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-galactosyl- ceramide (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 Ó2019 American Association for Cancer Research. recombinant monoclonal antibody (mAb) to Clec9a does not 952 Cancer Immunol Res; 7(6) June 2019 Downloaded from cancerimmunolres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 3, 2019; DOI: 10.1158/2326-6066.CIR-18-0650 Clec9a-aGC–Targeted Cancer Vaccine 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.