IL-12 as an adjuvant for cell-mediated immunityPhillip Scott* and Giorgio Trinchieri†

Recent studies have shown that IL-12 initiates the develop-ment of cell-mediated immunity following infection withcertain infectious agents. This attribute of IL-12 makes it apotentially effective adjuvant for vaccines. This reviewsummarizes our findings in the Leishmania system, out-lining the role of endogenous IL-12 in resistance to L. majorand the use of exogenous IL-12 in a vaccine against L.major, and also describes other examples in which IL-12 hasbeen shown to be effective as an adjuvant at inducingprotective immunity. The potential use of IL-12 as anadjuvant in therapeutic vaccines is also discussed.

Key words: adjuvant / IL-12 / leishmania / vaccine

©1997 Academic Press Ltd

THE ABILITY TO INDUCE protective immunity against apathogen by immunization is dependent upon theexpansion of a memory T-cell population, which uponsubsequent activation will be capable of rapidlyresponding, leading to the elimination of the patho-gen. Vaccines have traditionally been composed ofeither attenuated or killed whole organisms, althoughrecombinant proteins and peptides have been thesource of antigen in newer vaccines. The successfuluse of such subunit vaccines usually requires admini-stration with adjuvants, which are thought to act as adepot for the administered antigen. However, inorder to be effective adjuvants also need to providecues that help direct the immune response inparticular directions, such as towards cell-mediatedimmunity or humoral immunity. Such cues appear tobe provided by whole organisms, and presumablyhave evolved during interactions of pathogens withthe mammalian immune system, and in particularwith the innate immune system. However, the molec-ular basis accounting for the ability of pathogens tofavor particular types of immune responses is not yetcompletely defined.

The most important advance in the last 10 years inour understanding of how to direct the immuneresponse by vaccination has been the description, andsubsequent refinement, of the Th1/2 paradigm.1 Thisparadigm has provided the framework necessary toformulate basic questions related to defining the cuespathogens provide that shape the immune response.A major advance in this area came when it was shownthat IL-12 promoted the development of Th1 cells invitro.2,3 Thus, in naive T-cell populations exposure toantigen in the presence of IL-12 for several days,followed by restimulation with antigen alone, led tothe development of IFN-γ producing T cells. Moreo-ver, it was found that one could link together IL-12,the innate immune response, pathogens, and Th1 celldevelopment. This was done by showing that thebacterium, Listeria monocytogenes, induced Th1 celldevelopment, and that this occurred by stimulation ofmacrophages to produce IL-12.2 This observation hasled to the description of a common pathway leadingfrom the innate immune response to adaptive immu-nity, in which intracellular pathogens stimulate mac-rophages to produce IL-12, which promotes thedevelopment of Th1 cells from a naive cell popula-tion. An additional component of the pathway is theactivation of NK cells by IL-12. The role that NK cellsplay in shaping the adaptive immune response is likelyto be related to their production of cytokines, such asIFN-γ, rather than their cytotoxic activity.

IL-12 is a heterodimeric cytokine, composed of a40 kDa chain and 35 kDa chain, initially discoveredbecause of its ability to stimulate NK cells (henceoriginally termed natural killer cell stimulatory factor,NKSF).4 A similar molecule that influenced cytotoxicT cells (hence termed cytotoxic lymphocyte matura-tion factor) was found to be identical to NKSF,5 andboth are now called IL-12 (reviewed in refs 6, 7). Thiscytokine can be produced by macrophages, neu-trophils and dendritic cells, but does not appear to beproduced by normal B cells in appreciable amounts.8

IL-12 acts upon NK cells, T cells and B cells via areceptor that at present is known to comprise at leasttwo chains, termed IL-12Rb1 and IL-12Rb2.9,10 Themost important biological function mediated by IL-12appears to be the induction of IFN-γ production,

From the *Department of Pathobiology, University of Pennsylva-nia School of Veterinary Medicine, 3800 Spruce Street, Phil-adelphia, PA 19104 and †The Wistar Institute, 3601 SpruceStreet, Philadelphia, PA 19104, USA

seminars in IMMUNOLOGY, Vol 9, 1997: pp 285–291

©1997 Academic Press Ltd1044-5323/97/050285 + 07 $25.00/0/si970084

285

although IL-12 can augment the production of othercytokines, promote cell proliferation, and increasecytotoxic activity of cells.7

Th1 and Th2 cell development and the role ofIL-12 in leishmaniasis

Experimental murine leishmanial infections providedone of the first examples of the importance of Th1and Th2 cell subsets in disease. Because of the cleardifferential development of Th1 and Th2 cells in miceinfected with L. major, this model has been used todefine the factors that control the development ofCD4+ T-cell subsets, and regulate those subsets oncethey have developed.11-13 Leishmaniasis is a wide-spread and debilitating protozoal disease of man andanimals. Kala-azar, or visceral leishmaniasis, is oftenfatal in the absence of drug treatment. Cutaneousleishmaniasis, while not fatal, can leave disfiguringscars upon healing or, alternatively, lead to long-termchronic infections, such as diffuse cutaneous leishma-niasis (DCL), mucocutaneous leishmaniasis (MCL) orleishmaniasis recidivans (also called tuberculoid orlupoid leishmaniasis). Some of the more commonspecies of Leishmania causing cutaneous diseaseinclude L. major and L. tropica in the Old World, andL. mexicana, L. amazonensis and L. braziliensis in theNew World. Experimental infections with Leishmaniain mice can mimic several forms of human cutaneousleishmaniasis. Some strains of mice, such as BALB/c,are highly susceptible to L. major infection and fail todevelop a cell-mediated response to the parasite.Since these animals develop metastatic lesions, theyare often considered as a model for DCL. In contrast,other strains of mice, such as C3H, C57BL/6, C57BL/10 and 129, develop self-healing lesions associatedwith a strong cell-mediated immunity.11-13 The courseof infection in these animals is similar to that observedin patients with self-healing lesions.

In the late 1980s, it was shown by adoptive transferof Th1 and Th2 cell lines that Th1 cells mediateresistance, while Th2 cells mediate susceptibility.14

Furthermore, it was shown that infection in suscepti-ble mice was associated with the preferential produc-tion of IL-4, while cells from animals that wereresistant to L. major produced high levels of IFN-γ andlittle IL-4.15-17 Studies of the events leading to Th1 andTh2 cell development in L. major infected animalshave shown that IL-4 is important for Th2 celldevelopment, while IL-12 and IFN-γ promote Th1 cell

development. For example, L. major infection in C3Hmice is associated with increased levels of IL-12production, and neutralization of IL-12 with mAbtreatment demonstrated that this early IL-12 isrequired both for NK cell activation and Th1 celldevelopment.18 Consistent with these results arefindings demonstrating that treatment of L. majorinfected BALB/c mice with IL-12, given at the time ofinfection, enhances Th1 cell development and heal-ing.19,20 Conversely, neutralization of IL-4 in suscepti-ble BALB/c mice blocks Th2 cell development, andsuch mice develop a Th1 response and self-heal.15

These results are consistent with in-vitro studiesunrelated to leishmaniasis that demonstrate a role forIL-4 in Th2 cell differentiation, and IL-12 in Th1 celldifferentiation.21 Thus, at present, the simple modelfor both in-vitro and in-vivo Th cell subset develop-ment would be that the presence of IL-4 promotesTh2 development, while the presence of IL-12 pro-motes Th1 cell development.

IL-12 in a leishmanial vaccine

The study of the innate immune response to L. majorin the resistant C3H mouse suggested that IL-12 mightbe useful in a vaccine against leishmaniasis. To testthis possibility, BALB/c mice were immunized with asoluble leishmanial antigen in the presence orabsence of IL-12.22 The route of immunization wassubcutaneous, followed 10 days later by a boost givenintradermally. The immunized mice were then chal-lenged with L. major and the course of infectionmonitored. In contrast to the controls, mice immu-nized with leishmanial antigen and IL-12 were pro-tected against a normally fatal infection. These studieswere the first to establish the efficacy of using IL-12 ina vaccine requiring cell-mediated immunity. Morerecently, it was shown that a single antigen and IL-12could stimulate protective immunity.23 Thus, a mole-cule termed LACK was administered with IL-12 toBALB/c mice, in the same protocol as describedabove, and the animals were protected against chal-lenge infection. These results suggest that a singleantigen given with IL-12 will be sufficient to induce aprotective immune response in mice. This is animportant observation since it suggests that no addi-tional adjuvant is required other than a recombinantprotein and IL-12 to induce protection. It should bepointed out, however, that this antigen may besomewhat unique, since the type of initial immune

P. Scott and G. Trinchieri

286

response that develops to this single dominant leish-manial antigen — i.e. whether anti-LACK T cellsproduce IL-4 or IFN-γ — may be critical in determin-ing resistance and susceptibility to leishmaniasis.24,25

Our studies in C3H mice found that NK cells playeda role in the early development of Th1 cells followinginfection with L. major.26,27 Since IL-12 is associatedwith NK cell activation, we investigated what role NKcells might be playing in the development of vaccine-induced immunity. We found that administration ofsoluble leishmanial antigen and IL-12 induced an NKcell IFN-γ response in the lymph nodes draining thesite of immunization. In contrast, by 2 weeks afterimmunization the NK cell response had diminished,and was replaced by a CD4+ Th1 type response.22 ThisNK cell response was required for Th1 cell develop-ment following immunization with IL-12, since deple-tion of NK cells abrogated the development of a Th1response in immunized mice.22 Similarly, it was foundin a Schistosoma mansoni vaccine that the ability ofIL-12 to promote Th1 cells was dependent upon thepresence of an NK1.1 + cell population.28 Our currentinterpretation of these results is that NK cells produceIFN-γ, which promotes the production of endogenousIL-12 and thus enhances the development of a Th1response, and at the same time inhibits Th2 celldevelopment. This hypothesis is supported by ourfinding that depletion of IFN-γ at the time ofimmunization also abrogates Th1 celldevelopment.22

Use of IL-12 as an adjuvant in other systems

IL-12 has also been shown to be an effective adjuvantin several other systems. Significant protection againstbacteria and helminths has been observed in mousemodels when IL-12 has been part of the immunizationprotocol. For example, it was found that nonviableListeria monocytogenes administered with IL-12 elicitedprotective immunity against a lethal challenge infec-tion.29 Notably, the protection observed followingimmunization with Listeria antigen and IL-12 wasequivalent to that induced by sublethal infection withListeria, which was the first demonstration of protec-tion against Listeria in a vaccine using a non-viableorganism. Similarly, the acellular Bordetella pertussisvaccine was improved significantly when IL-12 wasused as an adjuvant.30 Thus, with IL-12 the efficacy ofthe Bordetella subunit vaccine was equivalent to thatobserved with the whole cell vaccine.30

In schistosomiasis, the efficacy of vaccines in amouse model using either attenuated organisms(irradiated cercariae) or antigen extracts wasincreased significantly when given with IL-12.28,31 Inboth cases, protection was associated with enhancedTh1 type responses, although the effects on antibodyisotype varied. Mice immunized with attenuatedcercariae exhibited enhanced levels of IgG1, whichwas unaffected by the presence of IL-12.31 In contrast,mice immunized with soluble extracts of the worms,in combination with IL-12, exhibited a dramaticreduction in IgG1 levels.28 In both cases, there was asignificant reduction in the levels of total IgE asso-ciated with the infection.

In viral systems, IL-12 was also found to be aneffective adjuvant. Thus, administration of IL-12enhanced type-1 immune responses against respira-tory syncytial virus and pseudorabies virus.32,33 In thepseudorabies system, the critical role that IFN-γ playswas directly demonstrated by showing that no protec-tion was obtained with IL-12 in mice lacking the IFN-γreceptor.32 Studies with Class I restricted peptidesstrongly suggest that peptides and IL-12 alone may besufficient to generate protective CTL activity, sincemice immunized with influenza NP peptides andIL-12 were shown to be protected against influenzachallenge infection.34

Studies carried out to examine the important andpractical issues of how best to administer IL-12 haveshown that IL-12 can be effective as an adjuvant whenadministered in combination with antigen by thesubcutaneous route, or when administered system-ically separate from the antigen.35 With the modelantigen, TNP-KLH, the nature of the memoryimmune response induced when IL-12 is used as anadjuvant was also investigated. Immunization ofBALB/c mice with TNP-KLH normally invokes a Th2response, while systemic administration of IL-12 — atdays –1, 0 and + 1, relative to subcutaneous injectionof TNP-KLH — promotes a Th1 response 7 dayslater.36 These results support the conclusion that IL-12alone, with an antigen, is sufficient to promote astable Th1 response. However, subsequent experi-ments have clouded this issue somewhat. For exam-ple, when mice given a primary immunization withTNP-KLH and IL-12 were rested for 30 days, and thenrechallenged with TNP-KLH, somewhat differentresults were obtained.37 Although these animals stillexhibited an IFN-γ response, spleen cells from thesemice produced IL-4 levels greater than that observedwhen mice were immunized with TNP-KLH alone.This observation has been interpreted as evidence

IL-12 as an adjuvant

287

that IL-12 can act as an effective adjuvant for both Th1and Th2 cells, although without protection studies-— which are not possible in this model — it is unclearhow biologically significant the levels of IL-4 observedare in this system. This leaves unresolved the issue ofwhether IL-12 acts as an effective adjuvant for bothTh1 and Th2 type responses in vivo. Certainly all ofthe in-vitro evidence, and most of the in-vivo evidence,suggests that IL-12 primarily augments Th1 responses.Other studies with defined antigens have shown thatIL-12 primarily augments Th1 responses. Other stud-ies with defined antigens have shown that IL-12 doesenhance the humoral immune response, althoughthe isotypes involved are those associated with a Th1type response. Thus, mice immunized with KLH-phospholipase A2 adsorbed to alum and IL-12 exhib-ited increased levels of IgG2a, IgG2b and IgG3antibodies, but no increase in IgG1 and a decrease inIgE responses.38 Nevertheless, the TNP-KLH experi-ments suggest that under certain circumstances IL-12will promote a Th2 response. One interpretation ofthe TNP-KLH results is that they suggest that IL-12and a single antigen are required, but not sufficient,to generate a stable and strong Th1 response. In all ofthese systems it is difficult to determine what levels ofIFN-γ constitute a strong Th1 response. Alternatively,the ability of IL-12 to act alone as an adjuvant solelyfor Th1 responses may depend on the natural bias ofthe immunogen — i.e. whether it favors a Th1 or aTh2 response, or neither, when given without IL-12.Thus, when used as an adjuvant with an immunogenbiased towards a Th2 response, which is the case withTNP-KLH, additional cofactors may be required to‘lock in’ a dominant Th1 response, while in the caseof immunogens that have no bias or are slightly biasedtowards a Th1 response, such cofactors may be lessimportant.

Several studies suggest that cytokines may beimportant in promoting the efficacy of IL-12 as avaccine adjuvant. The most well studied of theseinclude IL-1, IL-2 and TNF. Each of these canaugment IL-12 activity in vitro, and thus have thepotential to augment IL-12 activity in vivo. In theleishmanial system, it was found that depletion of IL-2compromised Th1 development, and inhibited NKcell activation.26 Other factors that may contribute tothe efficacy of IL-12 as an adjuvant are the costimula-tory molecules, B7.1 and B7.2. The ability of B7 tosynergize with IL-12 has been shown in vitro, both withhuman and murine cells.39,40 Moreover, the efficacy ofincorporating B7 into a vaccine has been amplydemonstrated in murine tumor systems.41-43

The ability of bacterial adjuvants to induce aTh1 response is dependent on IL-12

Adjuvants often include bacteria or their products,and their adjuvanticity may in part relate to the abilityof bacterial molecules to stimulate IL-12 production.For example, the most common of these bacterialadjuvants is complete Freund’s adjuvant, which con-tains killed Mycobacterium, and which has been usedextensively in experimental systems. Several bacteriacan stimulate IL-12 production by macrophages, andit is likely that the efficacy of bacterial adjuvants isrelated to increased levels of IL-12. Interestingly,however, optimal in-vitro production of IL-12 requirespriming of macrophages with IFN-γ, suggesting thatIFN-γ itself may promote a Th1 response by increasingIL-12 levels. In the leishmanial system, we found thatimmunization of mice with a soluble leishmanialantigen and another bacteria, Corynebacterium parvum,promoted resistance to infection with L. major.16

However, the immunization failed to induce completeprotection, and the animals eventually developedprogressive disease. In contrast, when mice wereimmunized with soluble leishmanial antigen, C. par-vum, and IFN-γ they were found to be completelyprotected against a normally fatal infection with L.major. At the time when these experiments were done,around 1990, the hypothesis was that IFN-γ promotedTh1 cell development, although the mechanisminvolved was undefined.

In order to investigate the role of IL-12 in the C.parvum and IFN-γ vaccine, we determined the abilityof such a vaccine to induce IL-12 production, andassessed whether IL-12 was required for the develop-ment of a Th1 response (Afonso, L. and Scott, P.,manuscript in preparation). For these studies we usedC57BL/10 mice, which focuses the studies on theability of the vaccine to upregulate a Th1 response,rather than downregulate a Th2 response, since incontrast to BALB/c mice C57BL/10 mice do notdevelop a dominant Th2 response following infectionwith Leishmania. When the innate immunologicalresponse induced by immunization was measured at 2days, lymph node cells from mice injected with C.parvum and leishmanial antigen exhibited a three-foldincrease in IFN-γ levels, and a two-fold increase in thenumber of cells producing IL-12 p40, compared withcells from animals immunized with antigen alone. Inboth cases, these increases were almost doubled whenmice were immunized with antigen, C. parvum, andIFN-γ. To determine if IL-12 was required for theenhanced IFN-γ responses observed when mice were

P. Scott and G. Trinchieri

288

immunized with antigen, C. parvum and IFN-γ, wetreated mice with anti- IL-12 antibodies prior toimmunization. In this case, the IFN-γ response wascompletely blocked. In addition, when the immuneresponse was measured in these animals at 10 days,treatment with anti- IL-12 completely blocked theinduction of a Th1 response (Afonso, L. and Scott, P.,manuscript in preparation). Our interpretation ofthese results is that C. parvum induces IL-12 produc-tion, and IFN-γ upregulates the IL-12 that is pro-duced. This is consistent with our findings that in-vitroC. parvum stimulated IL-12 production, and thatpriming of macrophages with IFN-γ augmented IL-12production (Afonso, L. and Scott, P., manuscript inpreparation).

Use of IL-12 as an adjuvant in therapeuticvaccines

IL-12 may play a role not only as an adjuvant inprophylactic vaccines, but also in therapeutic vac-cines. With such vaccines, it is necessary not only topromote a cell-mediated immunological response,but it often requires downregulation of an establishedand detrimental Th2 response. We investigated theability of IL-12 to switch an established Th2 response,and thus promote healing, in the L. major BALB/cmodel. BALB/c mice develop a stable Th2 responseby 2 weeks of infection, and efforts to alter thatresponse have been uniformly unsuccessful. Wetreated mice with IL-12 under various conditions at 3weeks of infection, and were unable to alter theeventual fatal nature of the disease, nor was IL-12 ableto promote the development of a Th1 response.44 Wethen investigated whether a combination of chemo-therapy with IL-12 might be more successful, andfound that while drug treatment had only a temporaryeffect on the course of infection, mice given IL-12during the time of drug treatment were able to switchtheir immunological response from a Th2 to a Th1response. Moreover, these animals were capable ofhealing their leishmanial infections, and were resis-tant to challenge infection.44 Thus, these experimentsindicate that in certain situations one may need toreduce the antigen load in order for IL-12 to be aneffective adjuvant.

IL-12 has also been shown to switch the dominantTh2 response associated with egg deposition in miceinfected with the helminth, S. mansoni. The principalpathology caused by this infection relates to thepresence of eggs in the liver by the worms, and the

subsequent immunological response leading to thedevelopment of a granuloma around each egg, andthe resulting fibrosis. These granulomas are charac-terized by the presence of high numbers of eosino-phils, and are considered a Th2 type response. IL-12treatment of animals prior to egg injection dramat-ically reduced granuloma size, and could even controlgranuloma development if the animals had beenpreviously sensitized to eggs.45 The control of granu-loma size extended to a reduction in tissue fibrosis,demonstrating that IL-12 can act as an adjuvant for avaccine that might control pathology.46

Conclusion

The description of the Th1/2 model, which explainshow selective immune responses — such as cell-medi-ated or humoral immunity — develop, provides thebasis for the rational design of new vaccines. Thus,whether or not the Th1/2 paradigm is more compli-cated than is often indicated, this paradigm passes theimportant test of providing a framework for practicaladvances in the treatment of disease. We have focusedon the first step, which has been to demonstrate thatIL-12 promotes Th1 cell development, and protectiveimmunity, in an experimental model of leishmaniasis.We have also shown that IL-12 can promote a switchfrom an established Th2 to a Th1 response, suggest-ing that IL-12 may be useful not only in prophylacticvaccines, but also with therapeutic vaccines. Pre-sumably, as our understanding of the innate andadaptive immune responses increases, we will be ableto incorporate more information into rational vaccinedesign. However, the success of vaccines designed toinduce particular types of immune responses isdependent upon knowing the effector mechanismsresponsible for protection. While this may be clearwith certain pathogens, such as Leishmania as well asother intracellular pathogens, the protective effectormechanisms for many bacteria, viruses and helminthsare not well defined. Thus, with the ability to directthe immune response in certain directions duringimmunization, it becomes even more important todefine the effector mechanisms that are necessary toeliminate infectious agents.

Vaccines have been the major contributor inpreventing a variety of infectious diseases, but thereremain many infectious diseases for which we have noeffective vaccines. As we increase our understandingof the immune response, it can be anticipated thatnewer vaccines incorporating this information will be

IL-12 as an adjuvant

289

developed. IL-12 is likely to play a major role in suchvaccines, both administered as a protein and as a DNAvaccine — the efficacy of which was recently shown inan experimental HIV vaccine47 — since it has beenshown to be an effective adjuvant in experimentalmurine vaccines directed against protozoa, bacteria,viruses and helminths. In many cases, previousattempts to vaccinate against some of these organismshave been either unsuccessful, or the protectionobserved was limited. These laboratory findings pro-vide a solid rationale for application of IL-12 tohuman vaccines.

Acknowledgements

This work was partially supported by NIH grant 35914. P.Scott is a recipient of the Burroughs Wellcome NewInvestigator Award in Molecular Parasitology.

References

1. Mosmann TR, Coffman RL (1989) TH1 and TH2 cells:different patterns of lymphokine secretion lead to differentfunctional properties. Annu Rev Immunol 7:145-173

2. Hsieh C-S, Macatonia SE, Tripp CS, Wolf SF, O’Garra A,Murphy KM (1993) Development of Th1 CD4+ T cells throughIL-12 produced by Listeria-induced macrophages. Science260:547-549

3. Manetti R, Parronchi P, Giudizi MG, Piccinni M-P, Maggi E,Trinchieri G, Romagnani S (1993) Natural killer cell stim-ulatory factor (NKSF/IL-12) induces Th1-type specific immuneresponses and inhibits the development of IL-4 producing Thcells. J Exp Med 177:1199-1204

4. Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S,Loudon F, Sherman B, Perussia B, Trinchieri G (1989)Identification and purification of Natural Killer cell stimulatoryfactor (NKSF), a cytokine with multiple biologic effects onhuman lymphocytes. J Exp Med 170:827-846

5. Stern AS, Podlaski FJ, Hulmes JD, Pan YE, Quinn PM, WolitzkyAG, Familletti PC, Stremlo DL, Truitt T, Chizzonite R, GatelyMK (1990) Purification to homogeneity and partial character-ization of cytotoxic lymphocyte maturation factor form humanB-lymphoblastoid cells. Proc Natl Acad Sci USA 87:6808-6812

6. Trinchieri G (1995) Interleukin 12: A proinflammatory cyto-kine with immunoregulatory functions that bridge innateresistance and antigen-specific adaptive immunity. Annu RevImmunol 13:251-276

7. Trinchieri G, Scot P (1995) Interleukin-12: a proinflammatorycytokine with immunoregulatory functions. Res Immunol146:423-431

8. Ma X, D’Andrea A, Kubin M, Aste-Amezaga M, Sartori A,Monteiro J, Showe L, Wysocka M, Trinchieri G (1995)Production of interleukin-12. Res Immunol 146:432-438

9. Chua AO, Wilkinson VL, Presky DH, Gubler U (1995) Cloningand characterization of a mouse IL-12 receptor-â component. JImmunol 155:4286-4294

10. Presky DG, Yang H, Minetti LJ, Chua AO, Nabavi N, Wu C-Y,Gately MK, Gubler U (1996) A functional interleukin 12receptor complex is composed of two â-type cytokine receptorsubunits. Proc Natl Acad Sci USA 93:14002-14007

11. Locksley RM, Scott P (1991) Helper T-cell subsets in mouseleishmaniasis: induction, expansion and effector function.Immunol Today 12:A58-A61

12. Scott P (1996) T helper cell development and regulation inexperimental cutaneous leishmaniasis. Chem Immunol63:98-114

13. Reiner SL, Locksley RM (1995) The regulation of immunity toLeishmania major. Annu Rev Immunol 13:151-177

14. Scott P, Natovitz P, Coffman RL, Pearce E, Sher A (1988)Immunoregulation of cutaneous leishmaniasis. T cell lines thattransfer protective immunity or exacerbation belong to differ-ent T helper subsets and respond to distinct parasite antigens.J Exp Med 168:1675-1684

15. Heinzel FP, Sadick MD, Holaday BJ, Coffman RL, Locksley RM(1989) Reciprocal expression of interferon gamma or IL-4during the resolution or progression of murine leishmaniasis.Evidence for expansion of distinct helper T cell subsets. J ExpMed 169:59-72

16. Scott P (1991) IFN-gamma modulates the early development ofTh1 and Th2 responses in a murine model of cutaneousleishmaniasis. J Immunol 147:3149-3155

17. Chatelain R, Varkila K, Coffman RL (1992) IL-4 induces a Th2response in Leishmania major-infected mice.. J Immunol148:1182-1187

18. Scharton-Kersten T, Afonso LCC, Wysocka M, Trinchieri G,Scott P (1995) IL-12 is required for Natural Killer cell activationand subsequent T helper 1 cell development in experimentalleishmaniasis. J Immunol 154:5320-5330

19. Heinzel FP, Rerko RM, Ahmed F, Pearlman E (1995) Endoge-nous IL-12 is required for control of Th2 cytokine responses. JImmunol 155:730-739

20. Sypek JP, Chung CL, Mayor SEH, Subramanyam JM, GoldmanSJ, Sieburth DS, Wolf SF, Schaub RG (1993) Resolution ofcutaneous leishmaniasis: Interleukin 12 initiates a protectiveTh1 immune response. J Exp Med 177:1797-1802

21. O’Garra A, Murphy K (1994) Role of cytokines in determiningT-lymphocyte function. Curr Opin Immunol 6:458-466

22. Afonso LCC, Scharton TM, Vieira LQ, Wysocka M, TrinchieriG, Scott P (1994) The adjuvant effect of interleukin-12 in avaccine against Leishmania major. Science 263:235-237

23. Mougneau E, Altare F, Wakil AE, Zheng S, Coppola T, WangZW, Waldmann R, Locksley RM, Glaichenhaus N (1995)Expression cloning of a protective Leishmania antigen. Science268:563-566

24. Julia V, Rassoulzadegan M, Glaichenhaus N (1996) Resistanceto Leishmania major induced by tolerance to a single antigen.Science 274:421-423

25. Reiner SL, Wang Z-E, Hatam F, Scott P, Locksley RM (1993)Common lineage of Th1 and Th2 subsets in leishmaniasis.Science 259:1457-1460

26. Scharton TM, Scott P (1993) Natural killer cells are a source ofIFN-γ that drives differentiation of CD4+ T cell subsets andinduces early resistance to Leishmania major in mice. J Exp Med178:567-577

27. Scharton-Kersten T, Scott P (1995) The role of the innateimmune response in Th1 cell development following Leishma-nia major infection. J Leukocyte Biol 57:515-522

28. Mountford AP, Anderson S, Wilson RA (1996) Induction ofTh1 cell-mediated protective immunity to Schistosoma man-soni by co-administration of larval antigens and IL-12 as anadjuvant. J Immunol 156:4739-4745

29. Miller MA, Skeen MJ, Ziegler HK (1995) Nonviable bacterialantigens administered with IL-12 generate antigen-specific Tcell responses and protective immunity against Listeria mono-cytogenes. J Immunol 155:4817-4828

30. Mahon BP, Ryan MS, Griffin F, Mills KHG (1996) Interleukin-12 is produced by macrophages in response to live or killedBordetella pertussis and enhances the efficacy of an acellular

P. Scott and G. Trinchieri

290

Pertussis vaccine by promoting induction of Th1 cells. InfectImmun 64:5295-5301

31. Wynn TA, Jankovic D, Hieny S, Cheever AW, Sher A (1995)IL-12 enhances vaccine-induced immunity to Schistosoma man-soni in mice and decreases T helper 2 cytokine expression, IgEproduction and tissue eosinophilia. J Immunol 154:4701-4709

32. Schijns VE, Haagmans BL, Horzinek MC (1995) IL-12 stim-ulates an antiviral type 1 cytokine response but lacks adjuvantactivity in IFN-gamma-receptor deficient mice. J Immunol155:2525-2532

33. Tang YW, Graham BS (1995) Interleukin-12 treatment duringimmunization elicits a T helper type-1 like response in micechallenged with respiratory syncytial virus and improves vaccineimmunogenicity. J Infect Dis 172:734-738

34. O’Toole M, Wooters J, Brown E, Swiniarski H, Cull G, Leger L,Herrmann S (1996) Interleukin-12 as an adjuvant in peptidevaccines. Ann NY Acad Sci 795:379-381

35. Bliss J, Maylor R, Stokes K, Murray KS, Ketchum MA, Wolf SF(1996) Interleukin-12 as vaccine adjuvant: Characteristics ofprimary, recall, and long-term resistance. Ann NY Acad Sci795:26-35

36. McKnight AJ, Zimmer GJ, Fogelman I, Wolf SF, Abbas AK(1994) Effects of IL-12 on helper T cell-dependent immuneresponses in vivo. J Immunol 152:2172-2179

37. Bliss J, VanCleave V, Murray K, Wiencis A, Ketchum M, MaylorR, Haire T, Resmini C, Abbas AK, Wolf S (1996) Interleukin-12as an adjuvant, promotes a T helper 1 but does not suppress aT helper 2 recall response. J Immunol 156:887-894

38. Germann T, Bongartz M, Dlugonska H, Hess H, Schmitt E,Kolbe L, Kolsch E, Podlaski FJ, Gately MK, Rude E (1995)Interleukin-12 profoundly up-regulates the synthesis of antigen-specific complement fixing IgG2a, IgG2b, IgG3 antibodysubclasses in vivo. Eur J Immunol 25:823-829

39. Murphy EE, Terres G, Macatonia SE, Hsieh C-S, Mattson J,Lanier L, Wysocka M, Trinchieri G, Murphy K, O’Garra A(1994) B7 and interleukin 12 cooperate for proliferation and

interferon γ production by mouse T helper clones that areunresponsive to B7 costimulation. J Exp Med 180:223-231

40. Kubin M, Kamoun M, Trinchieri G (1994) Interleukin 12synergizes with B7/CD28 interaction in inducing efficientproliferation and cytokine production of human T cells. J ExpMed 180:211-222

41. Coughlin CM, Wysocka M, Kurzawa HL, Lee WM, Trinchieri G,Eck SL (1995) B7-1 and interleukin 12 synergistically induceeffective antitumor immunity. Cancer Res 55:4980-4987

42. Zitvogel L, Robbins PD, Storkus WJ, Clarke MR, Maeurer MJ,Campbell RL, Davis CG, Tahara H, Schreiber RD, Lotze MT(1996) Interleukin-12 and B7.1 co-stimulation cooperate in theinduction of effective antitumor immunity and therapy ofestablished tumors. Eur J Immunol 26:1335-1341

43. Roa JB, Chamberlain RS, Bronte V, Carroll MW, Irvine KR,Moss B, Rosenberg SA, Restifo NP (1996) IL-12 is an effectiveadjuvant to recombinant vaccinia virus-based tumor vaccines:enhancement by simultaneous B7-1 expression. J Immunol156:3357-3365

44. Nabors GS, Afonso LCC, Farrell JP, Scott P (1995) Switch froma type 2 to a type 1 T helper cell response and cure ofestablished Leishmania major infection in mice is induced bycombined therapy with interleukin-12 and pentostam. ProcNatl Acad Sci USA 92:3142-3146

45. Wynn TA, Eltoum I, Oswald I, Cheever A, Sher A (1994)Endogenous interleukin 12 (IL-12) regulates granuloma forma-tion induced by eggs of Schistosoma mansoni and exogenousIL-12 both inhibit and prophylactically immunizes against eggpathology. J Exp Med 179:1551-1561

46. Wynn TA, Cheever AW, Jankovic D, Poindexter RW, Caspar P,Lewis FA, Sher A (1995) An IL-12 based vaccination method forpreventing fibrosis induced by schistosome infection. Nature376:594-596

47. Kim JJ, Ayyavoo V, Bagarazzi ML, Chattergoon MA, Dang K,Wang B, Boyer JD, Weiner DB (1997) In vivo engineering of acellular immune response by coadministration of IL-12 expres-sion vector with a DNA immunogen. J Immunol 158:816-826

IL-12 as an adjuvant

291