Research Article 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- phocytes with immune regulatory activity. Their ability to bridge the innate and adaptive immune systems has been studied using the glycolipid ligand a-galactosylceramide (aGC). To better harness the immune adjuvant properties of iNKT cells to enhance priming of antigen-specific CD8 þ T cells, we encapsulated both aGC and antigen in a Clec9a-targeted nanoemulsion (TNE) to deliver these molecules to cross- presenting CD8 þ dendritic cells (DC). We demonstrate that, even in the absence of exogenous glycolipid, iNKT cells supported the maturation of CD8a þ DCs to drive efficient cross-priming of antigen-specific CD8 þ T cells upon delivery of Clec9a/OVA-TNE. The addition of aGC to the TNE (Clec9a/OVA/aGC) further enhanced activation of iNKT cells, NK cells, CD8a þ DCs, and polyfunctional CD8 þ T cells. When tested therapeutically against HPVE7-expressing TC-1 tumors, long-term tumor suppression was achieved with a single administration of Clec9a/E7 peptide/aGC TNE. Anti- tumor activity was correlated with the recruitment of mature DCs, NK cells, and tumor-specific effector CD8 þ T cells to the tumor-draining lymph node and tumor tissue. Thus, Clec9a-TNE codelivery of CD8 þ T-cell epitopes with aGC induces alternative helper signals from activated iNKT cells, elicits innate (iNKT, NK) immunity, and enhances antitumor CD8 þ T-cell responses for control of solid tumors. Introduction Invariant natural killer T (iNKT) cells are a subset of preacti- vated innate immune cells that possess markers of both NK and T cells and play a role in cancer immunity. The activation of iNKT cells requires T-cell receptor recognition of processed glycolipids presented on an MHC class I–like molecule, CD1d. Conventional dendritic cells (cDC) are specialized antigen-presenting cells (APC) that prime the adaptive immune system. However, within this population of cells, CD8a þ cDCs are the dominant APC for cross-priming of antigen-specific T cells (1) and also glycolipid presentation on CD1d for the activation of iNKT cells (2). cDCs possess the appropriate machinery for capturing, processing, and presenting glycolipid antigens for the activation of iNKT cells (3). In turn, activation of iNKT cells provides helper signals via CD40–CD40L interactions (4) and chemokine signals (5) that augment DC maturation, production of IL12 and consequently induction of innate immunity, including rapid activation of NK cells, and promotion of adaptive T-cell responses. The discovery of an NKT-cell glycolipid antigen a-galactosyl- ceramide (aGC) more than 20 years ago has led to a better understanding of the immune adjuvant role of iNKT cells in augmenting simultaneous innate and tumor-specific adaptive immunity (transactivation). However, the use of soluble aGC as a therapeutic has its limitations. Hyporesponsiveness of iNKT cells upon secondary restimulation (6), acute liver toxicity (7), as well as the variability of iNKT cell numbers between individuals hinder clinical utility of aGC. A variety of methods aiming to overcome these limitations and optimize the adjuvanting effects of iNKT cells have been explored over the past decade. One such approach involves the adoptive transfer of aGC-loaded autolo- gous DCs, which can overcome iNKT cell hyporesponsive- ness (8–10) and promote lymphocyte infiltration into tumors (8). However, cellular-based vaccines can be costly and labor- intensive to produce and are usually specific to an individual patient (11). Therefore, in vivo targeting of DCs using inert delivery vectors has been explored to drive tumor-specific 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 concurrently toward the CD8a þ DC subset. Few endocytic recep- tors have shown specificity for the CD8a þ DC subset. However, an endocytic C type lectin receptor known as Clec9a is highly expressed by CD8a þ CD103 þ DCs and to a lesser extent by plasmacytoid DCs (pDC) in mice (13–15). In humans, Clec9a is expressed by the equivalent CD141 þ BDCA3 þ DC popula- tion (16). DC Clec9a regulates T-cell cross-priming, which involves recruitment of early endosomal components and enzymes colocalized with antigens for cross-presentation (17). In the absence of licensing or danger signals, delivery of a recombinant monoclonal antibody (mAb) to Clec9a does not The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia. Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). Corresponding Author: Stephen R. Mattarollo, The University of Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland 4102, Australia. Phone: 61-7-34436985; Fax: 61-7-31765946; E-mail: s.mattarollo@uq.edu.au Cancer Immunol Res 2019;7:952–62 doi: 10.1158/2326-6066.CIR-18-0650 Ó2019 American Association for Cancer Research. Cancer Immunology Research Cancer Immunol Res; 7(6) June 2019 952 Downloaded from http://aacrjournals.org/cancerimmunolres/article-pdf/7/6/952/2354627/952.pdf by guest on 21 March 2024