Proc. Nati. Acad. Sci. USAVol. 89, pp. 8308-8312, September 1992Immunology

Induction of antigen-specific class I-restricted cytotoxic T cells bysoluble proteins in vivoSYAMAL RAYCHAUDHURI*t, MICHELLE TONKS*, FRANK CARBONEt, THOMAS RYSKAMP*,W. JOHN W. MORROW*, AND NABIL HANNA**IDEC Pharmaceuticals Corporation, 11099 North Torrey Pines Road, La Jolla, CA 92037; and tDepartment of Pathology and Immunology, MonashUniversity, Alfred Hospital, Prahran, Victoria 3181, Australia

Communicated by Vernon R. Young, May 14, 1992

ABSTRACT Cytotoxic T lymphocytes (CTL) are inducedspecifically against viral and tumor antigens presented bymajor histocompatibility complex class I molecules on thesurface of infected or transformed cells. Intracellularly syn-thesized antigens are processed and associated with class Iantigens within cells before presentation on the cell surface.Because of this special requirement for CTL induction, exog-enous soluble antigens do not, in general, induce specific CTLresponses. To overcome this problem, various laboratorieshave resorted to the use of vaccinia virus and other replicatingexpression vectors for intracellular antigen delivery leading tothe stimulation of humoral and cell-mediated immunity tospecific proteins. However, for human use it is safer to usepurified and defined antigens for inducing immune responses.Using soluble ovalbumin and human immunodeficiency virusglycoprotein gpl20, we have explored the possibility ofusing anantigen formulation consisting of squalane and Tween 80 toelicit antigen-specific CTL responses in mice. We have dem-onstrated that this antigen formulation is a potent inducer ofCD8+, class I-restricted, antigen-specific CTLs. The CTLpriming induced by soluble antigen in squalane/Tween 80resembles the reported response to the vaccinia recombinantcontaining human immunodeficiency virus envelope proteinand by splenocytes cytoplasmically loaded with soluble oval-bumin. The ramifications of these findings for vaccine devel-opment are discussed.

Cytotoxic T lymphocytes (CTL; CD81) have been demon-strated to be the major host defense mechanism in responseto a variety of viral infections (1-11). These effector cellseliminate infected or transformed cells by recognizing anti-genic peptides in association with class I molecules on thesurface of these cells. A considerable body of evidencesuggests that class I and class II presentation relies on theconsequences of intracellular processing events (12-15). Ex-ogenous, nonreplicating agents and soluble antigens enter theendosomal pathway and are presented at the cell surface inassociation with class II molecules, activating CD4+ T cells.Alternatively, endogenously synthesized antigens (e.g., viralproteins) that are synthesized in infected cells enter througha different pathway. In this latter case, protein fragmentsassociate with class I molecules, and this complex activatesCD8+ CTLs. Experimental evidence has shown that cyto-plasmic loading of certain soluble proteins can induce anti-gen-specific CTLs, whereas immunization with soluble an-tigens alone was ineffective for inducing such responses(16-18). Therefore, several groups have resorted to the useof live expression vectors such as vaccinia (19), Salmonella(20), bacillus Calmette-Gudrin (BCG), and other systems (3,21, 22) for presenting foreign antigenic determinants to theimmune system. Live recombinant vaccine approaches,

however, may pose safety risks, especially in immunocom-promised individuals (23).

In this report, we describe a noninfectious, chemicallydefined formulation that, when injected in combination withsoluble antigens, induces CD8+ class I-restricted CTL re-sponses in two different protein antigen systems-namely,soluble ovalbumin (Ova) and recombinant human immuno-deficiency virus (HIV) gp120.

MATERIALS AND METHODSMice. Female C57BL/6 (H-2b) and BALB/c (H-2d) mice

were purchased from Harlan-Sprague-Dawley.Antigens. Ova (grade VII) was purchased from Sigma and

used in the native form. The gp120-IIIb was purchased fromAmerican Biotechnology (Cambridge, MA).Tumors and Transfectants. The tumor cells used were the

la- lines EL4 (C57BL/6, H-2b thymoma) and NIH 3T3 cells.Derivation of the Ova-producing EL4 transfectant, EG7-Ova, has been described (18). The gpl60-expressing trans-fectant, 15-12, has been described (24). The L cells express-ing Ld or DW were provided by T. Hensen (WashingtonUniversity, St. Louis).

Preparation of Antigen Formulation. The componentssqualane (Aldrich), Tween 80 (Aldrich), and pluronic L121(BASF, Parsippany, NJ) were dissolved in phosphate-buffered saline, pH 7.4, to form a crude oil-in-water emulsioncomprising 15% (wt/vol) squalane and 0.6% Tween 80 withor without pluronic L121 at a concentration of0.0045-3.75%.The crude emulsion was then cycled through a MilOTmicrofluidizer (Microfluidics, Newton, MA) six times at 40Cto obtain a stable homogeneous emulsion with a meanparticle size ranging from 250 to 300 nm. The resultantemulsion was sterile-filtered by using a 0.22-pm filter andstored under nitrogen before use. The emulsion was testedfor endotoxin by the Limulus amebocyte-lysate gel-clot as-say, and the particle size was determined by laser-diffractionparticle-size analysis.

Immunizations. Mice were immunized i.v. with a 200-.lIsuspension of 25 x 106 splenocytes after a cytoplasmicloading, as described (16, 17). For Ova-ST, 30 ,gg ofOva wasused per inoculation, whereas for gpl20, 1 jig was used.Before immunization, the triple-strength ST was diluted bymixing with an appropriate amount of soluble antigen to afinal 200-ulI vol and mixed for 30 sec. This process formed astable emulsion of soluble antigen in ST emulsion. The finalvolume was made up with Hanks' balanced salt solution

Abbreviations: Ova, chicken egg ovalbumin; ST, antigen formulationcontaining squalane and Tween 80; CTL, cytotoxic T lymphocyte(s);HIV, human immunodeficiency virus; mAb, monoclonal antibody;CFA, complete Freund's adjuvant; APC, antigen-presenting cell;MBP, myelin basic protein; SAFm, Syntex adjuvant formulation ofsqualane, pluronic L121, Tween 80, and muramyl dipeptide; HBSS,Hanks' balanced salt solution; E:T, effector-to-target cell ratio.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(HBSS). As a control, various adjuvants that are commer-cially available were used as indicated below. The SAFm, orSyntex adjuvant formulation (25) consisting of squalane,pluronic L121, Tween 80, and muramyl dipeptide, was mixedwith soluble antigens in a total volume of 200 1.d and injecteds.c. and into the tail base. Similarly, antigens were mixedwith alum (Pierce) or complete Freund's adjuvant (CFA).In Vitro Stimulation of CTL. Spleen cells (30 x 106) from

normal or immunized mice primed at least 14 days earlierwere incubated with 1.5 x 106 irradiated (20,000 rads; 1 rad= 0.01 Gy) EG7-Ova cells for Ova responses or 1.5 x 10615-12 cells treated with mitomycin C for gpl20 response in24-well plates at 370C in 7% C02/air. All tissue cultures wereperformed in a complete medium consisting of Iscove'smodified Dulbecco's medium supplemented with 10% fetalcalf serum, 2 mM glutamine, gentamycin sulfate, and 2 x 105M 2-mercaptoethanol. In some experiments, gpl20-immunized spleen cells were stimulated with a syntheticpeptide corresponding to the immunodominant CTL epitope(18IIIb)(RIQRGPGRAFVTIGK) from the V3 region ofgpl20(24).In Vitro Depletion ofLymphocyte Subpopulations. For the in

vitro depletion experiments, in vivo-primed or in vitro-stimulated spleen cells were treated with monoclonal anti-body (mAb) RL.172 (anti-CD4) or mAb 3.168 (anti-CD8) andcomplement for removal of CD4+ or CD8+ T cells. Theeffectiveness of mAb and complement-mediated depletionwas measured flow cytometrically by using fluorescein iso-thiocyanate-conjugated anti-CD4 or anti-CD8 antibodiesfrom Pharmingen (San Diego). Our method routinely yieldedcells that are >95% enriched for CD4+ or CD8+ cells. ThemAbs RL.172 and mAb 3.168 were from J. Sprent (ScrippsClinic and Research Foundation).

Cytotoxicity Assay. Target cells (1 x 106) were labeled with100 ACi (1 Ci = 37 GBq) of 51Cr for 60 min. For peptidesensitization of target cells, 50 ,ug of the desired peptides andradioactive sodium chromate were incubated with 1 X 106target cells for 60 min, as described (18). After being washed,104 labeled target cells and serial dilutions of effector cellswere incubated in 200 ,ul of RPMI medium/10% fetal calfserum/2 mM glutamine/gentamycin sulfate/2 X 10-5 M2-mercaptoethanol for 4 hr at 37°C. One hundred microlitersof supernatant was collected, and the specific lysis wasdetermined as follows: percentage specific lysis = 100 X[(release by CTL - spontaneous release)/(maximal release -spontaneous release)]. Spontaneous release in the absence ofCTL was <25% of maximal release by detergent in allexperiments.

Peptide Synthesis and Purification. Synthetic peptides cor-responding to amino acid sequences 253-276 (EQLESIIN-FEKLEWTSSNVMEER) of the Ova [Ova-(253-276)], aminoacid sequences 84-102 of the myelin basic protein [MBP-(84-102)] (DENPVVHFFKNIVTPRTPP), and amino acid se-quences 308-322 of gpl60 (1811IB) (RIQRGPGRAFVTIGK)were assembled by solid-phase peptide synthesis with anApplied Biosystems 430A synthesizer. The peptides weresynthesized as C-terminal amides with p-methylbenzhydry-lamine resin. Purified peptides were solubilized in HBSS at afinal concentration of 10 mg/ml and diluted to the desiredconcentration in the complete medium.

RESULTSIn the course of examining several adjuvant systems toinduce active immunity we observed that soluble antigensformulated in squalane/Tween 80 (ST) with or without plu-ronic L121 could induce class I-restricted CTL as well ashumoral responses in experimental animals. The componentsof ST have been used previously as part of the vehicle for themuramyl dipeptide-containing adjuvant SAFm (25). After

two to three immunizations, both Ova and gpl20 in STinduced antibody responses comparable with that induced bySAFm or CFA. In this study, we will report the classI-restricted CTL priming in mice induced by soluble antigensin ST after a single immunization. Because the murineOva-CTL system is well characterized (16-18), we first usedit as a model in this study before conducting experiments withHIV gp120.Ova-ST Induces Class I-Restricted CTL Responses in Mice.

C57BL/6 mice were inoculated once with 30 pg of Ova withor without ST. To generate effector cells, spleen cells weretaken from the immunized mice and stimulated in vitro withthe EG7-Ova transfectants. After in vitro culture with EG7-Ova, priming was assessed by the presence of Ova-specificeffectors capable of lysing EG7-Ova. Mice immunized with30 pug of Ova in ST showed an excellent CTL responsespecific for EG7-Ova (Fig. 1). The extent ofEG7-Ova killingby spleen cells from Ova-ST-immunized mice was compa-rable to that of mice immunized with cytoplasmically loadedspleen cells. Mice injected with soluble Ova in HBSS or Ovaadsorbed on alum showed no evidence of CTL priming (Fig.1). CTL priming was also absent in mice immunized with Ovain SAFm (Fig. 1).To determine whether soluble Ova in ST induced a CTL

response specific to the peptide Ova-(253-276) as that in-duced by transfectant cells or by Ova-cytoplasmically loadedsplenocytes (16, 18), spleen cells were prepared from immu-nized mice and stimulated in vitro with irradiated EG7-Ovacells. The effector cells were tested against EL4 cells coatedwith the Ova-(253-276) peptide or with a control peptidederived from myelin basic protein [MBP-(84-102)]. Table 1demonstrates that Ova-ST-primed effector cells could effec-tively lyse untransfected EL4 cells coated with Ova-(253-276) peptide but could not lyse EL4 cells coated with MBP-(84-102) peptide.

Because, in some situations, soluble antigens can induceCD4+CD8- class II-restricted CTL responses (26, 27), weinvestigated whether soluble Ova in ST induced CD8+ ef-fector T cells. Splenocytes from Ova-immunized mice werecultured for 5 days with irradiated transfectants in vitro. Cells

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FIG. 1. In vivo induction of CTL by Ova-ST. C57BL/6 micewere immunized once with 30 Ag of Ova in HBSS (A), alum (-),SAFm (o), or ST (o). As a positive control, mice were immunizedwith spleen cells cytoplasmically loaded with soluble Ova (A). Twoweeks after immunization, 30 x 106 spleen cells were mixed withirradiated (20,000 rads) EG7-Ova cells at a ratio of 20:1 in Iscove'smodified Dulbecco's medium/10%o fetal calf serum and distributed insix wells of a 24-well plate. The resulting effector cells were thentested for lysis of 51Cr-labeled EG7-Ova or EL4 cells. Only thecytotoxicity against 51Cr-labeled EG7-Ova is shown. Approximately6% cytotoxicity against 51Cr-labeled EL4 was seen at an effector-to-target cell ratio (E:T) of 33:1 (data not shown).

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Table 1. Recognition of synthetic peptide byOva-ST-induced CTL

Specific cytotoxicityof target cells, %EL4 + EL4 +

Immunization E:T EL4 Ova-(253-276) MBP484-102)Ova-HBSS 11:1 0 5 5

3:1 0 5 0Ova cytoplasmically

loaded 11:1 0 38 03:1 0 21 0

Ova-ST 11:1 0 59 23:1 0 40 2

Recognition of synthetic peptide by Ova-ST-induced CTL. Spleencells from C57BL/6 mice immunized with 30 ug of Ova-HBSS, 30jg of Ova-ST, or 25 x 106 syngeneic spleen cells loaded with ahypertonic solution ofOva at 10 mg/ml were stimulated in vitro withEG7-Ova. After 5 days of culture, effector cells were tested against51Cr-labeled EL4 or 51Cr-labeled EL4 sensitized with Ova2ssv276 or51Cr-labeled EL4 sensitized with MBP.

were then harvested and depleted of CD4+ or CD8+ T cellsand then tested against 51Cr-labeled transfectants. Fig. 2ademonstrates that in the Ova system, depletion of CD8+ Tcells abrogated cytolytic activity conferred by the wholeeffector-cell population. However, depletion of the CD4+T-cell population had no effect on lysis of EG7-Ova. Exper-iments were then done to map the major histocompatibilitycomplex restriction of the Ova-specific CTL with H-2Kb-transfected L cells. Table 2 shows that Ova-ST-inducedCTLs recognize the peptide Ova-(253-276) in the context ofmajor histocompatibility complex Kb. This finding is consis-tent with the specificity of CTL induced by Ova-transfectedcells or spleen cells cytoplasmically loaded with Ova (16, 18).HIV gpl2UIMb-ST Induces Class I-Restricted CTL Re-

sponses in Mice. The successful induction of Ova-specificCTL led to testing the applicability of ST to the induction ofCTL against a clinically relevant system involving solubleHIV-gpl20 antigen. To demonstrate the induction ofantigen-specific CTL responses in mice with gpl20-ST, we used asynthetic peptide (18111b), which corresponds to an immu-nodominant CTL epitope (RIQRGPGRAFVTIGK) from theV3 region of gpl20 for in vitro stimulation (28). Our results(Fig. 3) show that 1 gg of HIV gpl20-ST consistently givesan efficient CTL priming in vivo, as indicated by the abilityof the gpl20-ST-primed and in vitro peptide 181IIb-stimulated spleen cells to kill 15-12 cells (NIH 3T3 fibroblastsexpressing the HIV gpl60IIIb gene product). The samespleen cells can also kill P815 cells infected with recombinant

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Table 2. Ova-ST-induced CTL are H-2Kb restricted5lCr-labeled target Peptide Specific lysis,* %

L cells - 11L cells + 0L/Kb - 7L/Kb + 61

CTL effectors were generated by stimulat Ova-ST-primedspleen cells with irradiated EG7-Ova in vitro. After 5 days ofculture,effector cells were prepared and tested against Ova-253-276)-coatedL cells or L cells transfected with the H-2Kb gene. No cytotoxicitywas observed against MBP(84-102)-coated L/Kb.*E:T used was 17:1.

vaccinia-gpl60IIIb (data not shown). Specificity of the CTLresponse was further confirmed by the ability of the sameeffector cells to lyse NIH 3T3 fibroblast target cells coatedwith the 18EI1b peptide but not those cells coated with theMBP-derived peptide (Table 3). In contrast, similarly stim-ulated spleen cells from the soluble gpl20 emulsified in CFAor in SAFm showed no evidence ofCTL priming (Fig. 3). Fig.2b shows that, as with the Ova system, the gpl20-ST-induced CTL response is also mediated by CD8+ T cells.BecauseNIH 3T3 cells do not express a class II gene product,it can be concluded that the kiling induced by gpl20 immu-nization and effected by CD8+ T cells is class I-restricted.-Further major histocompatibility complex mapping studieswith gp120-ST-induced CTLs were done with L cells trans-fected with Ld or Dd. We observed that the CTLs areDd-restricted similar to those induced by vaccinia-gpl60recombinant (data not shown; ref. 28). Finally, the specificityofCTL priming in vivo was further shown by the inability ofspleen cells from gp12O-immunized mice to manifest a sec-ondary Ova-specific CTL response after in vitro culture withEG7-Ova. No cytotoxicity against 5'Cr-labeled EG7-Ovawas seen (data not shown).

Soluble Antigens in ST Primes Dirtl CD@+ Cells in Vivo.It might be argued that antigen-ST did not lead to the primingof class I-restricted precursor cells in vivo but mediated itseffect through the activation of class II-restricted helper Tcells, which, in turn, activated antigen-specific CTLs in invitro culture. To rule out this possibility, we determined, inthe Ova system, whether Ova-ST primed CD8+ T-cell pop-ulations in vivo and whether these cells were critical for an invitro secondary response. Thus, we depleted the CD4+ orCD8+ populations from spleens of Ova-ST-immunized miceand from naive mice before in vitro stimulation. These treatedspleen cell populations were then stimulated in vitro withirradiated EG7-Ova alone or in combination with CD4+ andCD8+ T cells from Ova-ST-immunized mice or in various

33:1 11:1 3:1 1:1E:T

FIG. 2. Ova-ST (a) or HIV gpl20-ST (b)-primed effector cells are CD8+ cells. In vivo-primed spleen cells (30 x 106) were stimulated in vitrowith 1.5 x 106 irradiated EG7-Ova cells (20,000 rads) or with 170 pg of gp120-derived 18ffb peptide, as described (28). After 5 days of culture,the effector cells were depleted ofCD4+ (r) or CD8+ (n) cells by specific mAb (RL172 or 3.168) and complement. Specific lysis was tested against51Cr-labeled EG7-Ova (a) or against 51Cr-labeled 15-12 cells (b). No lysis was seen against EL4 or NIH 3T3 cells (data not shown).

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FIG. 3. In vivo induction of HIV-1 envelope-specific CTL byrecombinant gpl20-ST. BALB/c mice were immunized with 1 Ag ofgpl20 in ST (o), CFA (m), or SAFm (o). Three weeks later, spleencells from gpl20-immunized mice were stimulated in vitro with the181B (gpl20Ib-derived) synthetic peptide for 5 days. Thereafter,resultant effector cells generated from gp120-ST-immunized micewere tested against the 15-12 transfectant or nontransfected NIH 3T3cells. The specific cytotoxicity against the 51Cr-labeled 15-12 cells is-shown. This percentage is calculated after subtracting the percentagekill against 51Cr-labeled 3T3 cells, which is usually 10-15% at the E:Tshown.

combinations of CD4+ or CD8+ T cells from naive mice. Fig.4 shows that in vivo-primed CD8+ cells are essential for themanifestation ofa secondary CTL response in vitro, whereasCD4+ T cells are required but do not need to be primed.Similarly, we have observed that CD8+ T cells are requiredfor the manifestation of gp120-specific secondary CTL re-sponses in vitro (data not shown).

DISCUSSIONUse ofwhole soluble antigens as immunogens to induce CTLresponses is an attractive and a safe approach for the devel-opment of vaccines and immunotherapeutics. The wholeantigens will contain all the potential CTL and T helperepitopes and may overcome the influence of immune re-sponse genes. This concept is particularly important in anoutbred situation, where the immune response to an antigenis heterogeneous with respect to HLA restriction and epitopespecificity and is directed at multiple epitopes (29). However,the approach to CTL induction by soluble antigen has notbeen encouraged due to the intrinsic difficulty in antigenpresentation via the class I pathway. Our work demonstratesthe induction of CD8+, class I-restricted, CTL responsesagainst soluble protein antigens. The CTL priming inducedby antigen-ST resembles the activity induced by transfec-

Table 3. gpl20-induced CTL recognize 18Illb peptide

Specific cytotoxicityof target cells, %

NIH 3T3 + NIH 3T3 +Immunization* E:T 18111b MBP-(84-102)

gpl2O-ST 33:1 59 2.911:1 57 03:1 29 0

*BALB/c mice were immunized once with 1 pg ofgpl20 in ST. Threeweeks after immunization, spleen cells were stimulated in vitro withgp120-derived 18Ilb peptide for 5 days, as described in Fig. 3legend. Thereafter, the resultant effector cells were tested againstNIH 3T3 cells sensitized with the 181Mb peptide containing thedominant CTL epitope or with control MBP484-102) peptide.

Q

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20

100:1 33:1E:T

11:1 3:1

FIG. 4. Ova-ST primes CD8+ T cells in vivo. Ova-ST-immunizedand unprimed spleen cells were either depleted of CD4+ cells orCD8+ cells with CD4-specific mAb or CD8-specific mAb pluscomplement. Mixtures of equal numbers of CD4+ cells from primedand CD8+ cells from unprimed (A), or equal numbers ofprimed CD8+cells and unprimed CD4+ cells (o), or primed CD4+ cells and primedCD8+ cells (n), or primed CD8+ cells alone (e), or primed CD4 cellsalone (o) were stimulated in vitro with irradiated EG7-Ova for 5 daysusing complete Iscove's modified Dulbecco's medium. The resultingeffector cells were tested against 5lCr-labeled EG7-Ova or EL4starting at an E:T of 100:1. No cytotoxicity was seen with 51Cr-labeled EL4 cells (data not shown).

tants and by splenocytes cytoplasmically loaded with solubleOva and by recombinant vaccinia containing HIV gp120. Inthe Ova system, EG7-Ova, cytoplasmically loaded Ovasplenocytes, and Ova-ST all induced (i) class I-restricted,antigen-specific CD8+ CTL; (ii) CTL that recognized targetssensitized with the Ova-(253-276) synthetic peptide; and (iii)a long-lived CTL response, lasting at least 4 months after onlyone immunization (data not shown). The gp120-ST-inducedCTL recognize the env-expressing cell line, 15-12, and un-transfected NIH 3T3 cells coated with an env-derived 181IIbsynthetic peptide but not the NIH 3T3 cells alone, or NIH3T3 cells coated with irrelevant peptide. These findings areanalogous to those seen with CTL primed by immunizationwith recombinant vaccinia-gp160IIIB virus (24, 28).

It could be argued that the commercially available antigenscould contain peptide fragments that might induce CTLdirectly. However, the following experiments indicated thatthe antigens we used do not contain degraded fragments: (i)both Ova and gpl20 were pure as judged by SDS/PAGE andimmunoblots, and (ii) in a functional assay, we sensitizedEL4 cells or NIH 3T3 cells with soluble antigens (as much as1 mg per 1 x 106 cells for soluble Ova) and tested as targetsagainst Ova or gp120-specific CTL lines we had generated.No significant killing was seen, suggesting that the antigensdo not contain functional peptides in sufficient quantity forthe observed immunization effect with whole antigens in ST.It should be mentioned that 0.01% contamination with pep-tide (10 ,ug of peptide in 1 mg of Ova solution) would beexpected to produce significant killing of Ova-coated targetswith an Ova-specific CTL line.The mechanism by which the ST can drive an exogenous

antigen into a processing pathway required for class I-re-stricted CTL response remains to be elucidated. It is possiblethat ST may help deliver the antigen into the cytoplasm and,therefore, the endogenous pathway. Alternatively, solubleantigens in ST may be taken up effectively by antigen-presenting cells (APC) in vivo, and the resulting peptides mayenter into the class I pathway (30). At this time, the nature ofthe APC involved in exogenous soluble antigen-mediatedclass I-restricted CTL priming is not known. In a recent

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study, Rock et al. (31) demonstrated the existence of spe-cialized APCs that express class II molecules and simulta-neously present antigen in class I- and class II-restrictedpathways. It is conceivable that, in our system, solubleantigen in ST could effectively be taken up by specializedAPCs and the antigen presented in the context of both classI and II molecules, as evidenced by the induction ofboth CTLand humoral (data not shown) responses. The nature of theAPC involved in soluble antigen-ST-induced CTL priming invivo needs further definition.The ramifications of our work on the development of safe

and effective vaccines and immunotherapies are potentiallyimportant. Recently it has been shown that soluble gpl60 andinfluenza hemagglutinin can prime for CTL response wheninjected in immunostimulating complexes (32). Because im-munostimulating complexes contain toxic saponin compo-nents, its use in humans is probably not feasible. In anotherstudy, Deres et al. (33) showed in vivo priming of virus-specific CTL induction with a synthetic peptide conjugated toa lipid "tail." Whether the same lipid moiety conjugated toa native protein antigen can induce class I-restricted CTLresponses is unclear. In conclusion, this report demonstratesthat soluble antigens in a chemically defined formulation caninduce class I-restricted CTL and humoral responses in mice.Moreover, the components of ST appear to be safe as the STcontaining pluronic copolymers has been administered inhuman and nonhuman primates as part of our internal-imageactive immunotherapy program (34, 35).

We thank Drs. W. Rastetter and D. Talmage for many insightfulideas and critical review of the manuscript, Drs. R. Germain and J.Berzofsky for the 15-12 transfectant, Drs. S. Gowda and E. Engel-man for the vaccinia-HIVgpl6OIIIb vector, and Marijo Hostetler forpreparing the manuscript. This work was supported by the New YorkLife Insurance Co.

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