion of CD4+ T cells with a mAb just before the tumor challenge resulted in the complete loss of tumor rejection (5). In these experiments, T cell help for CTL priming was fully available at the time of vaccination. This outcome suggests that CD4+ T cells play a more direct role in the effector phase of tumor rejection. Second, we have demonstrated previously that vaccination against tumor cells completely lacking MHC class I expression resulted in tumor rejection that was comparable to that seen against a challenge with an MHC class I positive variant of the same tumor (6). Depletion of either CD4+ T cells or NK cells, but not CD8+ T cells, resulted in the inability to reject the MHC class I negative variant, suggesting that CD4+ T cells may provide help as well as a measure of antigen specificity to effector cells of the immune response that do not themselves have the capacity for antigen-specific recognition.

To dissect the mechanism of CD4+ T cell effector function during tumor rejection, we studied immune responses induced by a genetically modified whole cell vaccine. A comparison of genetically modified whole cell vaccines, using the B16 melanoma (7) transduced with a large set of individual cytokine genes, demonstrated that GM-CSF– transduced vaccines generated potent and long-lasting systemic antitumor immunity that was dependent on both CD4+ and CD8+ T cells (5). Induction of immunity against the poorly immunogenic B16 tumor appears to be due to the ability of the locally produced GM-CSF to activate bone marrow–derived APCs during the priming phase to process and present tumor antigens to both CD4+ and CD8+ T cells (810). In the following studies, we were able to demonstrate that rather than simply providing help for CD8+ T cells, CD4+ T cells express both Th1 and Th2 cytokines and recruit other antitumor effector cells in addition to CD8+ T cells.

Materials And Methods

In Vivo Vaccination: Tumor Challenge Experiments.

6–8-wk-old female C57BL/6 mice were obtained from the National Cancer Institute (Bethesda, MD) and housed in the Johns Hopkins Oncology Center Animal Facility. In the same facility, CD4−/−, CD8−/−, γ-IFN−/−, IL-4−/−, IL-5−/−, X-CGD, and inducible nitric oxide synthase (iNOS−/−)1 mice that had been backcrossed to the C57BL/6 background for greater than seven generations were bred, and female offspring were used for experiments starting at 6–8 wk of age. CD4−/− and CD8−/− mice were gifts from Dr. Tak Mak (Ontario Cancer Institute, University of Toronto, Toronto, Canada). IL-4−/−, γ-IFN−/−, and iNOS−/− mice were obtained from The Jackson Laboratories (Bar Harbor, ME). IL-5−/− and X-CGD mice were gifts of Dr. Eric Pearlman (Case Western Reserve, Cleveland, OH) and Dr. Mary Dinauer (Indiana University School of Medicine, Indianapolis, IN), respectively. In brief, mice were injected subcutaneously in the left flank with 106 irradiated (50 Gy) B16 tumor cells transduced with the GM-CSF gene (B16-GM-CSF), resulting in the production of 420 ng GM-CSF/106 cells/24 h (5). 2 wk later, mice were challenged in the right flank with 105 live nontransduced B16 cells (B16-WT). B16-GM-CSF and B16 wild-type cells were provided by Dr. Glenn Dranoff (Dana Farber Cancer Institute, Boston, MA). Before injection, cells were harvested while in log phase growth from in vitro cell culture by trypsinization and were washed three times in serum-free 1× HBSS. All injections were in 0.1-ml vol. All experimental groups were matched for both age and sex. Data from the immunization challenge experiments are presented as Kaplan-Meier plots, representing the percentage of animals without detectable tumor. Mice were monitored twice weekly for tumor growth. All individual experiments included a minimum of 10 mice/group, and data from at least two repeat experiments are pooled in the Kaplan-Meier analyses.

Quantitative RT-PCR.

TRIZOL (GIBCO BRL, Gaithersburg, MD) was used to extract total RNA from lymphocytes draining the site of s.c. vaccination with irradiated B16-GM-CSF and from the site of s.c. challenge with live B16-WT cells. The SUPERSCRIPT Preamplification System (GIBCO BRL) was used to reverse transcribe total RNA to cDNA. The cDNA and cytokine competitor (a gift from Dr. Richard Locksley, University of California San Francisco, San Francisco, CA) were then PCR amplified for γ-IFN (forward: 5′-CATTGAAAGCCTAGAAAGTCTG-3′; reverse: 5′-CTCATGAATGCATCCTTTTTCG-3′), yielding 267- and 320-bp fragments, respectively. The cDNA and cytokine competitor were also PCR-amplified for IL-4 (forward: 5′-CATCGGCATTTTGAACGAGGTCA-3′; reverse: 5′-CTTATCGATGAATCCAGGCATCG-3′), yielding 240- and 360-bp fragments, respectively. The following cycling conditions were used: 94°C for 3 min, then 35 cycles at 94°C for 40 sec, 60°C for 20 sec, and 72°C for 40 sec, followed by a final extension at 72°C for 10 min. All reactions used PCR Gem 100 (Perkin-Elmer Corp., Norwalk, CT) for hot start and 32P-labeled primers for later determination of final product yield. The products were then resolved on a 6% polyacrylamide gel, dried on a Bio-Rad gel dryer (Bio-Rad Lab., Hercules, CA), and the final product yields were quantified on a PhosphorImager scanner (Molecular Dynamics, Sunnyvale, CA). The cytokine cDNA was held constant and the competitor concentration was varied until the yield of the two products were at least within threefold of each other. The initial concentration of the cytokine in the PCR reaction was then calculated by the following formula: initial cytokine level = (cytokine product signal/competitor product signal) × (starting concentration of competitor). The number of copies per microgram of total RNA was then calculated.

Immunohistochemistry for iNOS.

The biopsy of the tumor challenge site was removed and flash frozen in O.C.T. compound (Sakura Finetek USA, Inc., Torrance, CA) and dry-ice cooled isopentane. 5-μm tissue sections were fixed for 10 min in acetone and washed with TBS. Sections were quenched with a solution of 0.05% H2O2, 1% normal goat serum, and 0.5% milk for 30 min and then blocked with TBS/milk/1% normal goat serum for 10 min. The sections were then incubated with either α-iNOS antibody (11) or Mac-3 antibody (American Type Culture Collection, Rockville, MD) for 60 min. After washing, sections were incubated with biotinylated secondary antibody, goat α-rabbit IgG Fc for the α-iNOS antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and rabbit α-rat IgG (Vector Labs, Inc., Burlingame, CA) for the Mac-3 antibody, for 30 min. After washing, sections were incubated with ExtAvidin alkaline phosphatase (Sigma Chemical Co., St. Louis, MO) for 60 min. Slides were washed, and then a Fast Red substrate was used for detection.

Results

Antitumor Immunity Requires CD4+-dependent Effector Cells in Addition to CD8+ T Cells.

Wild-type, CD4 knockout (CD4−/−) (12) and CD8 knockout (CD8−/−) mice (13) were vaccinated subcutaneously in the left flank with 106 irradiated B16-GM-CSF (B16 cells transduced with GM-CSF) and challenged 2 wk later in the right flank with 105 wild-type (nontransduced) B16 melanoma cells. This dose of wild-type B16 cells is roughly 100-fold greater than the minimum dose that forms tumors in nonvaccinated C57BL/6 wild-type mice, but is rejected in the majority of animals previously vaccinated with B16-GM-CSF. In contrast to vaccinated wild-type mice, immunization of CD4−/− mice failed to prime a systemic immune response capable of rejecting this tumor challenge (Fig. 1). Interestingly, although similarly immunized CD8−/− mice (13) are impaired in their ability to reject this tumor challenge, a significant fraction of CD8−/− mice mounted a successful tumor rejection. In several experiments over a range of tumor challenge doses, the fraction of immunized CD8−/− mice that rejected a tumor challenge was roughly half of that observed in immunized wild-type mice (data not shown). In contrast, no measurable antitumor responses were ever demonstrated in CD4−/− mice. These observations directly demonstrate the existence of other CD4+ T cell–dependent effector mechanisms besides MHC class I restricted CD8+ CTL.

Vaccination with B16-GM-CSF Induces the Expression of Both Th1 and Th2 Cytokines.

The best characterized function of CD4+ T cells is the elaboration of cytokines that regulate downstream effector function of both cellular and humoral immunity. The division of CD4+ T cell or Th function into Th1 and Th2 phenotypes originally was based on the observation that murine T helper clones produced certain cytokines in a mutually exclusive fashion (1416). Subsequent studies indicated that Th1 cytokines, such as γ-IFN, as well as cytokines that promote Th1 differentiation, such as IL-12, inhibit Th2 development (17– 19), whereas Th2 cytokines, such as IL-4 and IL-10, inhibit Th1 development (2022). Taken together, these findings suggested that productive in vivo Th responses would develop uniquely along either a Th1 or a Th2 pathway and that the ensuing immunologic effector mechanisms would reflect the distinct pattern of cytokine production associated with the particular pathway of Th differentiation.

We used the B16 vaccination-challenge system to evaluate the expression of γ-IFN, the prototypical Th1 cytokine, and IL-4, the prototypical Th2 cytokine at the site of tumor challenge. Competitive quantitative PCR analysis of γ-IFN and IL-4 mRNA at the site of tumor challenge demonstrated that both of these messages were significantly increased, relative to naive mice (Fig. 2 A). The increase in γ-IFN and IL-4 mRNA was similar in vaccinated CD8−/− mice to that observed in wild-type mice. In contrast, γ-IFN and IL-4 mRNA were not increased at the tumor challenge site in vaccinated CD4−/− mice, indicating that both γ-IFN and IL-4 production are dependent upon activated CD4+ T cells. These results contrast with the findings in other studies on immune responses to parasites and intracellular bacteria that demonstrated that the production of one or the other cytokine ultimately dominates the effector phase of the response, leading to a polarized pattern of Th cytokine secretion that is associated with the ability to clear the infection (2329).

Vaccination with B16-GM-CSF Requires Both Th1 and Th2 Cytokines for Maximal Systemic Tumor Immunity.

To determine whether the Th1 cytokine, γ-IFN, or the Th2 cytokine, IL-4, was required for the induction of systemic antitumor immunity by B16-GM-CSF, vaccination-challenge experiments were performed in γ-IFN−/− and IL-4−/− mice (30, 31). As demonstrated in Fig. 2,B, protective immunity against B16 melanoma challenge was significantly decreased in both sets of cytokine gene knockout mice. In the case of γ-IFN−/− mice, protection against tumor challenge was completely eliminated, whereas in IL-4−/− mice, protection was reduced by ∼50% relative to vaccinated wild-type mice. Thus, in accordance with the PCR results in Fig. 2 A, and in contrast to the immune response in previous infectious models, induction of maximal systemic antitumor immunity was dependent on both the Th1 and the Th2 components of the immune response.

Eosinophils Are a Th2 Effector Cell.

To further define potential downstream effector mechanisms mediating tumor rejection, tissue from the tumor challenge site was examined. As seen in Fig. 3, eosinophils are one of the most prominent infiltrating cell types at the site of tumor challenge in vaccinated mice (in addition to macrophages and lymphocytes). Eosinophils represent a candidate Th2-dependent antitumor effector cell. Their differentiation from myeloid progenitors requires the Th2 cytokine IL-5 (32), and their recruitment from the blood into tissues is partially dependent on IL-4 (33). Indeed, the dense eosinophil infiltrate at the tumor challenge site is reminiscent of that seen in IL-4 transduced tumor cells after in vivo injection (34, 35). Eosinophils are completely absent from the tumor challenge site in vaccinated CD4−/− mice and are ∼80% reduced in vaccinated IL-4−/− mice, indicating that CD4+ T cell–derived IL-4 is critical for their recruitment. In contrast, the challenge site of vaccinated CD8−/− and γ-IFN−/− mice demonstrated an eosinophil infiltrate similar to that of wild-type mice.

Several lines of evidence support the notion that the infiltrating eosinophils are important Th2 dependent antitumor effectors, rather than simply inactive bystanders. First, protection against tumor challenge was significantly diminished in vaccinated IL-5−/− mice (Fig. 2,B) (36). Because IL-5 is critical for differentiation of bone marrow progenitors into eosinophils, mature eosinophils fail to develop in IL-5−/− mice. Second and most importantly, the loss of systemic antitumor immunity was associated with the absence of eosinophils at the tumor challenge sites in IL-5−/− mice (Fig. 3). Third, earlier studies of both mouse and human tissues revealed eosinophil degranulation at the tumor site, as demonstrated by the presence of eosinophil granule specific major basic protein in the interstitial space (37). This degranulation is a characteristic feature of activated eosinophils (38).

Tumor-specific CD8+ T Cells Are Not Sufficient for Maximal Tumor Immunity.

A number of potential antitumor effector mechanisms could depend on the Th1 component. The majority of studies on antitumor immunity have focused on CTL generation as the critical Th1-dependent effector mechanism. We wished to examine whether the priming of tumor-specific CTL was impaired in γ-IFN knockout mice, which might account for the marked dependence of successful tumor rejection on the ability to produce this cytokine. Interestingly, induction of CTL specific for an immunodominant MHC class I restricted B16 antigen derived from the tyrosinase related protein-2 protein (39) was identical in wild-type, IL-4−/−, and γ-IFN−/− mice (Fig. 4). Although CTL induction was normal in the cytokine gene knockout mice, tyrosinase related protein-2–specific CTL could not be detected in B16-GM-CSF vaccinated CD4−/− mice. This finding is consistent with recent reports that have identified CD154/CD40 interactions between CD4+ T cells and APCs as mediators of T cell help for CTL priming via the cross-priming pathway (4042). The fact that there was not a loss of antitumor CTL activity associated with the corresponding loss of in vivo tumor protection in the cytokine gene knockout mice does not mean that CTL are unimportant as antitumor effectors, but it does indicate that CTL alone are insufficient to eliminate tumor challenges.

Antitumor Immunity Requires Production of Nitric Oxide (NO) and Superoxide From Tumoricidal Macrophages.

Another potential Th1 effector mechanism is NO production by iNOS in activated macrophages. NO production by macrophages has been demonstrated to have tumoricidal activity both in vitro and in vivo (4354). Indeed, immunohistochemical staining at the challenge site of B16-GM-CSF vaccinated wild-type mice showed an abundant infiltration of macrophages (Fig. 5,B). This infiltrate was still evident in the cytokine gene knockout mice, but was absent in CD4−/− mice (Fig. 5,C). Infiltrating macrophages stained strongly positive for iNOS in vaccinated wild-type mice (Fig. 5,H). In contrast, despite the presence of large numbers of macrophages in B16-GM-CSF vaccinated γ-IFN−/− mice (Fig. 5,E), iNOS was virtually absent at the challenge site in γ-IFN−/− mice (Fig. 5,K). iNOS expression was present at the challenge site of IL-4−/− mice (Fig. 5 L), although levels were reduced relative to wild-type mice. The reduction in iNOS levels in IL-4−/− mice was somewhat unexpected, given that IL-4 inhibits iNOS expression in macrophages (5557). One possible explanation for this reduction in IL-4−/− mice could be the compensatory hyperexpression of other cytokines, such as IL-10, that indirectly inhibit iNOS expression through the inhibition of TNF-α expression, a costimulator for iNOS induction (21, 22, 5860). It is also possible that the decreased iNOS expression is secondary to the decreased tissue eosinophilia, as eosinophils themselves are known to express TNF-α (61, 62). Furthermore, eosinophils also express eosinophil peroxidase and macrophage inflammatory protein-1, both of which induce TNF-α release from macrophages (63, 64). Therefore, it is possible that eosinophils function as a positive modulator for iNOS release by macrophages. Furthermore, the dramatic absence of iNOS expression in γ-IFN−/− mice, which fail to reject tumor in response to vaccination, suggests that NO production by tumor infiltrating macrophages may represent an important in vivo antitumor effector mechanism.

Direct functional evidence for the role of iNOS in tumor rejection was sought in vaccination-challenge experiments in iNOS−/− mice. In accordance with the previous immunohistochemical staining, vaccinated iNOS−/− mice show a substantial decrease in protection against challenge with tumor (Table 1), thereby demonstrating the critical role for NO in antitumor killing.

Activated phagocytes can also mediate killing through the production of superoxide, which is produced by the NADPH oxidase–enzyme complex. Mutations in the CYBB gene which encodes the 91-kD glycoprotein component of the oxidase (gp91phox) account for the X-linked form of chronic granulomatous disease, an immunodeficiency syndrome characterized by recurrent infections with catalase-positive microorganisms. Phagocytes from chronic granulomatous disease patients or genetic knockout mice of gp91phox (X-CGD mice) (65) mount a severely defective respiratory burst with little or no generation of superoxide or hydrogen peroxide. X-CGD mice immunized with B16-GM-CSF showed a substantial decrease in protection against challenge with B16 tumor (Table 1), thereby implicating superoxide in the antitumor response. The loss of tumor protection in both the iNOS−/− and X-CGD mice indicates that both NO and superoxide involved in phagocytic killing play a major role in the antitumor effect induced by B16-GM-CSF.

Discussion

Taken together, the experiments presented here demonstrate that effective antitumor immunity is critically dependent upon the CD4+ T cell, which is responsible for orchestrating multiple immunologic effector arms, dependent on both Th1 and Th2 cytokines. We have demonstrated previously that the priming phase of the immune response to GM-CSF–secreting tumor cells involves the recruitment of bone marrow–derived APCs which process and present tumor antigens to both CD4+ and CD8+ T cells (10). In this study, we have examined the effector mechanisms required for the successful rejection of tumor found at distant sites. As the challenge tumor used in these studies is MHC class II negative, the effector phase of the response also requires processing of tumor antigens by infiltrating APCs for presentation to CD4+ T cells. These observations underscore the critical role of bone marrow–derived APCs in two phases of the antitumor immune response—priming of de novo T cell responses (thought to be mediated largely by activated dendritic cells) and amplification of the effector phase through the processing and presentation of tumor antigen to memory CD4+ T cells. Through the local release of cytokines, these cells direct the effector response, recruiting and activating tumoricidal macrophages, eosinophils, and the other populations seen histologically. This cooperative interaction between APC and memory CD4+ T cell is the hallmark of a classic delayed type hypersensitivity response.

The requirement for CD4+ T cells in the effector phase of an immune response to tumors that do not themselves express MHC class II is consistent with earlier results in adoptive transfer systems. Infusion of tumor-specific CD4+ T cells was shown to be capable of eradicating the MHC class II negative FBL-3 murine leukemia line (66). Class II positive macrophages were found to be required to present processed tumor antigens to CD4+ T cells (67). Furthermore, several vaccine strategies that directly enhance the priming of tumor-specific CD4+ T cells have been shown to augment the systemic rejection of MHC class II negative tumors (6871).

Previous studies in cancer immunology have focused largely on Th1 effector mechanisms, and particularly on CTL generation. Interestingly, CTL may play a smaller role in eradication of the B16 challenge tumor than in other cancer vaccine systems because of the expression of Fas ligand by B16 (as well as by many human melanomas), which may protect the tumor from direct CTL-mediated killing (72). Instead, it appears that both macrophages and eosinophils play a major role in the immune response against B16 and probably act together to achieve more efficient tumor killing. This is supported by previous studies showing that (a) eosinophil peroxidase can synergize with macrophage reactive oxygen intermediates to kill tumor cells (73) and (b) peroxidases can catalyze the oxidation of nitrite to generate further cytotoxic radicals (74). However, neither macrophages nor eosinophils have an intrinsic capacity for tumor specificity. Instead, the tumor specificity of these effectors is based on their activation by neighboring tumor-specific Th cells.

To date, the major effort in tumor antigen identification has focused almost exclusively on antigens recognized by tumor-specific, MHC class I restricted CTL. Several antigen-specific vaccine strategies incorporating class I restricted tumor antigens identified through these efforts are being explored currently in animal models and early phase clinical trials. In the absence of known MHC class II restricted tumor antigens, such strategies rely on the effects of adjuvants or incorporate surrogate class II antigens from infectious pathogens to provide help for CTL induction. The studies presented here suggest that although Th responses generated by this type of vaccination may enhance CTL induction during priming, they are unlikely to participate in the effector phase that requires tumor-specific CD4+ T cell help. Ultimately, the optimal tumor antigen–specific vaccine will ideally incorporate a panel of dominant tumor antigens recognized by both CD4+ and CD8+ T cells.

The finding that GM-CSF transduced whole cell vaccines simultaneously induce Th1 and Th2 differentiation contrasts with previous findings of in vivo immune responses to both intracellular bacteria and parasites. In resistant mouse strains, such as C3H or C57BL/6, infection with the intracellular parasite Leishmania major exclusively induces a Th1 response, characterized by high γ-IFN and low IL-4 production (2325). Furthermore, eradication of L. major has been shown to be dependent upon γ-IFN– dependent stimulation of iNOS in macrophages (7584). Conversely, in susceptible mouse strains, such as BALB/c, infection with L. major induces exclusively a Th2 immune response, characterized by high IL-4 and low γ-IFN production (2325, 28, 85). Based upon this altered pattern of Th differentiation and the direct inhibition of macrophage activation by IL-4 and IL-10 (22, 56, 60, 8688), BALB/c mice fail to mount effective immunity against L. major challenge. In contrast, protective immunity against helminth infections, such as Schistosoma mansoni and Nippostrogylus brasiliensis, is associated with Th2 development and the induction of IL-4 (26, 27, 89).

In each of these models, the definitive demonstration that particular Th1 or Th2 cytokines are critical in directing appropriate immune responses comes from the finding that elimination of a given cytokine, either by in vivo antibody depletion or by genetic knockout, results in a loss of protection against the particular pathogen. For example, γ-IFN blockade renders formerly resistant mice susceptible to L. major infection (90, 91). In addition, administration of anti–IL-4 antibody to BALB/c mice renders them resistant to L. major infection (92). Conversely, IL-4 blockade or IL-12 administration renders resistant mice susceptible to the helminth infections Trichuris muris, Heligmosomoides polygyrus, and N. brasiliensis infections (29, 93, 94).

In these models, a Th0 response, characterized by a mixture of Th1 and Th2 cytokines, is often seen initially. However, the immune response in these models typically commits along Th1 or Th2 lines by 1–2 wk after infectious challenge (90, 92, 9597). It is still not known whether the dual Th1/Th2 phenotype in this antitumor response is produced by one or two different populations of Th cells. However, because the RT-PCR cytokine data at the challenge site were obtained 18 d after vaccination, the mice most likely had already differentiated past the Th0 stage. Furthermore, the finding that complete rejection of challenge tumors introduced 2 wk after vaccination requires both IL-4 and γ-IFN–dependent responses suggests that the dual Th1/Th2 response persists for an extended period of time.

It also remains to be determined whether the dual Th1/ Th2 effector response seen here is a general characteristic of other cell-based tumor vaccines, or is unique to the mechanism by which paracrine GM-CSF production initiates immune responses. Interestingly, a similar Th1/Th2 pattern was seen in response to a defined tumor antigen, i.e., a B cell lymphoma idiotype protein after immunization with GM-CSF producing lymphoma cells (98). Further characterization of the Th response made by antigen-specific T cells in response to tumor vaccination is being performed currently using TCR transgenic mice specific for a defined tumor antigen. It is likely that one of the reasons for the superiority of paracrine GM-CSF vaccines relative to other cytokine-transduced vaccines relates to the central role of GM-CSF in inducing bone marrow progenitors to differentiate into dendritic cells. Currently, we are attempting to determine whether dendritic cells that differentiate under GM-CSF control are uniquely capable of inducing and maintaining Th1 and Th2 differentiation simultaneously.

These findings of induction and maintenance of simultaneous Th1 and Th2 responses are somewhat surprising, given that Th1-associated cytokines such as IL-12 and γ-IFN inhibit Th2 differentiation and Th2-associated cytokines such as IL-4 and IL-10 inhibit Th1 differentiation. Currently, we do not know whether the dual Th1/Th2 response is based on the development of Th cells that simultaneously produce Th1 and Th2 cytokines, or a mixture of separate Th1 and Th2 populations. Nonetheless, the finding that Th1 and Th2 effector mechanisms can actually collaborate with each other in directing an effective antitumor response rather than antagonizing each other has important implications for the development of cancer immunotherapies in general. Ultimately, the most effective cancer immunotherapies may indeed be those that can simultaneously marshal multiple Th1 and Th2 effector mechanisms that can cooperate in most effectively killing both the primary cancer and metastatic tumor deposits.

Acknowledgments

We would like to thank Dr. Tak Mak for providing the CD4−/− and CD8−/− mice, Dr. Eric Pearlman for the IL-5−/− mice, and Dr. Mary Dinauer for the X-CGD mice.

This work was supported by Public Health Service grant AI/AG37934-01 and gifts from the Topercer family. H. Levitsky is a Scholar of the Leukemia Society of America.

References

References
1
Pardoll
DM
Tumour antigens. A new look for the 1990s
Nature
1994
369
357
[PubMed]
2
Boon
T
,
van der Bruggen
P
Human tumor antigens recognized by T lymphocytes
J Exp Med
1996
183
725
729
[PubMed]
3
Kawakami
Y
,
Rosenberg
SA
Human tumor antigens recognized by T-cells
Immunol Res
1997
16
313
339
[PubMed]
4
Pardoll
DM
Cancer vaccines
Nat Med
1998
4
525
531
[PubMed]
5
Dranoff
G
,
Jaffee
E
,
Lazenby
A
,
Golumbek
P
,
Levitsky
H
,
Brose
K
,
Jackson
V
,
Hamada
H
,
Pardoll
D
,
Mulligan
RC
Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony–stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity
Proc Natl Acad Sci USA
1993
90
3539
3543
[PubMed]
6
Levitsky
HI
,
Lazenby
A
,
Hayashi
RJ
,
Pardoll
DM
In vivo priming of two distinct antitumor effector populations: the role of MHC class I expression
J Exp Med
1994
179
1215
1224
[PubMed]
7
Fidler
IJ
Biological behavior of malignant melanoma cells correlated to their survival in vivo
Cancer Res
1975
35
218
224
[PubMed]
8
Inaba
K
,
Inaba
M
,
Romani
N
,
Aya
H
,
Deguchi
M
,
Ikehara
S
,
Muramatsu
S
,
Steinman
RM
Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor
J Exp Med
1992
176
1693
1702
[PubMed]
9
Inaba
K
,
Steinman
RM
,
Pack
MW
,
Aya
H
,
Inaba
M
,
Sudo
T
,
Wolpe
S
,
Schuler
G
Identification of proliferating dendritic cell precursors in mouse blood
J Exp Med
1992
175
1157
1167
[PubMed]
10
Huang
AY
,
Golumbek
P
,
Ahmadzadeh
M
,
Jaffee
E
,
Pardoll
D
,
Levitsky
H
Role of bone marrow– derived cells in presenting MHC class I–restricted tumor antigens
Science
1994
264
961
965
[PubMed]
11
Lowenstein
CJ
,
Hill
SL
,
Lafond-Walker
A
,
Wu
J
,
Allen
G
,
Landavere
M
,
Rose
NR
,
Herskowitz
A
Nitric oxide inhibits viral replication in murine myocarditis
J Clin Invest
1996
97
1837
1843
[PubMed]
12
Rahemtulla
A
,
Fung-Leung
WP
,
Schilham
MW
,
Kundig
TM
,
Sambhara
SR
,
Narendran
A
,
Arabian
A
,
Wakeham
A
,
Paige
CJ
,
Zinkernagel
RM
et al
Normal development and function of CD8+cells but markedly decreased helper cell activity in mice lacking CD4
Nature
1991
353
180
184
[PubMed]
13
Fung-Leung
WP
,
Schilham
MW
,
Rahemtulla
A
,
Kundig
TM
,
Vollenweider
M
,
Potter
J
,
van Ewijk
W
,
Mak
TW
CD8 is needed for development of cytotoxic T cells but not helper T cells
Cell
1991
65
443
449
[PubMed]
14
Mosmann
TR
,
Cherwinski
H
,
Bond
MW
,
Giedlin
MA
,
Coffman
RL
Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins
J Immunol
1986
136
2348
2357
[PubMed]
15
Cher
DJ
,
Mosmann
TR
Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones
J Immunol
1987
138
3688
3694
[PubMed]
16
Cherwinski
HM
,
Schumacher
JH
,
Brown
KD
,
Mosmann
TR
Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies
J Exp Med
1987
166
1229
1244
[PubMed]
17
Fernandez-Botran
R
,
Sanders
VM
,
Mosmann
TR
,
Vitetta
ES
Lymphokine-mediated regulation of the proliferative response of clones of T helper 1 and T helper 2 cells
J Exp Med
1988
168
543
558
[PubMed]
18
Gajewski
TF
,
Fitch
FW
Anti-proliferative effect of IFN-γ in immune regulation. I. IFN-γ inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones
J Immunol
1988
140
4245
4252
[PubMed]
19
Gajewski
TF
,
Goldwasser
E
,
Fitch
FW
Anti-proliferative effect of IFN-γ in immune regulation. II. IFN-γ inhibits the proliferation of murine bone marrow cells stimulated with IL-3, IL-4, or granulocyte–macrophage colony-stimulating factor
J Immunol
1988
141
2635
2642
[PubMed]
20
Fiorentino
DF
,
Bond
MW
,
Mosmann
TR
Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones
J Exp Med
1989
170
2081
2095
[PubMed]
21
Fiorentino
DF
,
Zlotnik
A
,
Vieira
P
,
Mosmann
TR
,
Howard
M
,
Moore
KW
,
O'Garra
A
IL-10 acts on the antigen presenting cell to inhibit cytokine production by Th1 cells
J Immunol
1991
146
3444
3451
[PubMed]
22
Fiorentino
DF
,
Zlotnik
A
,
Mosmann
TR
,
Howard
M
,
O'Garra
A
IL-10 inhibits cytokine production by activated macrophages
J Immunol
1991
147
3815
3822
[PubMed]
23
Locksley
RM
,
Heinzel
FP
,
Sadick
MD
,
Holaday
BJ
,
Gardner
KD
Jr
Murine cutaneous leishmaniasis: susceptibility correlates with differential expansion of helper T-cell subsets
Ann Inst Pasteur Immunol
1987
138
744
749
24
Scott
P
,
Natovitz
P
,
Coffman
RL
,
Pearce
E
,
Sher
A
Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens
J Exp Med
1988
168
1675
1684
[PubMed]
25
Heinzel
FP
,
Sadick
MD
,
Holaday
BJ
,
Coffman
RL
,
Locksley
RM
Reciprocal expression of interferon γ or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets
J Exp Med
1989
169
59
72
[PubMed]
26
Street
NE
,
Schumacher
JH
,
Fong
TA
,
Bass
H
,
Fiorentino
DF
,
Leverah
JA
,
Mosmann
TR
Heterogeneity of mouse helper T cells. Evidence from bulk cultures and limiting dilution cloning for precursors of Th1 and Th2 cells
J Immunol
1990
144
1629
1639
[PubMed]
27
Pearce
EJ
,
Caspar
P
,
Grzych
JM
,
Lewis
FA
,
Sher
A
Downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni.
J Exp Med
1991
173
159
166
[PubMed]
28
Heinzel
FP
,
Sadick
MD
,
Mutha
SS
,
Locksley
RM
Production of interferon γ, interleukin 2, interleukin 4, and interleukin 10 by CD4+lymphocytes in vivo during healing and progressive murine leishmaniasis
Proc Natl Acad Sci USA
1991
88
7011
7015
[PubMed]
29
Urban
JF
Jr
,
Katona
IM
,
Paul
WE
,
Finkelman
FD
Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice
Proc Natl Acad Sci USA
1991
88
5513
5517
[PubMed]
30
Dalton
DK
,
Pitts-Meek
S
,
Keshav
S
,
Figari
IS
,
Bradley
A
,
Stewart
TA
Multiple defects of immune cell function in mice with disrupted interferon-γ genes
Science
1993
259
1739
1742
[PubMed]
31
Kopf
M
,
Le Gros
G
,
Bachmann
M
,
Lamers
MC
,
Bluethmann
H
,
Kohler
G
Disruption of the murine IL-4 gene blocks Th2 cytokine responses
Nature
1993
362
245
248
[PubMed]
32
Campbell
HD
,
Tucker
WQ
,
Hort
Y
,
Martinson
ME
,
Mayo
G
,
Clutterbuck
EJ
,
Sanderson
CJ
,
Young
IG
Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5)
Proc Natl Acad Sci USA
1987
84
6629
6633
[PubMed]
33
Schleimer
RP
,
Sterbinsky
SA
,
Kaiser
J
,
Bickel
CA
,
Klunk
DA
,
Tomioka
K
,
Newman
W
,
Luscinskas
FW
,
Gimbrone
MA
Jr
,
McIntyre
BW
et al
IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1
J Immunol
1992
148
1086
1092
[PubMed]
34
Tepper
RI
,
Coffman
RL
,
Leder
P
An eosinophil-dependent mechanism for the antitumor effect of interleukin-4
Science
1992
257
548
551
[PubMed]
35
Golumbek
PT
,
Lazenby
AJ
,
Levitsky
HI
,
Jaffee
LM
,
Karasuyama
H
,
Baker
M
,
Pardoll
DM
Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4
Science
1991
254
713
716
[PubMed]
36
Kopf
M
,
Brombacher
F
,
Hodgkin
PD
,
Ramsay
AJ
,
Milbourne
EA
,
Dai
WJ
,
Ovington
KS
,
Behm
CA
,
Kohler
G
,
Young
IG
,
Matthaei
KI
IL-5–deficient mice have a developmental defect in CD5+B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses
Immunity
1996
4
15
24
[PubMed]
37
Simons
JW
,
Jaffee
EM
,
Weber
CE
,
Levitsky
HI
,
Nelson
WG
,
Carducci
MA
,
Lazenby
AJ
,
Cohen
LK
,
Finn
CC
,
Clift
SM
et al
Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte–macrophage colony-stimulating factor gene transfer
Cancer Res
1997
57
1537
1546
[PubMed]
38
Kato
M
,
Abraham
RT
,
Okada
S
,
Kita
H
Ligation of the β2 integrin triggers activation and degranulation of human eosinophils
Am J Respir Cell Mol Biol
1998
18
675
686
[PubMed]
39
Bloom
MB
,
Perry-Lalley
D
,
Robbins
PF
,
Li
Y
,
el-Gamil
M
,
Rosenberg
SA
,
Yang
JC
Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma
J Exp Med
1997
185
453
459
[PubMed]
40
Bennett
SR
,
Carbone
FR
,
Karamalis
F
,
Flavell
RA
,
Miller
JF
,
Heath
WR
Help for cytotoxic-T cell responses is mediated by CD40 signalling
Nature
1998
393
478
480
[PubMed]
41
Schoenberger
SP
,
Toes
RE
,
van der Voort
EI
,
Offringa
R
,
Melief
CJ
T-cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions
Nature
1998
393
480
483
[PubMed]
42
Ridge
JP
,
Di Rosa
F
,
Matzinger
P
A conditioned dendritic cell can be a temporal bridge between a CD4+T-helper and a T-killer cell
Nature
1998
393
474
478
[PubMed]
43
Hibbs
JB
Jr
,
Taintor
RR
,
Chapman
HA
Jr
,
Weinberg
JB
Macrophage tumor killing: influence of the local environment
Science
1977
197
279
282
[PubMed]
44
Stuehr
DJ
,
Nathan
CF
Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells
J Exp Med
1989
169
1543
1555
[PubMed]
45
Li
LM
,
Kilbourn
RG
,
Adams
J
,
Fidler
IJ
Role of nitric oxide in lysis of tumor cells by cytokine-activated endothelial cells
Cancer Res
1991
51
2531
2535
[PubMed]
46
Kwon
NS
,
Stuehr
DJ
,
Nathan
CF
Inhibition of tumor cell ribonucleotide reductase by macrophage- derived nitric oxide
J Exp Med
1991
174
761
767
[PubMed]
47
Yim
CY
,
Bastian
NR
,
Smith
JC
,
Hibbs
JB
,
Samlowski
WE
Macrophage nitric oxide synthesis delays progression of ultraviolet light-induced murine skin cancers
Cancer Res
1993
53
5507
5511
[PubMed]
48
Xie
K
,
Huang
S
,
Dong
Z
,
Gutman
M
,
Fidler
IJ
Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastases in mice treated with liposomes containing lipopeptide CGP 31362
Cancer Res
1995
55
3123
3131
[PubMed]
49
Xie
K
,
Huang
S
,
Dong
Z
,
Juang
SH
,
Gutman
M
,
Xie
QW
,
Nathan
C
,
Fidler
IJ
Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells
J Exp Med
1995
181
1333
1343
[PubMed]
50
Bastian
NR
,
Yim
CY
,
Hibbs
JB
Jr
,
Samlowski
WE
Induction of iron-derived EPR signals in murine cancers by nitric oxide. Evidence for multiple intracellular targets
J Biol Chem
1994
269
5127
5131
[PubMed]
51
Cui
S
,
Reichner
JS
,
Mateo
RB
,
Albina
JE
Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or -independent mechanisms
Cancer Res
1994
54
2462
2467
[PubMed]
52
Dong
Z
,
Staroselsky
AH
,
Qi
X
,
Xie
K
,
Fidler
IJ
Inverse correlation between expression of inducible nitric oxide synthase activity and production of metastasis in K-1735 murine melanoma cells
Cancer Res
1994
54
789
793
[PubMed]
53
Tanguay
S
,
Bucana
CD
,
Wilson
MR
,
Fidler
IJ
,
von Eschenbach
AC
,
Killion
JJ
In vivo modulation of macrophage tumoricidal activity by oral administration of the liposome-encapsulated macrophage activator CGP 19835A
Cancer Res
1994
54
5882
5888
[PubMed]
54
Yim
CY
,
McGregor
JR
,
Kwon
OD
,
Bastian
NR
,
Rees
M
,
Mori
M
,
Hibbs
JB
Jr
,
Samlowski
WE
Nitric oxide synthesis contributes to IL-2–induced antitumor responses against intraperitoneal Meth A tumor
J Immunol
1995
155
4382
4390
[PubMed]
55
Liew
FY
,
Millott
S
,
Li
Y
,
Lelchuk
R
,
Chan
WL
,
Ziltener
H
Macrophage activation by interferon γ from host-protective T cells is inhibited by interleukin (IL)3 and IL4 produced by disease-promoting T cells in leishmaniasis
Eur J Immunol
1989
19
1227
1232
[PubMed]
56
Oswald
IP
,
Wynn
TA
,
Sher
A
,
James
SL
Interleukin 10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor α required as a costimulatory factor for interferon γ–induced activation
Proc Natl Acad Sci USA
1992
89
8676
8680
[PubMed]
57
Bogdan
C
,
Vodovotz
Y
,
Paik
J
,
Xie
QW
,
Nathan
C
Mechanism of suppression of nitric oxide synthase expression by interleukin 4 in primary mouse macrophages
J Leukocyte Biol
1994
55
227
233
[PubMed]
58
Gazzinelli
RT
,
Oswald
IP
,
Hieny
S
,
James
SL
,
Sher
A
The microbicidal activity of interferon γ–treated macrophages against Trypanosoma cruzi involves an l-arginine–dependent, nitrogen oxide–mediated mechanism inhibitable by interleukin 10 and transforming growth factor β
Eur J Immunol
1992
22
2501
2506
[PubMed]
59
Gazzinelli
RT
,
Oswald
IP
,
James
SL
,
Sher
A
IL-10 inhibits parasite killing and nitrogen oxide production by IFN-γ–activated macrophages
J Immunol
1992
148
1792
1796
[PubMed]
60
Oswald
IP
,
Gazzinelli
RT
,
Sher
A
,
James
SL
IL-10 synergizes with IL-4 and transforming growth factor β to inhibit macrophage cytotoxic activity
J Immunol
1992
148
3578
3582
[PubMed]
61
Costa
JJ
,
Matossian
K
,
Resnick
MB
,
Beil
WJ
,
Wong
DT
,
Gordon
JR
,
Dvorak
AM
,
Weller
PF
,
Galli
SJ
Human eosinophils can express the cytokines tumor necrosis factor α and macrophage inflammatory protein-1 α
J Clin Invest
1993
91
2673
2684
[PubMed]
62
Beil
WJ
,
Weller
PF
,
Tzizik
DM
,
Galli
SJ
,
Dvorak
AM
Ultrastructural immunogold localization of tumor necrosis factor-α to the matrix compartment of eosinophil secondary granules in patients with idiopathic hypereosinophilic syndrome
J Histochem Cytochem
1993
41
1611
1615
[PubMed]
63
Spessotto
P
,
Dri
P
,
Bulla
R
,
Zabucchi
G
,
Patriarca
P
Human eosinophil peroxidase enhances tumor necrosis factor and hydrogen peroxide release by human monocyte– derived macrophages
Eur J Immunol
1995
25
1366
1373
[PubMed]
64
Fahey
TJ
III
,
Tracey
KJ
,
Tekamp-Olson
P
,
Cousens
LS
,
Jones
WG
,
Shires
GT
,
Cerami
A
,
Sherry
B
Macrophage inflammatory protein 1 modulates macrophage function
J Immunol
1992
148
2764
2769
[PubMed]
65
Morgenstern
DE
,
Gifford
MA
,
Li
LL
,
Doerschuk
CM
,
Dinauer
MC
Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to Aspergillus fumigatus.
J Exp Med
1997
185
207
218
[PubMed]
66
Greenberg
PD
,
Kern
DE
,
Cheever
MA
Therapy of disseminated murine leukemia with cyclophosphamide and immune Lyt-1+,2- T cells. Tumor eradication does not require participation of cytotoxic T cells
J Exp Med
1985
161
1122
1134
[PubMed]
67
Kern
DE
,
Klarnet
JP
,
Jensen
MC
,
Greenberg
PD
Requirement for recognition of class II molecules and processed tumor antigen for optimal generation of syngeneic tumor-specific class I–restricted CTL
J Immunol
1986
136
4303
4310
[PubMed]
68
Ostrand-Rosenberg
S
,
Roby
CA
,
Clements
VK
Abrogation of tumorigenicity by MHC class II antigen expression requires the cytoplasmic domain of the class II molecule
J Immunol
1991
147
2419
2422
[PubMed]
69
Clements
VK
,
Baskar
S
,
Armstrong
TD
,
Ostrand-Rosenberg
S
Invariant chain alters the malignant phenotype of MHC class II+tumor cells
J Immunol
1992
149
2391
2396
[PubMed]
70
Baskar
S
,
Ostrand-Rosenberg
S
,
Nabavi
N
,
Nadler
LM
,
Freeman
GJ
,
Glimcher
LH
Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class II molecules
Proc Natl Acad Sci USA
1993
90
5687
5690
[PubMed]
71
Wu
TC
,
Guarnieri
FG
,
Staveley
OCKF
,
Viscidi
RP
,
Levitsky
HI
,
Hedrick
L
,
Cho
KR
,
August
JT
,
Pardoll
DM
Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens
Proc Natl Acad Sci USA
1995
92
11671
11675
[PubMed]
72
Hahne
M
,
Rimoldi
D
,
Schroter
M
,
Romero
P
,
Schreier
M
,
French
LE
,
Schneider
P
,
Bornand
T
,
Fontana
A
,
Lienard
D
et al
Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape
Science
1996
274
1363
1366
[PubMed]
73
Nathan
CF
,
Klebanoff
SJ
Augmentation of spontaneous macrophage-mediated cytolysis by eosinophil peroxidase
J Exp Med
1982
155
1291
1308
[PubMed]
74
van der Vliet
A
,
Eiserich
JP
,
Halliwell
B
,
Cross
CE
Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide–dependent toxicity
J Biol Chem
1997
272
7617
7625
[PubMed]
75
Green
SJ
,
Meltzer
MS
,
Hibbs
JB
Jr
,
Nacy
CA
Activated macrophages destroy intracellular Leishmania major amastigotes by an l-arginine–dependent killing mechanism
J Immunol
1990
144
278
283
[PubMed]
76
Liew
FY
,
Li
Y
,
Millott
S
Tumor necrosis factor α synergizes with IFN-γ in mediating killing of Leishmania majorthrough the induction of nitric oxide
J Immunol
1990
145
4306
4310
[PubMed]
77
Liew
FY
,
Li
Y
,
Millott
S
Tumour necrosis factor (TNF-α) in leishmaniasis. II. TNF-α–induced macrophage leishmanicidal activity is mediated by nitric oxide from l-arginine
Immunology
1990
71
556
559
[PubMed]
78
Liew
FY
,
Millott
S
,
Parkinson
C
,
Palmer
RM
,
Moncada
S
Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from l-arginine
J Immunol
1990
144
4794
4797
[PubMed]
79
Liew
FY
,
Li
Y
,
Moss
D
,
Parkinson
C
,
Rogers
MV
,
Moncada
S
Resistance to Leishmania majorinfection correlates with the induction of nitric oxide synthase in murine macrophages
Eur J Immunol
1991
21
3009
3014
[PubMed]
80
Mauel
J
,
Ransijn
A
,
Buchmuller-Rouiller
Y
Killing of Leishmania parasites in activated murine macrophages is based on an l-arginine–dependent process that produces nitrogen derivatives
J Leukocyte Biol
1991
49
73
82
[PubMed]
81
Evans
TG
,
Thai
L
,
Granger
DL
,
Hibbs
JB
Effect of in vivo inhibition of nitric oxide production in murine leishmaniasis
J Immunol
1993
151
907
915
[PubMed]
82
Assreuy
J
,
Cunha
FQ
,
Epperlein
M
,
Noronha-Dutra
A
,
O'Donnell
CA
,
Liew
FY
,
Moncada
S
Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major.
Eur J Immunol
1994
24
672
676
[PubMed]
83
Stenger
S
,
Thuring
H
,
Rollinghoff
M
,
Bogdan
C
Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major.
J Exp Med
1994
180
783
793
[PubMed]
84
Wei
XQ
,
Charles
IG
,
Smith
A
,
Ure
J
,
Feng
GJ
,
Huang
FP
,
Xu
D
,
Muller
W
,
Moncada
S
,
Liew
FY
Altered immune responses in mice lacking inducible nitric oxide synthase
Nature
1995
375
408
411
[PubMed]
85
Morris
L
,
Troutt
AB
,
McLeod
KS
,
Kelso
A
,
Handman
E
,
Aebischer
T
Interleukin-4 but not gamma interferon production correlates with the severity of murine cutaneous leishmaniasis
Infect Immun
1993
61
3459
3465
[PubMed]
86
Liew
FY
,
Li
Y
,
Severn
A
,
Millott
S
,
Schmidt
J
,
Salter
M
,
Moncada
S
A possible novel pathway of regulation by murine T helper type-2 (Th2) cells of a Th1 cell activity via the modulation of the induction of nitric oxide synthase on macrophages
Eur J Immunol
1991
21
2489
2494
[PubMed]
87
al-Ramadi
BK
,
Meissler
JJ
Jr
,
Huang
D
,
Eisenstein
TK
Immunosuppression induced by nitric oxide and its inhibition by interleukin-4
Eur J Immunol
1992
22
2249
2254
[PubMed]
88
Ding
L
,
Linsley
PS
,
Huang
LY
,
Germain
RN
,
Shevach
EM
IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the upregulation of B7 expression
J Immunol
1993
151
1224
1234
[PubMed]
89
Urban
JF
Jr
,
Madden
KB
,
Svetic
A
,
Cheever
A
,
Trotta
PP
,
Gause
WC
,
Katona
IM
,
Finkelman
FD
The importance of Th2 cytokines in protective immunity to nematodes
Immunol Rev
1992
127
205
220
[PubMed]
90
Belosevic
M
,
Finbloom
DS
,
Van Der Meide
PH
,
Slayter
MV
,
Nacy
CA
Administration of monoclonal anti–IFN-γ antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major.
J Immunol
1989
143
266
274
[PubMed]
91
Wang
ZE
,
Reiner
SL
,
Zheng
S
,
Dalton
DK
,
Locksley
RM
CD4+ effector cells default to the Th2 pathway in interferon γ–deficient mice infected with Leishmania major.
J Exp Med
1994
179
1367
1371
[PubMed]
92
Sadick
MD
,
Heinzel
FP
,
Holaday
BJ
,
Pu
RT
,
Dawkins
RS
,
Locksley
RM
Cure of murine leishmaniasis with anti-interleukin 4 monoclonal antibody. Evidence for a T cell–dependent, interferon γ–independent mechanism
J Exp Med
1990
171
115
127
[PubMed]
93
Finkelman
FD
,
Madden
KB
,
Morris
SC
,
Holmes
JM
,
Boiani
N
,
Katona
IM
,
Maliszewski
CR
Anti-cytokine antibodies as carrier proteins. Prolongation of in vivo effects of exogenous cytokines by injection of cytokine– anti-cytokine antibody complexes
J Immunol
1993
151
1235
1244
[PubMed]
94
Finkelman
FD
,
Madden
KB
,
Cheever
AW
,
Katona
IM
,
Morris
SC
,
Gately
MK
,
Hubbard
BR
,
Gause
WC
,
Urban
JF
Jr
Effects of interleukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites
J Exp Med
1994
179
1563
1572
[PubMed]
95
Chatelain
R
,
Varkila
K
,
Coffman
RL
IL-4 induces a Th2 response in Leishmania major–infected mice
J Immunol
1992
148
1182
1187
[PubMed]
96
Reed
SG
,
Scott
P
T-cell and cytokine responses in leishmaniasis
Curr Opin Immunol
1993
5
524
531
[PubMed]
97
Reiner
SL
,
Zheng
S
,
Wang
ZE
,
Stowring
L
,
Locksley
RM
Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+T cells during initiation of infection
J Exp Med
1994
179
447
456
[PubMed]
98
Levitsky
HI
,
Montgomery
J
,
Ahmadzadeh
M
,
Staveley-O'Carroll
K
,
Guarnieri
F
,
Longo
DL
,
Kwak
LW
Immunization with granulocyte–macrophage colony-stimulating factor–transduced, but not B7-1–transduced, lymphoma cells primes idiotype-specific T cells and generates potent systemic antitumor immunity
J Immunol
1996
156
3858
3865
[PubMed]
1

Abbreviations used in this paper: iNOS, inducible nitric oxide synthase; NO, nitric oxide.

Author notes

Address correspondence to Hyam Levitsky, Johns Hopkins University School of Medicine, 347 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205. Phone: 410-614-0552; Fax: 410-614-9705; E-mail: hy@welchlink.welch.jhu.edu