The Journal of Immunology

ISCOMATRIX Adjuvant Combines Immune Activation with Antigen Delivery to Dendritic Cells In Vivo Leading to Effective Cross-Priming of CD8+ T Cells

Peter Duewell,*,1 Ulrich Kisser,*,1 Klaus Heckelsmiller,* Sabine Hoves,* Patrizia Stoitzner,† Sandra Koernig,‡ Adriana B. Morelli,‡ Bjo¨rn E. Clausen,x Marc Dauer,{ Andreas Eigler,‖ David Anz,# Carole Bourquin,# Eugene Maraskovsky,‡ Stefan Endres,# and Max Schnurr*

Cancer aim to induce CTL responses against tumors. Challenges for design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous in- jection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-g production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8a+ DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8a+ DCs in VDLNs, whereas Langerin+ DCs and CD8a2 DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8a+ DCs in vivo for efficient induction of CTL responses. The Journal of Immunology, 2011, 187: 55–63.

umor vaccines seek to induce a CTL response against in vitro-generated DCs loaded with tumor Ag as an individualized tumors. To achieve efficient tumor cell killing, different tumor vaccine (1, 2). As DC activation has been recognized to be strategies have been evaluated for inducing both CD4+ Th of critical importance for the induction of productive immune T + cell and CD8 T cell responses against tumor Ags. Attention has responses, DC maturation was included in protocols focused on exploiting dendritic cells (DCs) as professional APCs (3, 4). However, the production of DC vaccines is labor- and cost- capable of presenting exogenous Ag not only on MHC class II, but intensive, as cytapheresis may be required and vaccines have to also on MHC class I, a process termed cross-presentation. Because be manufactured individually for each patient. A promising strategy of feasibility issues, most vaccination protocols have relied on circumventing the need of ex vivo DC generation and manipulation is targeting vaccines directly to DCs in vivo (5, 6). Moreover, the development of cell-free vaccines for off-the-shelf use will make *Medizinische Klinik Innenstadt, Ludwig-Maximilians-Universita¨t, 80336 Munich, vaccines accessible to more patients and reduce manufacturing costs Germany; †Department of Dermatology, Innsbruck Medical University, 6020 Inns- x (7). Challenges for the design of effective cancer vaccines are DC- bruck, Austria; ‡CSL Ltd., Parkville, Victoria 3052, Australia; Department of Im- munology, Erasmus Medical Center, University Medical Center, 3015 GE Rotterdam, specific Ag targeting, facilitating MHC class I epitope processing, The Netherlands; {Department of Medicine II, Saarland University Hospital, 66421 ‖ and identifying adjuvants that activate DCs in vivo for generating Homburg, Germany; Klinik fu¨r Innere Medizin I, Klinikum Dritter Orden, 80638 Munich, Germany; and #Division of Clinical Pharmacology, Ludwig-Maximilians and activating effector T cells as well as innate effector cells. University, 80336 Munich, Germany An important parameter for vaccine design is the choice of tumor 1P.D. and U.K. contributed equally to this work. Ag and its physical properties (i.e., peptide, protein, DNA or RNA). Received for publication December 20, 2010. Accepted for publication April 20, Most clinical trials have evaluated synthetic MHC class I- and/or 2011. MHC class II-restricted peptides derived from tumor Ags. A This work was supported by grants from the Deutsche Krebshilfe (to M.S.), Deutsche drawback of using preformed peptides is restricting treatment to Forschungsgemeinschaft (SCHN 664/3-1 to M.S. and GK1202 to M.S., C.B., D.A., patients with a limited number of MHC haplotypes. This limitation and S.E.), BayImmunet (to C.B. and S.E.), and Austrian Science Fund (P-214780 to P.S.). B.E.C. is a VIDI Fellow of the Netherlands Organization for Scientific Re- can be circumvented by using full-length proteins, which 1) contain search (917-76-365). This work is part of the doctoral thesis of U.K. at the University multiple MHC class I and MHC class II epitopes, 2) can be used of Munich. without knowledge of the patient’s MHC haplotype, and 3) are Address correspondence and reprint requests to Dr. Max Schnurr, Medizinische presented for prolonged periods on MHC molecules as compared Klinik Innenstadt, University of Munich, Ziemssenstrasse 1, 80336 Munich, Ger- many. E-mail address: [email protected] with peptides (8). A shortcoming may be that DCs cross-present The online version of this article contains supplemental material. soluble protein inefficiently on MHC class I. However, this limita- tion can be circumvented by using Ab–Ag conjugates that target DC Abbreviations used in this article: CpG-ODN1826, CpG-containing oligodeoxynucleo- tide 1826; DC, dendritic cell; DT, diphtheria toxin; DTR, diphtheria toxin receptor; surface receptors involved in Ag uptake, such as Fc receptors (8– ICS, intracellular cytokine staining; ISCOM, Ag formulated with ISCOMATRIX 11), members of the C-type lectin receptor family including CD205, adjuvant; LN, lymph node; VDLN, vaccine site-draining lymph node. the mannose receptor, and DC-specific intracellular adhesion mol- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ecule 3-grabbing nonintegrin (12–14). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1004114 56 IMMUNE ACTIVATION AND CROSS-PRESENTATION OF ISCOM VACCINE

Alternatively, Ag can be formulated with chemically defined Transgenic OT-1 mice were provided by Prof. T. Brocker (Department of delivery systems, such as ISCOMATRIX adjuvant, which is de- Immunology, University of Munich, Munich, Germany). Animal experi- rived from the immunostimulating complex (ISCOM) first de- ments were approved by the local regulatory agencies. For vaccination, mice were injected s.c. into the lower hind leg with 50– scribed by Morein et al. (15). They observed enhanced immune 100 ml PBS containing either OVA (30 mg) with or without adjuvants, responses to protein formulated with a mixture of , ISCOMATRIX adjuvant (5 mg), or OVA/ISCOM vaccine (containing 0.3 phospholipids, and cholesterol that forms cage-like structures mg OVA/5 mg ISCOM). Vaccination was repeated on day 7 (prime-boost (15). ISCOMATRIX vaccines that contain only a purified fraction regimen). of quillaia saponin were shown to be safe, well tolerated, and LN preparation highly immunogenic in humans, generating Ab and T cell re- sponses (16). An ISCOM vaccine has the Ag incorporated into the Mice were anesthetized with 1-chlor-2,2,2-trifluorethyl-difluormethyl ether structure during manufacture, whereas an ISCOMATRIX vaccine (Forene; Abbott, Wiesbaden, Germany) and killed by cervical dislocation. The VDLNs were removed and processed into single-cell suspensions by is made by mixing Ag with preformed ISCOMATRIX adjuvant. passing through a 70-mm cell strainer. Adjusted cell numbers were pro- Both entities are otherwise identical. Interestingly, ISCOM vac- cessed for phenotypic and functional analysis. cines facilitate cross-presentation by DCs via translocation of Ag from endosomes into the cytosol (8, 17). Moreover, ISCOMA- mAbs and flow cytometry TRIX adjuvant-based tumor vaccines containing full-length tumor For flow cytometry, cell suspensions were incubated for 20 min at 4˚C with Ag-induced humoral and cellular immune responses in mouse the following Abs: CD3ε (clone 145-2C11), CD4 (clone GK1.5), CD8a models (18, 19) as well as in cancer patients (20). Taken together, (clone 53-6.7), CD11b (clone M1-70), CD11c (clone HL3), IL-12p40 these studies demonstrate the high clinical potential of ISCO- (clone C15.6), CD19 (clone 1D3), CD86 (clone GL1), NK1.1 (clone PK136; all from BD Biosciences, San Jose, CA); CD69 (clone H1.2F3) MATRIX adjuvant in tumor vaccines. and IFN-g (clone XMG1.2; both from Caltag Laboratories, Carlsbad, CA); Little is known about the immunological effects mediated by and Ly6G (clone RB6-8C5; eBioscience, San Diego, CA) and MHC class ISCOM and ISCOMATRIX vaccines on DCs and other leukocyte II (clone NIMR-4; SouthernBiotech, Birmingham, AL). Samples were populations in vivo. Understanding the mechanisms of action un- acquired on a FACSCalibur (BD Biosciences, Heidelberg, Germany) and derlying these vaccines will be important for optimal use in the analyzed using FlowJo version 7.2.1 (Tree Star, Ashland, OR). clinic and further optimization. In this study, we investigated the Intracellular cytokine staining and pentamer staining effects of ISCOMATRIX adjuvant and an ISCOM vaccine con- taining the model Ag OVA on cytokine milieu and on phenotype To assess cytokine expression by DCs, NK cells, and NKT cells, cell suspensions of VDLNs were incubated with 1 mg/ml brefeldin A (Fluka, as well as function of innate and adaptive immune effector cells Munich, Germany) for 4 h at 37˚C and subsequently stained for surface in vaccine site-draining lymph nodes (VDLNs). We further studied markers, fixed with 2% paraformaldehyde, incubated with mAb against IL- Ag uptake and presentation by distinct DC populations in VDLNs 12p40 or IFN-g in 0.5% saponin in PBS, and analyzed by flow cytometry. and analyzed the induction of cellular immune responses in wild- To measure OVA-specific CTL response, PBMCs of 100–200 ml peripheral type mice and mice in which distinct DC populations were depleted. blood were stimulated with SIINFEKL peptide (1 mg/ml) for 1 h at 37˚C before addition of 1 mg/ml brefeldin A. Three hours later IFN-g in- tracellular cytokine staining (ICS) was performed as described above. Alternatively, the number of OVA-specific CTLs was determined using Materials and Methods b Cell culture media and reagents SIINFEKL/H-2K pentamer (ProImmune, Oxford, U.K.) together with mAbs against CD8a, CD19, and NK1.1. Unspecific binding of pentamer by dead cells and CD19+ B cells/NK1.1+ NK cells was excluded by gating Primary cells were cultivated in RPMI 1640 media supplemented with heat- 2 2 2 inactivated 10% FCS (Life Technologies BRL, Paisley, U.K.), 2 mM L- on 7-aminoactinomycin D CD19 NK1.1 cells before analysis as rec- glutamine, penicillin (100 U/l), streptomycin (0.1 mg/ml), 100 mM non- ommended by the manufacturer’s protocol. essential amino acids, 1 mM sodium pyruvate (all PAA, Linz, Austria), and 50 mM 2-ME. The OVA/ISCOM vaccine was generated by associating Cytokine measurement palmitified OVA (Sigma-Aldrich, St. Louis, MO), which in some experi- ments was labeled with the fluorochrome Alexa 488, into the ISCOM VDLNs were shock frozen in liquid nitrogen, processed using mortar and structure by formulation in the presence of ISCOPREP saponin, phos- pestle, and lysed with 50 ml protein lysis buffer (Bio-Rad, Munich, Ger- 3 pholipid, and cholesterol, as previously described (17). ISCOMATRIX many). Samples were vortexed for 30 s, centrifuged for 15 min at 12,000 g adjuvant was made in the same way without the OVA. The H2-Kb– and 4˚C, and supernatant was used for cytokine analysis. Samples were adjusted to 2 mg protein/ml as determined by Bradford assay (Bio-Rad). restricted peptide OVA257–264 (SIINFEKL) was purchased from Jerini Peptide Technologies (Berlin, Germany). CpG-containing oligodeox- IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, TNF-a, IFN-g, and GM-CSF concentrations were determined by a Bio-Rad multiplex ynucleotide 1826 (CpG-ODN1826; InvivoGen, San Diego, CA) and Pam3- Cys (InvivoGen) were used at 5 mg, LPS (LPS 0111:B4; Chondrex, suspension array according to the manufacturer’s instructions. For Western Redmond, WA) at 1 mg, and aluminum hydroxide (Aluhydrogel; Superfos blot analysis, adjusted protein samples were separated by SDS-PAGE. IL- Biosector, Frederikssund, Denmark) at 100 mg per injection. IFA (Bacto/ 1b was detected using a primary goat anti-mouse IL-1b Ab (R&D Sys- Difco, Mount Pritchard, Australia) was used at a 50:50 dilution of the tems) followed by a secondary HRP-coupled anti-goat Ab (Santa Cruz stock solution. Biotechnology, Heidelberg, Germany). b-actin served as a loading control. IL-1b in supernatants of bone marrow-derived DCs was measured by Mice and ELISA (BD Biosciences). DCs (106 cells/ml) were treated with 200 ng/ml LPS for 2 h before addition of 10 mM caspase-1 inhibitor z-YVAD-fmk Female C57BL/6 mice, 6–8 wk old, were purchased from Harlan Win- (Calbiochem,Darmstadt,Germany)for1hfollowedby6hincubation kelmann (Borchen, Germany). Langerin-diphtheria toxin receptor (DTR)/ with 5 mg/ml ISCOMATRIX adjuvant. enhanced GFP knock-in mice on C57BL/6 background expressing a DTR and enhanced GFP under the Langerin promoter were provided by Dr. B.E. In vitro cross-presentation assay Clausen (21). For depletion of Langerin+ DCs we administered 500 ng DT (Sigma-Aldrich) 1 d prior to . A week later DT injection was Total DCs were enriched by Percoll density gradient centrifugation (1.077 repeated to keep Langerin+ DCs absent throughout the experiment. For g/cm3; Biochrom) after mechanical and enzymatic processing of spleens. depletion of CD11c+ DCs, transgenic mice expressing the DTR fused with Untouched CD8a+ DCs were obtained by combining the Ab mixture of the GFP under control of the CD11c promoter (CD11c-DTR mice) were used mouse DC isolation kit (Invitrogen) with biotinylated anti-CD4 (clone (22). Irradiated C57BL/6 mice were reconstituted with bone marrow from RM4-5) and anti-B220 (clone RA3-6B2; both from BioLegend). Finally, CD11c-DTR transgenic mice, rendering DTR expressing CD11c+ DCs cells were enriched by magnetic separation using streptavidin-coupled from hematopoietic origin sensitive to DT treatment. Reconstituted mice beads (Invitrogen). Purity was usually .90% as determined by flow were administered three doses of DT every 3 d from day 0. An 80% de- cytometry. CD8a+ DCs were incubated with the indicated amounts of OVA pletion of CD11c+ DCs was confirmed before the first immunization. or OVA/ISCOM vaccine for 90 min and washed four times in serum-free The Journal of Immunology 57 media. Finally, 2.5 3 104 DCs were used as stimulators for 5 3 104 mice. Tumor size was determined using a caliper, and mice were culled enriched CFSE-labeled OT-I T cells. Proliferation from viable CD8+ when the tumor size reached 200 mm2. T cells was determined after 3 d (23). Statistical analysis In vivo T cell proliferation assay A Student t test was applied to reveal significant differences between Splenocytes from OT-1 mice were suspended at 5 3 107 cells/ml in PBS/ groups. A p value , 0.05 was accepted as the level of significance. 0.1% BSA containing 10 mM CFSE (Invitrogen/Molecular Probes, Eu- gene, OR) for 10 min at 37˚C. Cells were washed twice with cold RPMI Results 1640/10% FCS followed by two washes in PBS. CFSE-labeled OT-1 cells (2 3 106) in 200 ml PBS were injected into the tail vein of mice that were Subcutaneous injection of an OVA/ISCOM vaccine and vaccinated once with OVA, OVA/ISCOM vaccine, or PBS on the same day. ISCOMATRIX adjuvant leads to recruitment and activation of Two days later, peripheral blood was collected and CFSE dilution of innate and adaptive immune effector cells in VDLNs proliferating OT-1 CD8+ T cells was analyzed by flow cytometry. To characterize the immunostimulatory effect of ISCOM vaccines Ex vivo cross-presentation by DC subpopulations on leukocyte populations in vivo, we injected mice s.c. with OVA, the OVA/ISCOM vaccine, or ISCOMATRIX adjuvant without Ag Twenty-four hours after s.c. immunization, VDLNs were processed into a single-cell suspension with a collagenase/DNAase solution. DCs were and removed VDLNs for analysis of leukocyte composition and enriched by Nycodenz density gradient centrifugation (1.082 g/cm3; activation marker expression. Both ISCOMATRIX adjuvant and Nycomed Pharma, Oslo, Norway), followed by depletion of non-DCs and the OVA/ISCOM vaccine induced a visible enlargement of the plasmacytoid DCs with an Ab mixture containing anti-CD3 (clone KT3), VDLNs, reaching a maximum after 24 h (Supplemental Fig. 1A). anti–Thy-1 (cloneT24/31.7), anti-CD19 (clone ID6), anti-Gr1 (clone RB6- 8C5), and Ter-119 and magnetic beads coated with anti-rat IgG (Dynal At 24 h the total cell number had increased 5- to 6-fold as com- beads M450; Invitrogen). Enriched DCs were stained with mouse anti– pared with VDLNs of mice injected with PBS or OVA alone (Fig. CD11c-allophycocyanin, anti–CD8-FITC, and anti–CD205-PE (BD Bio- 1A). FACS analysis of VDLN cell suspensions revealed a signifi- sciences) and FACS sorted into three DC subpopulations (FACSAria; BD cant increase of B cell, CD4+ T cell, and CD8+ T cell numbers . Biosciences). A purity of 93% was obtained routinely. DCs were cul- (Fig. 1B); overall, the CD4/CD8 ratio was not affected (data not tured with CFSE-labeled OT-I cells at 10,000:50,000 DC/OT-I cell ratio, and proliferation of OT-I cells was measured 48 h later by flow cytometry. shown). Additionally, we observed a dramatic increase in the numbers of NK1.1+CD32 NK cells (13-fold) and NK1.1+CD3+ In vivo cytotoxicity assay NKT cells (11-fold) (Fig. 1C). Interestingly, VDLNs contained Mice were injected i.v. with a mixture of splenocytes differentially labeled a population with high sideward light scatter property and coex- + + with CFSE (2, 20, or 200 nM) loaded with 1, 10, or 100 nM SIINFEKL pression of Ly6G and CD11b , which was absent in control LNs peptide, respectively, and unloaded spleen cells were labeled with 10 mM (Fig. 1D). These cells could be identified as granulocytes using chloromethyl-benzoyl-aminotetramethyl-rhodamine (Invitrogen/Molecular H&E staining (Supplemental Fig. 1B). Importantly, this immune 3 6 Probes). A total of 12 10 cells per mouse were injected, consisting of stimulatory effect was limited to the VDLNs, since contralateral a mixture containing each target cell population. Inguinal LNs draining the immunization site were collected 24 h after injection of target cells. LNs and spleens did not differ in cell number or composition from Presence of viable injected target cells was determined using exclusion by control animals (data not shown). 7-aminoactinomycin D. Percentage killing was calculated using the for- Next, we characterized the activation status of innate and adap- mula as described (24). tive immune effector cells in VDLNs by measuring CD69 and Tumor induction intracellular IFN-g expression. The OVA/ISCOM vaccine induced significant CD69 upregulation on B cells, CD4+ T cells, and CD8+ The OVA-expressing thymoma EG7 was obtained from the American Type T cells (Fig. 2A). Additionally, we observed upregulation of CD69 Culture Collection. The B16-OVA melanoma was a gift from Dr. J. Hess (Erlangen, Germany). PancOVA was generated from the pancreatic cancer expression and IFN-g production by NK cells and NKT cells (Fig. line Panc02 (18). For tumor induction, 106 tumor cells in 100 ml PBS were 2B). Overall, we observed no difference between ISCOMATRIX injected s.c. into the flank of age-matched vaccinated and unvaccinated adjuvant alone and the OVA/ISCOM vaccine with regard to

FIGURE 1. ISCOM vaccine leads to the recruitment of innate and adaptive immune cells into VDLNs. Mice were vaccinated s.c. with PBS, OVA, OVA/ ISCOM, or ISCOMATRIX adjuvant (IMX) alone. VDLNs were processed into single- cell suspensions after 24 h. A, Cell count of total LN cells. B, Effect on numbers of CD19+ B cells, CD4+ T cells, and CD8+ T cells, as assessed by FACS analysis. C, Effect on numbers of CD32NK1.1+ NK cells, CD3+NK1.1+ NKT cells, and D, Ly6G+CD11b+ cells. E, Effect on num- bers of CD11c+ CD8a+ and CD8a2 DC populations. Each graph depicts means 6 SD of one representative out of two in- dependent experiments with five animals per group. *p , 0.05. 58 IMMUNE ACTIVATION AND CROSS-PRESENTATION OF ISCOM VACCINE

duction of Th1-driving cytokines by DCs, such as IL-12 (26). Thus, we analyzed expression of the costimulatory molecule CD86 and IL-12 production by DCs in VDLNs. Both the OVA/ ISCOM vaccine and ISCOMATRIX adjuvant induced a significant upregulation of CD86 by DCs in vivo. This effect was most pronounced for CD8a+ DCs (Fig. 2C) and was similarly effective as the TLR9 ligand CpG-ODN1826 (data not shown). Additionally, a fraction of CD8a+ DCs in VDLNs produced IL-12, whereas CD8a2 DCs expressed no IL-12p40 above background levels (Fig. 2C). The OVA/ISCOM vaccine targets Ag to DCs in VDLNs and induces effective cross-presentation for CTL activation To assess in vivo whether the OVA/ISCOM vaccine targets Ag to DCs, mice were injected with Alexa 488-labeled OVA, either as soluble protein or formulated as an OVA/ISCOM vaccine. VDLNs and contralateral LNs were removed after 24 h and Ag uptake by CD11c+ DCs was studied by flow cytometry. Analysis revealed Ag uptake by ∼15% of CD8a2 DCs and 20% of CD8a+ DCs in the VDLNs. Interestingly, uptake of the ISCOM vaccine was more efficient than uptake of soluble protein by CD8a2 DCs. A similar trend was observed for CD8a+ DCs, but this was not statistically significant (Fig. 3A). To evaluate the capacity of DCs to cross-present the Ag in vivo, we transferred CFSE-labeled T cells from OT-1 mice, which recognize the H2-Kb–restricted epitope SIINFEKL, into mice vaccinated with either soluble OVA (30 mg) or the OVA/ISCOM FIGURE 2. Innate and adaptive immune cells in VDLNs upregulate vaccine (containing 0.3 mg OVA). OT-1 T cell proliferation and activation marker expression and IFN-g production. Mice were vaccinated IFN-g production were assessed after 3 and 10 d in the VDLNs by s.c. with PBS, OVA, OVA/ISCOM, or ISCOMATRIX adjuvant alone and flow cytometry. As shown in Fig. 3B, the OVA/ISCOM vaccine VDLNs were processed into single-cell suspensions after 24 h for flow induced a potent T cell proliferative and IFN-g response. In cytometry. A, Expression of the activation marker CD69 by B cells, CD4+ contrast, even 100-fold higher concentrations of soluble protein + 2 T cells, and CD8 T cells. B, Expression of CD69 and IFN-g by CD3 induced only minor T cell proliferation. Interestingly, cross- NK1.1+ NK cells and CD3+NK1.1+ NKT cells. C, Expression of CD86 and + 2 presenting capacity of APCs in VDLNs was lasting for a period IL-12p40 by CD8a and CD8a DC populations. Results from one rep- of up to 10 d after vaccination. To assess which DC population resentative experiment of two are shown as means 6 SD comprising of five was most efficient in CD8+ T cell activation, we isolated LN- animals per group. *p , 0.05. resident CD8a+DEC205+ DCs, LN-resident CD8a2DEC2052 DCs, and migratory CD8a2DEC205+ DCs from VDLNs by FACS recruitment of leukocyte populations and their activation, indi- and cocultured the DC subsets with CFSE-labeled OT-I T cells. + cating that these effects were mediated by the immunostimulatory This experiment revealed that CD8a DCs were most efficient capacity of ISCOMATRIX adjuvant. in cross-presenting the OVA/ISCOM vaccine in vivo (Fig. 3C). Cross-presentation efficacy was further studied with splenic The OVA/ISCOM vaccine recruits DCs to VDLNs and induces CD8a+ DCs pulsed with either soluble OVA or OVA/ISCOM DC activation in vivo in vitro. These experiments confirmed highly effective cross- DCs comprise a heterogeneous family of APCs. Lymphatic tissues presentation of the vaccine Ag by this DC subset and further in mice contain three main DC types, which express CD11c and demonstrated a .100-fold Ag-sparing effect as compared with 2 2 can be discriminated as CD8a+CD205+, CD4+CD8a , and CD4 soluble Ag (Fig. 3D). CD8a2 DCs. In skin-draining LNs, two additional DC pop- b ulations, CD205+Langerin+ DCs and CD205+CD11b+ interstitial The OVA/ISCOM vaccine induces high levels of IL-1 and IL-6 DCs, are found. Of these, CD8a+ DCs are highly specialized in as well as a mixed Th1/Th2 cytokine profile in VDLNs cross-presentation and CTL induction (25). To assess the influence To evaluate the cytokine response mediated by the ISCOM vaccine, of the OVA/ISCOM vaccine and ISCOMATRIX adjuvant on DCs, we vaccinated mice and removed the VDLNs after 6, 12, and 24 h. we quantified CD11c+ DCs in LN preparations of vaccinated VDLNs of mice injected with either PBS or OVAserved as controls. mice. A significant increase of CD11c+ DCs was observed after Concentrations of IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, 24 h for both ISCOMATRIX adjuvant and the OVA/ISCOM IL-12p70, GM-CSF, and TNF-a were measured by multiplex assay. vaccine. Because of the low number of DCs in VDLNs, we lim- Strikingly, high levels of IL-1b and IL-6 were produced in VDLNs 2 ited our analysis to CD8a+ and CD8a DC subsets. The increase as early as 6 h after injection of the vaccine, indicative of an early of total DCs was mainly due to recruitment (or retention) of inflammatory response (Fig. 4A). Additionally, the vaccine induced 2 CD8a DCs, which made up ∼90% of CD11c+ DCs in the the production of GM-CSF and IL-12p40, as well as low levels of VDLNs. Additionally, the vaccine induced a 4- to 5-fold increase IL-4 and IL-5, indicative of a mixed Th1/Th2 type response. No in CD8a+ DCs (Fig. 1E). significant changes in IL-2, IL-10, IL-12p70, or TNF-a production The importance of DC activation for CTL induction has been were observed. To confirm that IL-1b was secreted in its bioactive demonstrated in DC-based therapy studies (3, 4). In this respect, form (17 kDa) we performed Western blot analysis of VDLN Th1 responses are required, which are dependent on the pro- lysates. As shown in Fig. 4B, p17 was detected in VDLNs but not The Journal of Immunology 59

FIGURE 3. ISCOM vaccine targets Ag to DC populations in the VDLNs leading to effi- cient cross-presentation by CD8a+ DCs. A,Ag uptake of Alexa 488-labeled soluble OVA or OVA/ISCOM vaccine by CD8a+ and CD8a2 DC populations in VDLNs was analyzed by flow cytometry 24 h after vaccination. Results from one representative experiment of two are shown as means 6 SD of five animals. *p , 0.05. B, T cell proliferation of adoptively transferred CFSE-labeled OT-I CD8+ T cells in recipient mice vaccinated with soluble OVA (30 mg) or OVA/ISCOM vaccine (0.3 mg OVA). CD45.2+ T cells were transferred at day 1 and day 7 after vaccinations of CD45.1+ mice. T cell proliferation and IFN-g production were assessed in VDLNs 3 d after transfer on days 4 and 10 by flow cytometry by gating on viable CD45.2+ CD8+ T cells. Representative data of three independent experiments are shown. C, Ex vivo cross-presentation of OVA/ISCOM vaccine and IFN-g production by different DC populations isolated from VDLNs by FACS sorting cocultured with CFSE-labeled OT-I CD8+ T cells. D, In vitro cross-presentation of OVA and OVA/ISCOM vaccine by CD8a+ DCs isolated from spleen. T cell proliferation was determined from viable CFSElowCD8+ T cells after 3 d coculture using calibration beads. In C and D, representative data from three indepen- dent experiments performed as triplicates are shown and data are given as means 6 SD.

control LNs (PBS). IL-1b secretion in response to the ISCOM DCs in CD8+ T cell cross-priming, we vaccinated irradiated mice vaccine was confirmed in LPS-primed DCs in vitro and could be reconstituted with bone marrow from CD11c-DTR mice, in which blocked with z-YVAD, an inhibitor of caspase-1 (Fig. 4C). Thus, CD11c+ DCs can be ablated by injection of DT (22). The vaccina- ISCOM vaccines induce caspase-1–dependent cleavage of pro–IL- tion protocol and depletion efficacy are shown in Supplemental Fig. 1b to active IL-1b in DCs. 2B. Importantly, non-CD11c–expressing APCs such as radio- resistant Langerhans cells, B cells, and monocytes are not de- The OVA/ISCOM vaccine induces priming of CTL capable of pleted and can still perform APC functions after DT treatment. As tumor cell killing shown in Fig. 5B, depletion of CD11c+ DCs almost completely We analyzed the vaccine-induced immune response using a prime- abolished CTL induction. To study the role of Langerhans cells and boost regimen in which mice were vaccinated twice with OVA/ Langerin+ dermal DCs in T cell priming, we made use of Langerin- ISCOM vaccine (0.3 mg OVA) or soluble OVA (30 mg) in a DTR mice, in which Langerin+ cells can be depleted by repeated weekly interval. To compare the potency of the ISCOM vaccine injections of DT (the vaccination protocol and depletion efficacy are formulation with other adjuvant systems, we also vaccinated mice depicted in Supplemental Fig. 2A). Depletion of Langerin+ APCs with OVA (30 mg) together with either TLR ligands (Pam3-Cys for had no impact on CTL priming or cytotoxic activity in vivo, in- TLR3, LPS for TLR4, CpG-ODN1826 for TLR9), IFA, or alum. On dicating that these DC subsets are dispensable for ISCOM vaccine- day 14, priming of OVA-specific CTLs was assessed by measuring mediated CTL induction (Fig. 5C,5D). These data strengthen the the frequency of splenic CD8+ T cells specific for the H2-Kb–re- hypothesis that CTL priming by ISCOM vaccines is predominantly stricted epitope SIINFEKL by IFN-g ICS. The OVA/ISCOM vac- mediated by conventional DCs, with other APCs playing a less cine induced highly efficient cross-priming of OVA-specific CTLs important role. with an average CTL frequency of 8.5% of total CD8+ T cells (Fig. The functional activity of the CTL was studied in an in vivo cy- 5A). Neither OVA alone or in combination with Pam3-Cys, IFA, or totoxicity assay using adoptively transferred SIINFEKL-pulsed and alum induced a detectable CTL response. Of the TLR ligands, CpG- CFSE-labeled splenocytes. Specific target cell lysis was 80 and 60% ODN1826 and LPS induced the best CTL responses with a frequency on days 10 and 35 after the second vaccination, respectively, in- of 1.8 and 0.7% of CD8+ T cells, respectively. To analyze the role of dicative of T cell memory induction (Fig. 6A). No significant killing 60 IMMUNE ACTIVATION AND CROSS-PRESENTATION OF ISCOM VACCINE

FIGURE 4. ISCOM vaccine induces a mixed Th1/ Th2 type response and high levels of IL-1b in a cas- pase-1–dependent manner. A, Mice were injected s.c. with PBS, OVA, or OVA/ISCOM and VDLNs were removed after 6, 12, and 24 h. Cytokine content was quantified by Multiplex cytokine array in lysates of pooled VDLNs from three mice per group. A repre- sentative experiment of two is shown. B, Western blot analysis of IL-1b p17 (active form) and p35 (pro–IL- 1b) in lysates of VDLNs following s.c. administra- tion of PBS or OVA/ISCOM. C, Bone marrow-de- rived DCs were primed in vitro with LPS for 2 h and subsequently incubated with ISCOMATRIX adjuvant (IMX) or OVA/ISCOM (5 mg/ml) in the absence or presence of z-YVAD-fmk (caspase-1 inhibitor). IL- 1b secretion was analyzed by ELISA and Western blot. Representative data from three independent experiments performed as triplicates are shown.

was observed in mice vaccinated with soluble OVA. Moreover, VDLNs in mice. Additionally, we found increased levels of IL-5 vaccinated mice were protected against challenge with OVA ex- and IL-12, indicative of a mixed Th1/Th2 response. IL-1b is pressing tumor cells, such as EG-7 thymoma, PancOVA pancreatic a proinflammatory cytokine that mediates recruitment of leuko- cancer, or B16-OVA melanoma cells (Fig. 6B). Vaccinated animals cytes to sites of inflammation and the secretion of other proin- remained tumor free during an observation period of 100 d after flammatory cytokines, such as IL-6. The secretion of bioactive IL- tumor induction. Mice did not develop tumors after a second tumor 1b is regulated by caspase-1–activating inflammasome complexes, challenge, indicative of long-term memory induction (data not such as the NLRP3 inflammasome (28). Interestingly, we observed shown). secretion of bioactive IL-1b by DCs incubated with ISCOMA- TRIX adjuvant in vitro, which occurred in a caspase-1–dependent Discussion manner. Assessing the role of different inflammasome types, such ISCOMATRIX adjuvant-based vaccines hold promise for gene- as the NLRP3 inflammasome, on the adjuvant properties of rating effective CTL responses against tumors (19). In this study, ISCOMATRIX adjuvant is the focus of ongoing studies. we investigated mechanisms of action of ISCOMATRIX adjuvant The immunostimulatory effect of ISCOMATRIX adjuvant was and an OVA/ISCOM vaccine. We used the ISCOM vaccine rather further demonstrated by the recruitment of activated innate and than the ISCOMATRIX vaccine to be sure that the Ag and adju- adaptive effector cells to VDLNs. The increase of total cell com- vant were associated, thereby enabling labeling of the Ag to track position within VDLNs was predominantly due to recruitment of the formulation. We found that ISCOM vaccines 1) induce potent B cells and T cells, expressing high levels of the early activation immune activation in VDLNs, 2) effectively target Ag to DCs marker CD69. Additionally, a marked increase of NK cells and in vivo, 3) induce DC maturation and IL-12 production in vivo, 4) NKT cells producing IFN-g as well as an influx of granulocytes facilitate Ag cross-presentation by CD8a+ DCs, and 5) mediate was observed. A cross-talk between NK cells and DCs has been a cellular immune response leading to tumor protection and T cell suggested to play an important role in anti-tumor immunity. Ma- memory induction. ture DCs can induce IFN-g expression in NK cells (29). Re- Immune activation by an ISCOMATRIX vaccine has been ciprocally, NK cells have the capacity to induce DC maturation demonstrated by lymphatic cannulation in sheep with the transient and IL-12 production (30). A similar interplay between gran- appearance of proinflammatory cytokines in efferent lymph, in- ulocytes and DCs leading to DC maturation and enhanced Ag cluding IFN-g, IL-6, IL-8, and IL-1b (27). Consistent with this presentation has been reported (31, 32). Indeed, VDLNs contained report, we found high levels of bioactive IL-1b and IL-6 in increased numbers of CD8a+ DCs expressing high levels of The Journal of Immunology 61

FIGURE 5. ISCOM vaccine induces highly efficient CTL priming mediated by CD11c+ DCs, but not Langerin+ DCs. A, Mice were vaccinated twice at a weekly interval with either PBS, OVA (30 mg), or OVA/ISCOM vaccine (0.3 mg OVA). For comparison with other adjuvant systems, mice were also vaccinated with OVA combined with either Pam3-Cys, LPS, CpG-ODN1826, IFA or alum. The frequency of OVA-specific CTLs in spleens was determined on day 14 by intracellular cytokine staining for IFN-g of CD8+ T cells stimulated with SIINFEKL peptide ex vivo. Results are shown as means 6 SD of four to five animals per group. *p , 0.01. B, C57BL/6 or lethally irradiated mice reconstituted with bone marrow from CD11c-DTR transgenic mice were vaccinated as above with DT or without injection and CTL frequency in the spleen was determined by SIINFEKL/H-2Kb tetramer staining. Results are shown as means 6 SD of four to five animals per group. *p , 0.002. C and D, Langerin-DTR transgenic mice were vaccinated as above with or without DT injection, and CTL frequency in peripheral blood was determined by SIINFEKL/H-2Kb pentamer staining (C). In vivo cytotoxicity of CTLs in Langerin- DTR mice was determined after adoptive transfer of CFSE/CTO-labeled target cells (D). Graphs show means 6 SD of four to five animals per group. Representative data of three independent experiments are shown. The experimental setups for B–D are depicted in Supplemental Fig. 2. costimulatory molecules, MHC class II (data not shown), and IL- in vivo. Whether other DC populations, such as Langerhans cells, 12p40. Taken together, these results indicate that ISCOMATRIX can also cross-prime CTLs is controversial (33, 34). In this study, adjuvant is a potent immune modifier, linking innate and adaptive we show that the ISCOM vaccine is effectively taken up by both immunity and creating a favorable milieu for Th1 type immune CD8a+ and CD8a2 DCs (including skin-derived migratory DCs) responses. in VDLNs, indicative of effective Ag targeting to DCs in vivo. Because CD8a+ DCs are highly specialized in cross-priming Cross-presentation was highly effective as demonstrated by the CTL responses, tumor vaccines should aim at targeting this DC potent proliferative response of adoptively transferred OT-1 subpopulation (and the corresponding DC population in humans) T cells. FACS sorting of DC populations in VDLNs revealed

FIGURE 6. ISCOM vaccine induces CTLs capable of effective target cell killing and mediating tumor protection. Mice were vaccinated twice at a weekly interval with either OVA (30 mg) or OVA/ISCOM (0.3 mg OVA). A, In vivo killing of adoptively transferred CFSE-labeled and SIINFEKL-pulsed target cells by vaccine-induced CTLs 10 and 35 d after the second vaccination. Results are shown as means 6 SD of three to four animals per group. B, Mice were challenged s.c. with 1 3 106 tumor cells of either EG-7 (OVA-expressing thymoma), B16-OVA (melanoma), or PancOVA (pancreatic cancer) 30 d after the second vaccination. Tumor growth curves are shown as means 6 SD of four to five animals per group. Representative data of at least two independent experiments are shown. 62 IMMUNE ACTIVATION AND CROSS-PRESENTATION OF ISCOM VACCINE that cross-presenting capacity was mainly attributed to CD8a+ exogenous antigen by dendritic cells induces potent antitumor T helper and CTL responses. 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