NKT Cell–Driven Enhancement of Antitumor Immunity Induced By

Published OnlineFirst May 3, 2019; DOI: 10.1158/2326-6066.CIR-18-0650

Research Article Cancer Immunology Research NKT Cell–Driven Enhancement of Antitumor Immunity Induced by Clec9a-Targeted Tailorable Nanoemulsion Pui Yeng Lam, Takumi Kobayashi, Megan Soon, Bijun Zeng, Riccardo Dolcetti, Graham Leggatt, Ranjeny Thomas, and Stephen R. Mattarollo

Abstract

Invariant natural killer T (iNKT) cells are a subset of lym- (Clec9a/OVA/aGC) further enhanced activation of iNKT þ þ phocytes with immune regulatory activity. Their ability to cells, NK cells, CD8a DCs, and polyfunctional CD8 T cells. bridge the innate and adaptive immune systems has been When tested therapeutically against HPVE7-expressing TC-1 studied using the glycolipid ligand a-galactosylceramide tumors, long-term tumor suppression was achieved with a (aGC). To better harness the immune adjuvant properties of single administration of Clec9a/E7 peptide/aGC TNE. Anti- þ iNKT cells to enhance priming of antigen-specific CD8 T cells, tumor activity was correlated with the recruitment of þ we encapsulated both aGC and antigen in a Clec9a-targeted mature DCs, NK cells, and tumor-specific effector CD8 nanoemulsion (TNE) to deliver these molecules to cross- T cells to the tumor-draining lymph node and tumor tissue. þ þ presenting CD8 dendritic cells (DC). We demonstrate that, Thus, Clec9a-TNE codelivery of CD8 T-cell epitopes with even in the absence of exogenous glycolipid, iNKT cells aGC induces alternative helper signals from activated iNKT þ supported the maturation of CD8a DCs to drive efficient cells, elicits innate (iNKT, NK) immunity, and enhances þ þ cross-priming of antigen-specific CD8 T cells upon delivery antitumor CD8 T-cell responses for control of solid of Clec9a/OVA-TNE. The addition of aGC to the TNE tumors.

Introduction The discovery of an NKT-cell glycolipid antigen a-galactosylceramide (aGC) more than 20 years ago has led to a better Invariant natural killer T (iNKT) cells are a subset of preacti- understanding of the immune adjuvant role of iNKT cells in vated innate immune cells that possess markers of both NK and augmenting simultaneous innate and tumor-specific adaptive T cells and play a role in cancer immunity. The activation of iNKT immunity (transactivation). However, the use of soluble aGC as cells requires T-cell receptor recognition of processed glycolipids a therapeutic has its limitations. Hyporesponsiveness of iNKT presented on an MHC class I–like molecule, CD1d. Conventional cells upon secondary restimulation (6), acute liver toxicity (7), as dendritic cells (cDC) are specialized antigen-presenting cells well as the variability of iNKT cell numbers between individuals (APC) that prime the adaptive immune system. However, within þ hinder clinical utility of aGC. A variety of methods aiming to this population of cells, CD8a cDCs are the dominant APC for overcome these limitations and optimize the adjuvanting effects cross-priming of antigen-specific T cells (1) and also glycolipid of iNKT cells have been explored over the past decade. One such presentation on CD1d for the activation of iNKT cells (2). cDCs approach involves the adoptive transfer of aGC-loaded autolo- possess the appropriate machinery for capturing, processing, gous DCs, which can overcome iNKT cell hyporesponsive- and presenting glycolipid antigens for the activation of iNKT ness (8–10) and promote lymphocyte infiltration into tumors (8). cells (3). In turn, activation of iNKT cells provides helper signals However, cellular-based vaccines can be costly and labor- via CD40–CD40L interactions (4) and chemokine signals (5) that intensive to produce and are usually specific to an individual augment DC maturation, production of IL12 and consequently patient (11). Therefore, in vivo targeting of DCs using inert delivery induction of innate immunity, including rapid activation of NK vectors has been explored to drive tumor-specific responses. cells, and promotion of adaptive T-cell responses. Various studies have utilized vectorized aGC and antigen in various passive and active applications (12). Few have explored active and specific targeting strategies delivering aGC and antigen The University of Queensland Diamantina Institute, The University of þ concurrently toward the CD8a DC subset. Few endocytic recep- Queensland, Translational Research Institute, Brisbane, Queensland, Australia. þ tors have shown specificity for the CD8a DC subset. However, an Note: Supplementary data for this article are available at Cancer Immunology endocytic C type lectin receptor known as Clec9a is highly Research Online (http://cancerimmunolres.aacrjournals.org/). þ þ expressed by CD8a CD103 DCs and to a lesser extent by Corresponding Author: Stephen R. Mattarollo, The University of Queensland, plasmacytoid DCs (pDC) in mice (13–15). In humans, Clec9a Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland þ þ is expressed by the equivalent CD141 BDCA3 DC popula- 4102, Australia. Phone: 61-7-34436985; Fax: 61-7-31765946; E-mail: [email protected] tion (16). DC Clec9a regulates T-cell cross-priming, which involves recruitment of early endosomal components and Cancer Immunol Res 2019;7:952–62 enzymes colocalized with antigens for cross-presentation (17). doi: 10.1158/2326-6066.CIR-18-0650 In the absence of licensing or danger signals, delivery of a 2019 American Association for Cancer Research. recombinant monoclonal antibody (mAb) to Clec9a does not

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þ appear to drive cross-priming of antigen-specific CD8 T-cell Antibodies responses in vivo (15, 18). Anti-CD40 (14, 19) and agonists Fluorochrome-conjugated mouse mAbs to murine CD3e forTLR3 (19) and TLR9 (20) are necessary for the induction of (145-2C11), CD8a (53-6.7), CD8b (YTS156.7.7), CD19 antigen-specific CTL. Thus, targeting the Clec9a receptor to reg- (1D3 or 6D5), CD4 (RM4-5), CD40 (3/23), CD44 (IM7), ulate T-cell responses became attractive for translational CD45.1 (A20), CD45.2 (104), CD69 (H1.2F3), CD80 (16- approaches in humans. However, only one study has demon- 10A1), CD86 (GL-1), IFNg (XMG1.2), NK1.1 (PK136), TCRb þ strated the feasibility of codelivering aGC and antigens to CD8a (H57-597), CD11c (HL3), I-A/I-E (M5/114.15.2), TNFa (MP6- DCs in nanoparticle systems via the Clec9a receptor. In that study, XT77), and associated isotype control antibodies were pur- a Clec9a-decorated anionic poly(lactic-coglycolic acid; PLGA) chased from BioLegend, BD Biosciences, or eBioscience. Gol- þ nanoparticle system generated iNKT cell–driven CD8 T-cell giPlug was purchased from BD Biosciences. Flow-count fluoro- responses against tumor (21) through the simultaneous delivery spheres were purchased from Beckman Coulter. aGC-loaded of aGC and antigen to the same DC (22). CD1d tetramer was generously provided by Prof. D. Godfrey We reported that cationic Clec9a antigen–targeted nanoemul- (University of Melbourne, Australia). DAMP4 fused with anti- sions (TNE) carrying whole antigen can promote cross-priming of Clec9a and isotype control (mAb-DAMP4) was kindly provided þ þ antigen-specific CD8 T cells through CD4 T cell–mediated by Dr. I. Caminschi and A/Prof. M. Lahoud at the Burnet induction of IFNa and CD40 signaling, resulting in tumor sup- Institute (Melbourne, Australia), with DAMP4 previously pression (23). Using this technology, we sought to investigate the described as an interfacial anchor on TNE (24). feasibility of this "oil-in-water" nanoemulsion system as a safe delivery vector for the simultaneous delivery of lipophilic Flow cytometry adjuvants, such as aGC, with tumor antigens for cancer Single-cell suspensions were prepared from tissues by mechan- immunotherapy. ical dissociation or from blood followed by red blood lysis for 1 or 15 minutes at room temperature, respectively. For analyses of DC subsets in spleens, whole spleens were incubated with 2 mg/mL of Materials and Methods collagenase D and 20 mg/mL of DNAse I for 15 minutes at 37C Mice before mechanical dissociation. Cells were antibody labeled at C57BL/6 and B6.SJL (CD45.1) mice were purchased from the predetermined optimal concentrations of antibodies for 45 min- Animal Research Centre (Perth, WA). iNKT cell–deficient Ja18 utes at 4C in PBS containing 2% FCS and 2 mmol/L EDTA. Flow- knockout mice were bred and maintained onsite at the Transla- count fluorospheres were added to the samples to calculate cell tional Research Institute Biological Research Facility. Mice were numbers upon acquisition. Intracellular cytokine staining was used at the ages of 6–12 weeks, sex-matched, and housed under preceded by the addition of GolgiPlug to the cells for 4 hours to specific pathogen-free conditions. All experiments were con- prevent cytokine release from the Golgi/ER complex, unless ducted following the animal ethics guidelines provided by the otherwise stated. Cells were permeabilized and fixed using BD National Health and Medical Research Council of Australia and Cytofix/Cytoperm kit, following the manufacturer's instructions. approved by the University of Queensland–Health Sciences Ani- Labeled cells were acquired on Gallios (Beckman Coulter). mal Ethics Committee (ethics number 301/15). Preparation of protein and peptide antigens in oil dispersion Reagents and peptides and TNE The following peptides were custom synthesized by GL Ovalbumin (OVA), SIINFEKL, and RAHYNIVTF (hereafter Biochem to a final purity of >95%: AM1 (Ac-MKQLADSLHQ- gf001) peptide solutions (10 mg/mL), and TNEs were prepared LARQVSRLEHA-NH2), OVA257–264 peptide (SIINFEKL), HPV.E7 as previously described (23, 24). Briefly, endotoxin-free OVA or peptide (RAHYNIVTF, gf001 peptide), and WH peptide SIINFEKL/RAHYNIVTF peptide were dissolved in ultrapure water (WPRFHSSVFHTHGGGK; ref. 20). Peptide concentration was (10 mL) and sonicated 2:1 with Cithrol GMO HP or Span80 determined by using HPLC analysis. Miglyol 812 (Cremer Oleo- solution (1%, w/v) in hexane solution before lyophilization. The chemicals) was a gift from IMCD Australia Limited. Cithrol GMO antigen-Cithrol GMO HP pellet was dissolved in Miglyol 812 to HP was a gift from Croda Europe Ltd. An alternative surfactant 5 mg/mL and used as oil phase. Clec9a-mAb or Ig decorated Sorbitan monooleate (Span80) was purchased from Sigma- TNEs were prepared as previously described (24). To make aGC- Aldrich. Reagent-grade 4-(2-hydroxyethyl)-1-piperazineethane- containing TNE, 2 mLofaGC was added to a final concentration of sulfonic acid (HEPES), zinc chloride (ZnCl2), n-hexane, and 10 mg/mL and 20 mL of Miglyol 812 was added to an oil volume dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. fraction of 2% (v/v). For TNEs containing OVA/SIINFEKL, 20 mL Endotoxin-free albumin from chicken egg white (OVA; 98% of protein/peptide in oil was added instead. WH peptide– purity, <1 EU/mg) was purchased from Hyglos (Bernried/ functionalized targeted emulsions were made in a similar fashion. Germany). mPEG-NHS (MW 5,000, protein dispersibility index Briefly, WH peptide was first conjugated to DSPE-PEG-NHS. <1.08, purity >95%) was purchased from Nanocs. RPMI-1640, WH-PEG conjugate was first synthesized by mixing WH peptide DMEM, and fetal calf serum (FCS) were purchased from GIBCO. with DSPE-PEG-NHS in 25 mmol/L HEPES solution, pH8.0 at 1:1 a-Galactosylceramide (aGC) was purchased from Avanti Polar molar ratio at 4C for 24 hours. WH-PEG conjugate was then Lipids. CellTrace Violet (CTV), CellTrace CFSE, LIVE/DEAD subjected to dialysis into water using a centrifugal dialysis unit of Fixable Aqua Dead Cell Stain was purchased from Molecular 3000 MWCO. WH-PEG conjugate in water was frozen rapidly in Probes. Collagenase D and DNase I were purchased from Roche. dry ice at an angle for 1 hour before lyophilization overnight. Amicon Ultra-0.5 Centrifugal Filter Units (MWCO 3000) were Dry-frozen WH-PEG conjugate was reconstituted in 25 mmol/L purchased from Merck Millipore. DAMP4 fused with antibody HEPES solution (pH 8.0) to a final concentration of 400 nmol/L (mAb-DAMP4) was generated as previously described (13). before use. To make WH-decorated TNE, antigen in Miglyol 812,

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aGC glycolipid, WH-PEG conjugate, and AM1 peptide were subjected to antibody staining, including permeabilization for added into an Eppendorf tube to a final concentration of intracellular staining of IFNg and TNFa. 25 mg/mL in oil volume fraction of 2% v/v, 2.5 mg/mL, 5% v/v, and 400 mmol/L, respectively. The mixture was sonicated for four Lymphocyte depletion in vivo þ 1-minute pulses at 60 W to form oil-in-water emulsions. Particle For in vivo depletion of CD8 T cells, 200 mg of anti-CD8b size was measured by a Zetasizer Nano ZS (Malvern Panalytical). (53-5.8; Bio X Cell) was administered by intraperitoneal injec- Data analysis with DTS software used the nonnegativity con- tions to vaccinated tumor-bearing mice on days 6 and 10 relative þ strained least-squares fitting algorithm. Dispersant refractive to tumor inoculation. CD8 T cell–depletion efficacy was greater þ index and viscosity of the dispersant were assumed to be 1.45 than 95%. For in vivo depletion of CD4 cells, 100 mg of anti-CD4 and 1.02 centipoise. Each sample had 10 runs of 10 seconds. (GK1.5; Bio X Cell) was administered by intraperitoneal injec- tions to na€ve mice one and 4 days prior to immunization. þ Tumor cell line and analysis of tumor growth in vivo Depletion efficacy of CD4 T cells and NKT cells were greater TC-1 cells are a lung epithelial cell line derived from C57BL/6 than 95%. For controls, equivalent doses of 2A3 control immu- mice, immortalized by HPV16 E6/E7, and transformed with an noglobulin (cIg) were administered. activated ras oncogene, a gift originally from T.C. Wu (John Hopkins University, Baltimore, MD; ref. 25). Authentication was Statistical analysis determined by reactivity of E7-specific T cells. Cells were sourced Results are presented as mean SE. Kaplan–Meier plots were from a batch of Mycoplasma-free stock, tested by PCR prior to used to analyze mouse survival, and a log-rank test was performed cryopreservation in 2012. Cells were prepared by passaging twice to assess the statistical significance of differences between survival in vitro for 4 days in complete DMEM containing 5 mmol/L HEPES curves. For all other data, Student t test, one-way ANOVA before inoculation into C57BL/6 mice. Mice were subcutaneously were used to assess differences between two and more groups (s.c.) inoculated with 2 105 of TC-1 cells in the right flank region (GraphPad Prism 7 Software). (day 0). Mice were then intravenously given 200 mL of antigen (5 mg) and aGC (5–500 ng) in soluble or TNE vector on day 7. Tumors were measured every 2 to 3 days with a digital caliper. Results þ Calculation of tumor volume (mm3) was as per formula: DC maturation and antigen-specific CD8 T-cell responses [length (mm) width2 (mm2)]/2. Mice were sacrificed when enhanced by activated iNKT tumor reached a size of 1,000 mm3. We generated nanoemulsions that targeted DCs by attaching an þ antibody against Clec9a, which is expressed on CD8a DC cell Antigen-specific CTL lysis assay surface, as in ref. 23. Clec9a/OVA TNE can support antigen- þ þ To assess the in vivo antigen-specific CD8 cytotoxic T-cell specific CTL induction by activating Clec9a DCs (26, 27) and response, 6 days after immunization with TNE, mice were each iNKT cells can be activated by and further amplify DC-mediated þ injected with 1 107 congenic CD45.1 splenocytes unloaded or signals (28). We therefore investigated whether Clec9a/OVA TNE loaded with 10 12 to 10 6 mol/L of relevant CD8 peptide for activates iNKT cells and whether iNKT cells contribute to the 90 minutes at 37C in complete RPMI and labeled for 10 minutes self-adjuvanting properties of the TNE. In wild-type (WT) mice, þ at 37C with CFSE or CTV (0.5 or 5 mmol/L) in 1 PBS at 1:1 CD1d-Tet iNKT and NK cells were activated by Clec9a/OVA TNE ratios. Twelve to 20 hours later, mice were sacrificed and spleno- immunization. The frequency of iNKT and NK cells producing cytes from the recipients were analyzed by flow cytometer to IFNg was increased, as compared with untreated or soluble assess peptide-specific killing. The percentage peptide-specific OVA-treated mice; however, expression per cell was not changed lysis was calculated as follows: % specific killing ¼ 100 (Fig. 1A). In contrast, fewer NK cells produced IFNg upon Clec9a/ / [1 – ((Experimentalloaded/Experimentalunloaded)/(Controlloaded/ OVA TNE immunization of iNKT-deficient Ja18 mice Controlunloaded))]. (Fig. 1A), demonstrating the partial dependence of NK cell function on iNKT cell activation upon Clec9a/OVA TNE immuniza- þ Ex vivo restimulation of tumor-specific CD8 T cells tion. CD69 is an early activation marker for lymphocytes. Clec9a/ þ þ To assess tumor-specific CD8 T-cell responses, tumor tissue, OVA TNE treatment saw an increase in frequency in CD69 iNKT and tumor-draining lymph nodes were isolated from TC-1 tumor- cells and overall expression of this molecule (Fig. 1B). Similarly, in þ bearing mice 7 days after vaccination and processed under sterile WT mice, an increase in the frequency CD69 NK and T cells and conditions. Briefly, tumor-draining lymph nodes and tumors expression of CD69 was observed upon Clec9a/OVA TNE treat- were mechanically dissociated with collagenase D (2 mg/mL) ment (Fig. 1B). These increases were negated in both cell types in and DNAse I (20 mg/mL) to create single-cell suspensions. For the absence of iNKT cells, indicating the activation of NK and T ex vivo restimulation, cell suspensions were then seeded into 24- cells is iNKT cell dependent. Activation of iNKT cells induces DC well plates and stimulated with 1 mg/mL of gf001 peptide and 1 in maturation (29). Clec9a/OVA TNE treatment induced upregula- þ 1,000 dilution (1 mg/mL) of GolgiPlug in complete RPMI media, tion of CD86 and CD80 but not CD40 expression on CD8a DCs along with control unstimulated group for 5 hours, unless oth- in an iNKT cell–dependent manner (Fig. 1C). These observations erwise stated. To assess long-term effector memory responses in coincide with the proportion of activated T cells expressing CD69 vaccinated mice, mice were bled via the retro-orbital sinus and (Fig. 1B). To understand the extent to which cross-priming of þ subjected to red blood cell lysis. Lymphocytes were then seeded antigen-specific CD8 T cells was affected in the absence of into 96-well plates and were either left unstimulated or restimu- activated iNKT cells, immunized WT and Ja18 / mice were lated with 5 mg/mL of gf001 peptide for 5 hours in the presence of challenged with dye-labeled target cells pulsed with various con- 1 in 1,000 dilution GolgiPlug in complete RPMI media. Cells were centrations of SIINFEKL peptide as a readout for CTL activity then collected from each well into polypropylene FACS tubes and in vivo. CTL activity was reduced in the absence of iNKT cells after

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Figure 1. Activation of NK cells, DC maturation, and antigen-specific CD8þ T-cell function is enhanced by iNKT cells after immunization with Clec9a/OVA TNE. To determine iNKT and NK cell activation upon Clec9a/OVA TNE treatment, C57BL/6 and Ja18-deficient mice (iNKT cell deficient) were treated with soluble endotoxin-free OVA, Clec9a/OVA TNE, or left untreated. Groups of mice were then (A) sacrificed at 3 hours to determine intracellular production of IFNg in (left) þ þ þ iNKT (gated as CD19 TcRb Cd1d-tetramer ) and (right) NK cells (gated as CD19 TcRb NK1.1 )or(B) sacrificed at 24 hours for analysis of CD69 expression on (left) iNKT, (middle) NK cells, or (right) T cells. C, The activation markers CD40, CD86, and CD80 on CD8aþ DCs (gated as CD11cþMHCIIþCD8aþ cells) were also examined 24 hours after immunization. D, To analyze the effect of iNKT cells on antigen-specific CD8þ T-cell induction in vivo, 6 days after TNE administration, mice were intravenously challenged with a mixture of CFSE/CTV labeled, CD45.1þ splenic cells loaded with OVA peptide (SIINFEKL) at the indicated concentrations. Sixteen hours later, spleens were analyzed for specific lysis of target cells. Statistical analyses between groups were carried out using two-way ANOVA test: , P < 0.05; , P < 0.005; , P 0.0005; , P < 0.0001. All data are representative of two independent experiments with n ¼ 4 per group. CTL, cytotoxic T lymphocytes; DC, dendritic cells; NK, natural killer; OVA, ovalbumin.

Clec9a/OVA TNE immunization but was not completely abro- Clec9a/OVA TNE and Clec9a/OVA/aGC TNE (Supplementary þ gated (Fig. 1D). CD4 T helper cells have also been shown to play Fig. S2a), whereas surface charge was increased with inclusion a role in the induction of CTL cross-priming upon Clec9a TNE of aGC (Supplementary Fig. S2b). WT mice were administered i.v. þ treatment (23). CD4 T cells were depleted in vivo 1 and 4 days Clec9a/OVA TNE, Clec9a/OVA/aGC TNE, soluble OVA, or OVA prior to TNE immunization (Supplementary Fig. S1a). As a plusaGC in PBS. Spleens were analyzed 3 and 24 hours after þ proportion of iNKT cells also expresses CD4, we observed that immunization, for iNKT cell and NK cell activity as well as CD8a þ 2 doses of anti-CD4 were sufficient to deplete CD4 T cells as well DC maturation. As expected, treatment of mice with soluble OVA þ as CD4 iNKT cells in the blood (Supplementary Fig. S1b) and in þ aGC but not OVA alone activated iNKT cells and NK cells, as the spleen (Supplementary Fig. S1c). To examine the effect of demonstrated by the production of intracellular IFNg and TNFa þ depletion of CD4 cells on CTL cross-priming, mice were chal- (Fig. 2A), and increased CD69 expression (Fig. 2B). Production of lenged with dye-labeled target cells pulsed with SIINFEKL. After IFNg and CD69 expression in splenic iNKT and NK cells in mice 24 hours, no significant difference in CTL activity was observed administered Clec9a/OVA/aGC TNE increased to a similar extent between nondepleted and CD4-depleted Clec9a/OVA TNE- after soluble OVA þ aGC (Fig. 2A and B). Clec9a/OVA/aGC TNE treated mice (Supplementary Fig. S1d). These observations dem- administration significantly enhanced the frequency of CD40-, þ þ onstrate that iNKT cells enhance NK cell activation and CD8a CD86-, and CD80-expressing CD8a DCs and average expression DC maturation after Clec9a/OVA TNE immunization. In addi- of these molecules, as compared with Clec9a/OVA TNE (Fig. 2C). þ tion, iNKT cells also enhance cross-priming of antigen-specific CD8a DC maturation was similar in mice treated with soluble þ þ CD8 T cells, and this augmentation is independent of CD4 T OVA þ aGC or Clec9a/OVA/aGC TNE (Fig. 2C). These results þ cells or CD4 iNKT cells in this setting. demonstrate that incorporation of aGC promotes iNKT/NK activation and DC maturation and that aGC-containing TNE are as Codelivery of a-galactosylceramide in Clec9a/OVA TNE effective as nonencapsulated aGC for innate immune cell enhances innate cell activities activation. Given the adjuvant capacity of iNKT cells in response to Clec9a/ þ OVA immunization, we sought to enhance the adjuvant effects of CD8 T-cell cytotoxicity is enhanced upon delivery of antigen þ iNKT cells by incorporating the NKT cell-stimulating glycolipid and aGC to Clec9a DCs þ ligand, aGC into the Clec9a/OVA TNE (Clec9a/OVA/aGC TNE). We next examined the development of antigen-specific CD8 Physical size and stability of the TNE did not differ between T-cell responses. Antigen-specific CTL activity, measured by lysis

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Figure 2. Clec9a/OVA/aGC TNE enhance innate immune cell activation. WT mice were administered soluble OVA, Clec9a/OVA TNE, soluble OVA þ aGC, or Clec9a/OVA/ aGC TNE and their spleens were analyzed after 3 hours for (A) intracellular cytokine production in (top) NKT and (bottom) NK cells. B, Additional treated mice þ were examined at 24 hours to assess activation markers CD69 on (top) iNKT and (bottom) NK cells, and (C) CD40, CD86, and CD80 on CD8a DCs (gated as þ þ þ CD11c MHCII CD8a ). Statistical analysis comparing treatment groups was done by one-way ANOVA: , P < 0.05; , P < 0.005; , P < 0.0005; , P < 0.0001. Data from A and B are representative of two independent experiments with n ¼ 5 per group. Data from C are pooled from two independent experiments with n ¼ 4 per group. IL, interleukin; MFI, mean ﬂuorescent intensity.

of target cells in vivo, was significantly higher in mice given Clec9a/ antigen-specific responses (5, 22). iNKT cell–induced DC matu- OVA/aGC TNE than in mice receiving Clec9a/OVA TNE and in ration requires CD40–CD40L interactions occurring in parallel to þ mice given soluble OVA þ aGC (Fig. 3a). As expected, CTL activity CD4 T-cell help (4, 30, 31). This interaction facilitates cross- þ was largely absent in mice receiving soluble OVA alone (Fig. 3a). priming of antigen-specific CD8 T-cell responses (5, 32). To Thus, activation of iNKT cells through Clec9a/OVA/aGC TNE confirm that activation of iNKT cells can promote CTL activity in þ þ enhances cross-priming of CD8 T cells and supports previous the absence of CD4 T-cell help, mice were immunized with the studies indicating the benefit for simultaneous delivery of aGC minimal MHC I-binding OVA epitope (SIINFEKL), which stimu- þ þ and antigen to cross-presenting CD8a DCs to drive enhanced lates a CD8 T-cell response. Clec9a/SIINFEKL/aGC TNE-treated

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Figure 3. þ þ Enhanced antigen-specific CD8 T-cell responses is achieved with the codelivery of antigen and aGC in Clec9a-targeted TNEs. Antigen-specific CD8 T-cell lytic activity was determined in mice administered soluble OVA, Clec9a/OVA TNE, soluble OVA þ aGC, or Clec9a/OVA/aGC TNE. Six days after TNE administration, mice received an intravenous injection of congenic, CFSE/CTV-labeled, CD45.1þ splenic target cells loaded with the indicated concentrations of peptide. Twelve to 14 hours later, (A) spleens were analyzed for antigen-specific clearance of the target cells. B, To determine in vivo CTL activity in mice administered Clec9a/OVA peptide/aGC TNE, Clec9a/OVA peptide TNE, soluble OVA peptide þ aGC, Clec9a/gf001 TNE or soluble gf001 þ aGC measured against SIINFEKL-pulsed targets or gf001-pulsed targets. Statistical analyses between groups were carried out using one-way ANOVA: , P < 0.05; , P < 0.001; , P < 0.0001. Data from A are pooled from five independent experiments with n ¼ 4 per group. Data from B are pooled from three independent experiments with n ¼ 4 per group.

þ mice could generate a CTL response, whereas CTL responses in Tumor suppression is associated with magnitude of CD8 T-cell mice treated with Clec9a/SIINFEKL TNE were minimal (Fig. 3B). responses at the tumor site Similarly, mice receiving soluble SIINFEKL þ aGC also induced To determine whether the TC-1 tumor suppression by þ minimal CTL activity in vivo (Fig. 3B). These results are consistent WH/gf001/aGC TNE was dependent on CD8 T-cell activity, þ with the concept that codelivery of peptide and aGC to the same CD8 T cells were depleted from tumor-bearing mice prior to þ Clec9a DC is beneficial for CTL induction. Likewise, enhanced treatment with WH/gf001/aGC500ng TNE. The therapeutic effect þ CTL activity was also observed with a second MHC I-binding was abrogated in the absence of CD8 T cells (Fig. 5A). Because peptide, gf001, derived from HPV16E7 protein incorporated into mature DCs are involved in the recruitment and activation of þ Clec9a/aGC TNE, relative to soluble gf001 þ aGC. These obser- antitumor CD8 tumor-infiltrating leukocytes (TIL), tumor- þ vations demonstrate that inclusion of CD4 T helper epitopes is draining lymph nodes (TdLN) and tumor tissue were analyzed þ þ þ not required in Clec9a/CD8 peptide/aGC TNE to induce CD8 for the presence of mature DCs and tumor-specific CD8 T cells þ CTLs capable of antigen-specific target cell killing. 7 days after treatment. Indeed, the number of CD80 DCs þ correlated with the number of effector CD8 T cells in both Tumor growth is reduced by Clec9a/CD8 epitope/aGC TNE TdLNs and in the tumors of vaccinated mice (Fig. 5B). Recruit- þ In view of the efficient generation of antigen-specific CTL, we ment of DCs and CD8 TILs was most prominent in mice tested the therapeutic effect of TNE in the HPV16 E7-expressing vaccinated with WH/gf001/aGC500ng TNE (Fig. 5B). The num- TC-1 tumor model. We functionalized TNE with WH peptide that ber of NK cells also correlated with the number of mature DCs targets Clec9a, as described (20, 23). CTL lytic capacity was similar in the TdLN and in tumor tissue of WH/gf001/aGC TNE- when comparing Clec9a/gf001/aGC and WH/gf001/aGC TNE vaccinated mice (Supplementary Fig. S3), suggesting that acti- (Fig. 4A). One dose of WH/gf001/aGC TNE containing varying vation of iNKT cells may help recruit NK cells into tumors. concentrations of aGC at 5, 50, and 500 ng was administered However, NK cell recruitment did not differ between mice intravenously 7 days after subcutaneous TC-1 inoculation. As receiving WH/gf001/aGC at different doses of aGC. To deter- controls, WH/aGC TNE or soluble aGC with gf001 peptide were mine the proportion of functional antigen-specificCTL,lym- administered. Mice vaccinated with WH/gf001/aGC TNE phocytes from the TdLN and tumor were restimulated with achieved the greatest tumor growth suppression, and this antitu- gf001 peptide in vitro and intracellular amounts of IFNg and mor effect was aGC dose dependent (Fig. 4B). The reduction in TNFa were assessed. The largest proportion of cytokine- þ tumor growth required TNE encapsulation of both peptide and producing CD8 T cells was detected in the TdLN and in tumor aGC, as TNE encapsulation of either component alone failed to tissue of mice treated with WH/gf001/aGC500ng TNE, followed reduce tumor growth. Furthermore, encapsulation in TNE was by WH/gf001/aGC5ng TNE and soluble gf001 þ aGC500ng more effective than soluble gf001 þ aGC. The reduced tumor (Fig.5C).Thus,thequantityofpolyfunctional antigen-specific þ growth in WH/gf001/aGC-treated mice was associated with effector CD8 T cells correlated with tumor suppression improved survival (Fig. 4C). induced by the vaccine.

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Figure 4. Tumor growth is suppressed by therapeutic vaccination with Clec9a-targeted-TNEs delivering tumor-associated antigen and aGC. In vivo lytic activity induced by Clec9a/gf001/aGC TNE or WH/gf001/TNE were compared with soluble gf001 and aGC administration in C57BL/6 mice. A, In vivo lysis was analyzed using gf001 peptide pulsed target cells. B, To test the antitumor efﬁcacy of WH-TNE, C57BL/6 mice (n ¼ 5/group) were inoculated subcutaneously with 2 105 E7- expressing (TC-1) tumor cells, then treated 7 days after with either 5 (top), 50 (middle), or 500 ng (bottom) of aGC with 5 mg gf001 peptide each in PBS or encapsulated in WH-TNE. Tumor volume was measured over time and both individual mice and averaged tumor volumes are shown. Survival curves for each group of vaccinated tumor-bearing mice are plotted over time. C, The endpoint for tumor-bearing mice was reached when tumor volume was 1 cm3. Statistical analyses between two groups were carried out using unpaired t test: , P < 0.01, and between multiple groups using one-way ANOVA: , P < 0.05; , P < 0.01 (WH/gf001/aGC vs. gf001þ aGC); #, P < 0.05; ##, P < 0.01 (gf001þ aGC vs. gf001). The survival curves were analyzed with log-rank test: , P < 0.005 (WH/ gf001/aGC vs. gf001þ aGC at equivalent aGC dose); #, P < 0.05 (vs. WH/gf001/aGC5ng). All data are representative of two independent experiments with n ¼ 4 (A)andn ¼ 5(B and C) per group.

þ þ Increased circulating CD8 T cells improves long-term survival Upon restimulation, a population of tumor-specific CD8 T cells þ The use of aGC can augment survival of CD8 T memory cell was observed in mice vaccinated with WH/gf001/aGC500ng TNE subsets and support recall responses (33, 34). To assess antigen- (Fig. 6). These observations indicate that Clec9a/CD8 epitope/ þ specific memory CD8 T-cell responses in circulation, surviving aGC TNE promote the long-term survival of polyfunctional þ mice were bled 30 days after WH/gf001/aGC TNE treatment. tumor-specific CD8 T cells and the efficiency of recall response Blood leucocytes were restimulated with gf001 peptide for 5 to tumor antigens. þ hours and analyzed for CD8 T-cell IFNg and TNFa production. Without restimulation, a small but distinct population of circu- þ lating CD8 T cells produced both IFNg and TNFa in WH/gf001/ Discussion aGC500ng TNE-treated mice, but not in surviving mice receiving Over the past decade, the understanding of how to harness the WH/gf001/aGC5ng TNE or soluble gf001þaGC500ng (Fig. 6). immunostimulatory properties of iNKT cell glycolipids for

958 Cancer Immunol Res; 7(6) June 2019 Cancer Immunology Research

Clec9a-aGC–Targeted Cancer Vaccine

Figure 5. Therapeutic tumor responses induced by Clec9a/antigen/aGC nanoemulsions are dependent on CD8þ T cells. C57BL/6 mice were inoculated s.c. with 2 105 TC-1 cells on day 0 then treated 7 days later with WH/gf001/aGC TNE and gf001 or left untreated. To deplete CD8þ T cells, anti-CD8b (200 mg/mouse) was administered intraperitoneally on days 6 and 10 after tumor inoculation (!). A, Tumor volume and survival were assessed over time. C57BL/6 mice (n ¼ 4) were inoculated s.c. with 2 105 TC-1 cells, then treated 7 days later as shown. Seven days later, mice were sacrificed and TdLNs and tumors were analyzed by flow þ þ þ þ þ þ þ þ þ þ cytometry. B, The correlation between CD80 DCs (CD45.2 CD80 CD11c MHCII ) and CD44 CD8 T cells (CD45.2 CD44 CD8b ) in the TdLN and tumor is shown with regression line. Numbers of CD80þDCs and effector CD8þ TILs, expression of CD80 (MFI) on DCs in TdLN and tumors (right). C, The functional þ þ activity of CD44 CD8 T cells was quantified using intracellular staining for IFNg and TNFa after gf001 peptide restimulation for 5 hours (1 mg/mL) in vitro. Statistical analysis between two groups was carried out using an unpaired t test: , P < 0.05; , P < 0.01 (WH/gf001/aGC TNE (intact) vs. WH/gf001/aGC TNE (depleted)). Survival curves were compared using a log-rank test: , P < 0.005 (A). Statistical analyses between multiple groups were carried out using Kruskal–Wallis test: , P < 0.05; , P < 0.01; , P < 0.001. Correlation analysis was carried out with Pearson correlation coefficient test: , P < 0.01 (B and C). Data are representative of two independent experiments with n ¼ 4 per group.

þ therapy in conditions such as viral infections and tumors has tumor-specific CD8 T-cell responses. Our study (i) provides improved. The drawbacks associated with soluble administration insight into the role for iNKT cells in the self-adjuvanting of aGC, including iNKT anergy and acute liver toxicity, prompted responses induced by Clec9a/OVA TNE, (ii) validates Clec9a þ the development of glycolipid carrier techniques. Some of these as an entry receptor into CD8a DCs for the combinatorial nanocarriers such as lipid-based octaarginine-modified (35) or processing of antigens and aGC for cancer immunotherapy, (iii) cationic liposomal nanocarriers (36) may improve the delivery confirms the adjuvanting effects of iNKT cells for cancer vaccines, and function of hydrophobic aGC (37), and have been used to and (iv) validates TNE as a biocompatible delivery vector for codeliver aGC with antigen to induce antitumor immunity. Here, glycolipids. we improved on passive targeting systems by functionalizing oil- We showed that Clec9a-targeted TNE encapsulating protein in-water TNE for the delivery of aGC and tumor-associated antigen or pooled CD4 and CD8 epitope peptides could induce þ þ þ antigen directed to Clec9a CD8a DCs for the optimization of antigen-specific CD8 T-cell responses and tumor control in the

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Lam et al.

Figure 6. þ Higher doses of aGC within WH/gf001 peptide TNE favors the development of long-term CD8 T-cell recall responses. Blood was obtained from healthy na€ve (n ¼ 2) and surviving tumor-bearing mice one month (day 37 after tumor inoculation) after vaccination (gf001 þ aGC500ng: n ¼ 1, WH/gf001/aGC5ng: n ¼ 1, WH/gf001/aGC500ng: n ¼ 4). Blood cells were restimulated with gf001 peptide (5 mg/mL) or left unstimulated for 5 hours. Intracellular production of both IFNg and TNF by overall CD8þ T cells was analyzed by ﬂow cytometry with representative plots shown. Data are representative of two independent experiments with n ¼ 5 per group.

absence of adjuvant. This self-adjuvanting function was found to the concept of simultaneous delivery of aGC and antigen to the þ be dependent on CD4 T-cell help and IFNa production for DC same DCs for optimal CTL induction (22), but may also suggest maturation and cross-priming of CTL (23). Other studies have how alternative T cell-helper signals from iNKT cells synergize also demonstrated that TLR9 stimulation, the production of IFNa, with other innate signals such as type I IFNs to enhance cross- and TLR9-mediated production of charged sphingolipids can priming (5, 23, 45). promote iNKT cell activation (26, 28, 38). Here we show that Cytotoxic tumor-specific T cells are useful for cancer vaccine Clec9a/OVA TNE activate iNKT cells, and NK cells, and that in the immunotherapy. In an oncogenic virus HPV16-E7–driven tumor þ absence of iNKT cells, activation of NK cells as well as CD8a DCs model TC-1, in which oncogenesis is dependent on the conserved þ and antigen-specific CD8 T cells was reduced. This observation E7 protein (46), we demonstrate the adjuvanting effects of demonstrates that iNKT cells play a role and suggests they enhance WH/gf001/aGC TNE for iNKT cell stimulation, driving systemic þ the self-adjuvanting effect of Clec9a/OVA TNE (28). iNKT cells polyfunctional E7-specific CD8 T cells. Ghinnagow and collea- provide maturation signals to DCs via CD40–CD40L interac- gues delivered multiple doses of Clec9a-targeted PLGA nanopar- tion (4, 22). The absence of CD86 and CD80 upregulation 24 ticles to treat a subcutaneous model of B16F10 tumor (21). Here hours after TNE immunization and the reduced lytic activity of we investigated the efficacy of a single i.v. administration of CTLs in iNKT cell–deficient mice suggest that a lack of DC CD40 WH/gf001/aGC TNE with increasing concentrations of aGC. engagement by iNKT cells prevents the enhanced induction of Previously, WH/protein TNE distribution to the spleen, liver, and þ T-cell costimulation and IL12 production (4). In the absence tumor indicated targeting of WH-TNE to CD8 cross-presenting þ of iNKT cells, persistent CD40 upregulation may also have been DCs in these tissues (23). Here we show that CD8 T-cell tumor þ mediated by other innate signals such as proinflammatory cyto- infiltration, E7-specific CD8 tumor T-cell responses, circulating þ kines, including TNFa and IFNa (39, 40). Our data thus support antigen-experienced memory CD8 T cells, and overall mouse the evidence that iNKT cells influence the development of func- survival correlate with the concentration of administered þ tional CD8a DCs necessary for CTL induction (41). To then aGC delivered in WH/peptide TNE. Thus, our method of iNKT þ þ address the contribution of CD4 T-cell help in the promotion of cell activation promotes a CD8 T-cell response profile of CTL cross-priming after Clec9a/OVA TNE treatment in this setting, protective long-term immunity (47). In sum, our observations þ including a subset of CD4 iNKT cells, anti-CD4 depletion was demonstrate how aGC-dependent iNKT cell activation sup- þ þ performed prior to TNE treatment. Depletion of CD4 cells did ports the generation of memory CD8 T cells involved in solid not affect CTL activity induced by Clec9a/OVA TNE. Our data tumor control. þ therefore indicate that CD4 iNKT cells are required for the Mature DCs stimulate CD8 T cells associated with production of immunostimulatory IFNa, which was shown pre- effector antitumor responses (48). In a variety of tumors, viously to be necessary for DC activation and CTL activity after tumor-infiltrating (Ti-) DCs were often phenotypically immature Clec9a/OVA TNE treatment (23, 42). and functionally suppressive through a variety of mechanisms in Clec9a/peptide/aGC TNE and soluble aGC þ peptide similarly the tumor microenvironment (49). By directing aGC to tumor- þ þ þ led to activation of iNKT cells, NK cells, and CD8a DCs in the infiltrating CD8 DCs, TiDCs were CD80 . Their frequency and spleen. However, antigen-specific CTL activity in vivo was intensity of CD80 expression correlated with that of polyfunc- þ enhanced in mice immunized with Clec9a/OVA/aGC TNE, as tional effector CD8 T cells in an aGC dose–dependent manner. compared with soluble aGC and OVA. This observation is sup- Therefore, WH/peptide/aGC targeting of tumor-infiltrating þ ported by previous studies using targeted or nontargeted PLGA CD8 DCs promotes DC activation from a normally immature nanoparticles encapsulating aGC and OVA antigen (12, 43, 44). or immune-suppressed state (50). This behavior is associated In addition, we also show that iNKT cells promote cross-priming with cross-talk with activated iNKT cells. Although the effector þ of minimal CD8 T-cell epitopes. Our results therefore support role of NK cells is not investigated here and long-term suppression

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Clec9a-aGC–Targeted Cancer Vaccine

of TC-1 tumors appears NK cell independent, recruited NK cells Writing, review, and/or revision of the manuscript: P.Y. Lam, T. Kobayashi, þ likely activate and recruit DCs and CD8 T cells to the site of B. Zeng, G. Leggatt, R. Thomas, S.R. Mattarollo tumor (51, 52). Thus, WH/aGC/peptide TNE codeliver antigen Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P.Y. Lam, M. Soon and innate immune signals that disrupt tolerizing tumor condi- Study supervision: R. Dolcetti, R. Thomas, S.R. Mattarollo tions and drive enhanced tumor-specific systemic responses syn- onymous with the hallmarks of successful immunotherapy (21). Acknowledgments þ Thus, targeting aGC and peptide to CD8 DCs using Clec9a/ The authors acknowledge the Translational Research Institute for providing aGC/peptide TNE is a flexible and translatable delivery approach an excellent research environment and core facilities that enabled this research with greater efficacy than approaches using soluble glycolipids. to be conducted. We particularly thank Siok Min Teoh and Daniel Kerage for technical assistance, and the Biological Resources and Flow Cytometry Core Disclosure of Potential Conflicts of Interest Facilities for technical support. Gratitude is extended to Associate Professors Mirelle Lahoud and Irina Caminschi (Monash University, Melbourne) for the R. Thomas reports receiving other commercial research support from and provision of the anti-mouse Clec9a IgG2a mAb. This work was supported from has received honoraria from speakers bureau of Merck & Co. No potential grant (1087691) jointly funded by Cancer Australia and Cure Cancer Australia. fl con icts of interest were disclosed by the other authors. P.Y. Lam was supported by a University of Queensland International Scholar- ship. S.R. Mattarollo was supported by an NHMRC Career Development Authors' Contributions Fellowship (1061429). Conception and design: P.Y. Lam, B. Zeng, G. Leggatt, R. Thomas, S.R. Mattarollo The costs of publication of this article were defrayed in part by the payment of Development of methodology: P.Y. Lam, B. Zeng, R. Thomas, S.R. Mattarollo page charges. This article must therefore be hereby marked advertisement in Acquisition of data (provided animals, acquired and managed patients, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. provided facilities, etc.): P.Y. Lam, T. Kobayashi, M. Soon, B. Zeng Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P.Y. Lam, R. Dolcetti, G. Leggatt, R. Thomas, Received September 17, 2018; revised November 13, 2018; accepted April 22, S.R. Mattarollo 2019; published first May 3, 2019.

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962 Cancer Immunol Res; 7(6) June 2019 Cancer Immunology Research

NKT Cell−Driven Enhancement of Antitumor Immunity Induced by Clec9a-Targeted Tailorable Nanoemulsion

Pui Yeng Lam, Takumi Kobayashi, Megan Soon, et al.

Cancer Immunol Res 2019;7:952-962. Published OnlineFirst May 3, 2019.

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