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PDF file - 22K, Increased proliferation of follicular helper T cells following exosome injection.

PDF file - 32K, Germinal center B cells and Plasma cells, but not Follicular B cells are proliferating in response to aGC-loaded exosomes.

PDF file - 136K, List of antibodies used in this study.

PDF file - 54K, Late onset treatment using aGC-loaded exosomes leads to T cell infiltration into the tumor, increased survival and decreased tumor growth.

PDF file - 22K, Early activation of marginal zone B cells following exosome administration.

PDF file - 38K, aGC-loaded exosomes activate innate immune cells.

Components of the tumor microenvironment (TME) are known to play an essential role during malignant progression, but often in a context-dependent manner. In bone and soft tissue sarcomas, disease-regulatory activities in the TME remain largely uncharacterized. This chapter introduces the cellular, structural, and chemical composition of the sarcoma TME from a pathobiological and therapeutic perspective.Sarcomas are malignant tumors with diverse features when it comes to primary tumor appearance, metastatic potential, and response to treatment. Many of the classic subtypes are mainly composed of malignant cells and are therefore assumed to be committed to autocrine signaling. Some of the tumors are infiltrated by immune cells and contain necrotic areas or excessive amounts of extracellular matrix (ECM) that regulates tissue stiffness and interstitial fluid pressure. Vascular invasion and blood vessel characteristics can in some instances be considered in the prognostic setting.Further insights into the disease-regulatory activities of the sarcoma TME will provide essential knowledge on how to develop successful combination treatments targeting not only malignant cells, but also their routes of nutrition and ability to shield themselves toward existing therapy.

Simple Summary The incidence of metastatic melanoma has been increasing over the past years with current therapies showing limited efficacy to cure the disease. Therefore, other options are being investigated, such as adoptive cell therapy (ACT) where activated immune cells are infused into a patient to attack melanoma. Natural killer (NK) cells are part of the innate immune system and extremely suitable for this kind of therapy since they show minimal toxicities in the clinical setting. In this review, we focus on current strategies for NK cell therapy and the development of new approaches that hold great promise for the treatment of advanced melanoma. Abstract Adoptive cell therapy (ACT) represents a promising alternative approach for patients with treatment-resistant metastatic melanoma. Lately, tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR)-T cell therapy have shown improved clinical outcome, compared to conventional chemotherapy or immunotherapy. Nevertheless, they are limited by immune escape of the tumor, cytokine release syndrome, and manufacturing challenges of autologous therapies. Conversely, the clinical use of Natural Killer (NK) cells has demonstrated a favorable clinical safety profile with minimal toxicities, providing an encouraging treatment alternative. Unlike T cells, NK cells are activated, amongst other mechanisms, by the downregulation of HLA class I molecules, thereby overcoming the hurdle of tumor immune escape. However, impairment of NK cell function has been observed in melanoma patients, resulting in deteriorated natural defense. To overcome this limitation, “activated” autologous or allogeneic NK cells have been infused into melanoma patients in early clinical trials, showing encouraging clinical benefit. Furthermore, as several NK cell-based therapeutics are being developed for different cancers, an emerging variety of approaches to increase migration and infiltration of adoptively transferred NK cells towards solid tumors is under preclinical investigation. These developments point to adoptive NK cell therapy as a highly promising treatment for metastatic melanoma in the future.

Components of the tumor microenvironment (TME) are known to play an essential role during malignant progression, but often in a context-dependent manner. In bone and soft tissue sarcomas, disease-regulatory activities in the TME remain largely uncharacterized. This chapter introduces the cellular, structural, and chemical composition of the sarcoma TME from a pathobiological and therapeutic perspective.

MHC-I epitope presentation to CD8 + T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptide-loading complex, the peptide editor tapasin is key to the selection of MHC-I–bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

Harris and collaborators show that neutropenia results in increased formation of plasma cells and elevated antibody production., Prolonged infections or adjuvant usage can trigger emergency granulopoiesis (EG), leading to dysregulation in neutrophil blood counts. However, the impact of EG on T and B cell function remains largely unknown. In this study, to address this question, we used a mouse model of neutropenia and studied immune activation after adjuvant administration. The initial neutropenic state fostered an environment of increased dendritic cell activation and T cell–derived IL-17 production. Interestingly, neutropenic lysozyme 2–diphtheria toxin A mice exhibited striking EG and amplified neutrophil recruitment to the lymph nodes (LNs) that was dependent on IL-17–induced prostaglandin activity. The recruited neutrophils secreted a B cell–activating factor that highly accelerated plasma cell generation and antigen-specific antibody production. Reduction of neutrophil functions via granulocyte colony-stimulating factor neutralization significantly diminished plasma cell formation, directly linking EG with the humoral immune response. We conclude that neutrophils are capable of directly regulating T cell–dependent B cell responses in the LN.

Introduction of chimeric antigen receptors (CARs) to natural killer (NK) cells has so far been the only practical method for specific targeting of NK cells against surface antigens. In contrast, T-cell receptor (TCR) gene therapy can supply large populations of cytotoxic T-lymphocytes (CTL) genetically modified to express a TCR that can also target intracellular antigens. However, the mispairing of endogenous and genetically transferred TCR subunits constitutes a bottleneck in the development of safe therapies as it often leads to formation of TCRs with unknown specificity. In order to overcome this obstacle and enable intracellular antigen targeting, we propose the use of NK cells for TCR gene therapy. In this study, we approach the obstacles associated with TCR gene therapy from a unique perspective that results in MHC-I-restricted epitope-specific targeting of tumors cells through expression of a functional TCR complex on NK cells. Our results show that the ectopic expression of CD3δ, CD3γ, and CD3ϵ chains along with TCR α/β gene delivery to NK cells enables the functional expression of a TCR specific to the HLA-A2-restiricted tyrosinase-derived melanoma epitope, Tyr368-379. NK cells expressing a functional TCR exhibit the capacity to degranulate in an antigen-specific manner in response to engagement of the cognate peptide/MHC ligand on target cells. In addition, upon engagement of their TCR, NK cells are fully capable of producing proinflammatory cytokines IFNγ and TNF-α, a signature mark of NK cell activation and immune cell recruitment. Finally, NK-TCR cells exhibit MHC-I-restricted antigen detection and antigen-specific lysis of tumor cells both in vitro and in vivo. Antigen-specific targeting of NK cells via TCR gene delivery stands out as a unique discovery providing a novel tool in the field of adoptive immunotherapy that can also overcome the major hurdle of “mispairing” in TCR gene therapy. Clinical trials using NK cells, including genetically modified NK cells expressing activating receptors or CARs, have clearly demonstrated a significant benefit in patients with various malignancies. The overall safety profile and promising clinical benefits of NK cells combined with the exclusive antigen specificity of the TCR, all together provide a novel approach in the design of efficient antigen-specific adoptive immunotherapy. Citation Format: Ayhan Parlar, Ece C. Sayitoglu, Cevriye Pamukcu, Anna-Maria Georgoudaki, Didem Ozkazanc, Mertkaya Aras, Benjamin Josey, Michael Chrobok, Suzanne Branecki, Pegah Zahedimaram, Lolai Ikromzoda, Evren Alici, Batu Erman, Tolga Sutlu, Adil D. Duru. Engineering antigen-specific natural killer cells against the melanoma-associated antigen tyrosinase via TCR gene transfer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A21.

Sarcomas are rare malignancies of mesenchymal origin for which current treatment options are very limited. Due to the vast heterogeneity of the various subtypes, the development of new targeted therapies requires a customized approach. Checkpoint blockade therapies are a promising alternative that still have not been systematically investigated for sarcoma patients. Thus, utilizing the infrastructure of the Cell Therapy Core Facility (CTC) at Nova Southeastern University (NSU), we developed a clinic-to-bench pipeline that enables the processing of fresh tumor material after surgical excision to generate primary sarcoma cell lines for biobanking and research purposes. Using this pipeline, we have extensively characterized mRNA, miRNA and cell surface expression profiles from more than 40 sarcoma cell lines to date, as well as validated their tumorigenicity by assessing expression of sarcoma/cancer associated genes such as Birc5, Birc2, Bcl2, Ewsr1-fli1, Syt/Ssx and Pcna. Moreover, we assessed the expression of immunomodulatory molecules that may suppress antitumor responses, such as PD-L1, PD-L2, OX40L and CD40L, using multicolor flow cytometry. Detailed characterization of the immune profile of 7 bone-related and 7 soft tissue sarcoma cell lines, assessed after 12 weeks of in vitro serial passaging, revealed a common signature in the cell surface expression of proliferative marker PCNA, and lymphocyte ligands CD112 and CD115. Moreover, immunoprofiling of the PCNA/CD112/CD155+ sarcoma cell lines (n=14) demonstrated persistent expression of PD-L2 in all cell lines and the potential applications of currently clinically approved anti-PD-1 checkpoint inhibitors for sarcoma treatment. Importantly, the conserved expression of these proteins enabled the distinction of sarcoma tumor cells from other cells in the fresh primary tumor mix directly after isolation. Thus, we next performed a comparative analysis of the immune profile of 6 freshly isolated sarcoma tumors and their respective cell lines. Comparison of the surface expression profiles of the generated cell lines and the corresponding primary tumor cells showed that the initial phenotype was preserved after more than 12 weeks of expansion and after cryopreservation. This proves that the generated cell lines are an accurate tool that can be used for the development of novel personalized immunotherapies. Most importantly, our results enabled the design of a translational research pipeline that allows the systematic characterization of the immune profile of understudied sarcomas for the first time in order to gain insight into which patients may directly benefit from available immunotherapies and which may be eligible to enroll in clinical trials assessing the efficacy of checkpoint inhibitors. In summary, the generated sarcoma biobank serves as a unique and highly representative primary resource that can be utilized to identify potential candidates for novel targeted and personalized immunotherapies for the treatment of sarcomas. Citation Format: Anna-Maria Georgoudaki, Robin Krueger, Michelle Hartman, Tamara Chinn, Dustin Tran, Reneé Potens, Wendy Weston, H. Thomas Temple, Adil D. Duru. Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A23.

Sarcomas are a broad category of cancers arising from cells of mesenchymal origin that occur in bone and soft tissues. They affect humans of all ages, and standard treatment options remain ineffective at increasing overall survival. Targeted immunotherapy is a rapidly developing field that offers extremely promising therapeutic prospects for many cancers. However, sarcomas are vastly understudied in this field. Natural killer (NK) cells are immune cells with inherent tumor-killing abilities that are a promising option for cancer immunotherapy. However, tumors can dysregulate the expression of NK cells ligands to escape immune detection, and the details of these escape mechanisms are not well known in sarcomas. Thus, a better understanding of NK cell-sarcoma interactions is necessary for the development of novel immunotherapies. In order to address this, we generated 20 cell lines from primary sarcoma tumors and characterized a group of known cancer-related NK cell receptor ligands. Our results show that all primary sarcoma cell lines express the inhibitory NKp44 ligand, PCNA, as well as CD112 and/or CD155, both of which are ligands for the NK cell activating receptor DNAM-1. However, NK cell cytotoxicity against sarcoma cells is still relatively low. Therefore, we generated a cell-based screening platform in which genetically modified (GM) NK cells that overexpress one activating receptor at a time are tested for degranulation against different sarcoma cell lines (n=12). Our results indicated patient-specific involvement of multiple NK cell receptors. In line with the conserved expression of CD112 and CD155 on sarcoma cells, we observed that DNAM-1+ GM-NK cells display significantly enhanced antitumor responses against all 12 cell lines when compared to wild-type (WT)-NK cells. We further confirmed that this enhanced NK cell response was indeed DNAM-1-dependent by interfering with the CD155/DNAM-1 interaction using blocking antibodies targeting either the receptor or its ligand. Additionally, the response of DNAM-1+ versus WT-NK cells was tested against various cancer cell lines, including those derived from a metastatic prostate carcinoma (LNCaP), primary pancreatic ductal adenocarcinoma (Capan-2), primary colorectal adenocarcinoma (Caco-2), primary lung alveolar basal epithelial adenocarcinoma (A549), metastatic neuroblastoma (SH-Sy5y), metastatic nerve sheath tumor (SNF02.2), two melanomas (A375 and DM6), and two leukemias (K562 and THP-1). In conclusion, DNAM-1 overexpression elicited a dynamic increase in NK cell degranulation against all cancer cell lines tested, including those that failed to induce a notable response in WT-NK cells, supporting the broad potential applicability of the DNAM-1+ GM-NK cells for the treatment of several malignancies. Furthermore, tumors expressing CD112 and/or CD155 can be targeted by DNAM-1+ GM-NK cells, and our GM NK cell screening platform can be used for rapid screening against not only sarcomas but also several cancers. Citation Format: Ece C. Sayitoglu, Michael Chrobok, Anna-Maria Georgoudaki, Benjamin J. Josey, Michelle Hartman, Esha Vallabhaneni, Rajeev Herekar, Savannah Bergeron, Robin Krueger, Tolga Sutlu, Evren Alici, Harry T. Temple, Adil D. Duru. Natural killer cells genetically modified to overexpress DNAM-1 exert enhanced antitumor responses against CD112/CD155+ sarcomas and other malignancies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A55.

Exosomes and the invariant NKT (iNKT) immune cell ligand α-galactosylceramide (αGC) may offer novel tools for cancer immunotherapy. In this study, we investigated whether exosomes loaded with αGC can activate iNKT cells and potentiate a cancer-specific adaptive immune response. αGC loaded exosomes readily activated iNKT cells both in vitro and in vivo. Exosomes loaded with αGC plus the model antigen ovalbumin (OVA) induced potent NK and γδ T-cell innate immune responses, and they also synergistically amplified T- and B-cell responses that were OVA specific. In contrast to soluble αGC, which anergizes iNKT cells, we found that αGC/OVA-loaded exosomes did not induce iNKT cell anergy but were more potent than soluble αGC + OVA in inducing adaptive immune responses. In an OVA-expressing mouse model of melanoma, treatment of tumor-bearing mice with αGC/OVA-loaded exosomes decreased tumor growth, increased antigen-specific CD8+ T-cell tumor infiltration, and increased median survival, relative to control mice immunized with soluble αGC + OVA alone. Notably, an additional injection of αGC/OVA-loaded exosomes further augmented the treatment effects. Our findings show that exosomes loaded with protein antigen and αGC will activate adaptive immunity in the absence of triggering iNKT-cell anergy, supporting their application in the design of a broad variety of cancer immunotherapy trials. Cancer Res; 73(13); 3865–76. ©2013 AACR.

In allogeneic hematopoietic stem cell transplantation, donor-derived T cells are key players for early immune reconstitution and efficient engraftment, as well as the graft-versus-leukemia and graft-versus-infection effects. However, a severe and quite common life-threatening complication is the development of graft-versus-host disease, during which the alloreactive donor T cells attack the host. Controlling graft-versus-host disease while preserving the benefits of graft-versus-leukemia still constitutes a challenge. A promising approach for the control of graft-versus-host disease is suicide gene therapy, which involves the ex vivo genetic modification of donor T cells with a suicide gene that allows for the selective elimination of the cells in vivo if graft-versus-host disease occurs. This article presents an overview of such approaches with special reference to lessons learned from previous clinical experiences, as well as a discussion of critical factors in suicide gene therapy.

Newly generated monoclonal antibodies directed against murine CD226 define CD226/CD155-interaction in NK-mediated killing of immature dendritic cells. The adhesion receptor CD226 (DNAM-1) is a member of the Ig superfamily possessing two extracellular V-like domains. In humans, CD226 was shown to be expressed by NK as well as T cells. During T cell priming, CD226-mediated costimulatory signals may skew the subsequent differentiation into the Th1 pathway. In addition, CD226 expressed on NK and cytotoxic T cells is engaged by its counter-receptor CD155, present on target cells, thereby triggering their elimination. We established mAb specifically recognizing mCD226, demonstrating that CD226 is expressed by precursor and mature but not developing T cells. In contrast, NK cells are distinguished by a rather heterogeneous CD226 expression profile. In addition, expression of CD226 appears coupled to that of other NK cell receptors, as high expression of CD226 was found to correlate with decreased proportions of Ly49D and H positive NK cells. Upon injection into mice, the anti-CD226 antibodies caused selective depletion of CD8+ T cells. Moreover, these antibodies as well as a naturally occurring CD226 splice variant lacking the outermost V-like domain were instrumental in determining that CD226 adheres to CD155 via its first domain. In addition, antibodies were identified as capable of blocking the CD226/CD155 interaction and to prevent NK-driven killing of immature DC. CD226 is thus the first mNK receptor identified to be essential for the elimination of this particular cell type.

Marginal zone macrophages (MZM) are strategically located in the spleen, lining the marginal sinus where they sense inflammation and capture Ag from the circulation. One of the receptors expressed by MZM is scavenger receptor macrophage receptor with collagenous structure (MARCO), which has affinity for modified self-antigens. In this article, we show that engagement of MARCO on murine macrophages induces extracellular ATP and loss of CD21 and CD62L on marginal zone B cells. Engagement of MARCO also leads to reduction of Ag transport by marginal zone B cells and affects the subsequent immune response. This study highlights a novel function for MZM in regulating Ag transport and activation, and we suggest that MARCO-dependent ATP release regulates this through shedding of CD21 and CD62L. Because systemic lupus erythematosus patients were shown to acquire autoantibodies against MARCO, this highlights a mechanism that could affect a patient’s ability to combat infections.

Previous studies have suggested that NK cells may limit T cell responses by their ability to eradicate dendritic cells, as demonstrated by NK cell-mediated killing of dendritic cells generated from mouse bone marrow cells or human monocytes with GM-CSF. In the present study, we demonstrated that conventional dendritic cells, generated in vitro with Flt3 ligand or from spleens, were resistant to NK cell-mediated lysis. However, upon stimulation with GM-CSF, NK cells could mediate lysis of these dendritic cells. GM-CSF-stimulated Flt3 ligand dendritic cells or splenic dendritic cells increased surface expression of costimulatory molecules and known NK cell ligands. Likewise, NK cells could target dendritic cells in vivo, which could be inhibited, in part, by anti-GM-CSF antibodies. The blocking of CD54 or CD226 inhibited NK cell-mediated cytotoxicity of the GM-CSF-stimulated Flt3 ligand conventional dendritic cells. Furthermore, the CD226+NKG2A− subset of NK cells was selectively better at targeting GM-CSF-stimulated Flt3 ligand conventional dendritic cells. However, CD155, a known ligand for CD226, could also act as an inhibitor of NK cell-mediated lysis, as dendritic cells lacking CD155 were more sensitive to NK cell-mediated lysis than wild-type dendritic cells. We hypothesize that by only permitting a subset of NK cells to target activated dendritic cells during inflammation, this would allow the immune system to balance between dendritic cells able to drive adaptive immune responses and dendritic cells targeted for elimination by NK cells to hinder, e.g., spread of infection.

Signaling lymphocytic activation molecule (SLAM) receptors have an important role in the development of immune responses because of their roles, for exampe, in NK cell cytotoxicity and cytokine production by NK, T cells and myeloid cells. The SLAM receptor CD244 (2B4, SLAMf4) is expressed on a variety of immune cell types but most of its functions have been examined on NK and T cells. In the present study, we investigated expression and function of CD244 in murine subsets of dendritic cells (DCs). We report that all subsets of murine DCs examined expressed CD244, although the expression levels of CD244 varied between subsets. Splenic and resident mesenteric lymph node (MLN) DCs from CD244(-/-) mice expressed lower levels of CD86 and MHC class II compared with wild-type mice. Upon Toll-like receptor (TLR) stimulation, no differences in surface expression of these molecules were observed between DCs from CD244(-/-) and wild-type mice. However, splenic DCs from CD244(-/-) mice upon stimulation with TLR binding ligands lipopolysaccharide (LPS) and CpG produced significantly higher levels of pro-inflammatory cytokines. In addition, DCs from CD244(-/-) mice elicited increased NK cell activation in vitro. These data add CD244 to a growing list of immuno-modulatory receptors found on DCs.

Autoimmune polyendocrine syndrome Type I (APS I) results in multiple endocrine organ destruction and is caused by mutations in the Autoimmune regulator gene ( AIRE ). In the thymic stroma, cells expressing the AIRE gene dictate T cell education and central tolerance. Although this function is the most studied, AIRE is also expressed in the periphery in DCs and stromal cells. Still, how AIRE regulated transcription modifies cell behaviour in the periphery is largely unknown. Here we show that AIRE is specifically expressed by 33D1 + DCs and dictates the fate of antibody secreting cell movement within the spleen. We also found that AIRE expressing 33D1 + DCs expresses self-antigens as exemplified by the hallmark gene insulin. Also, as evidence for a regulatory function, absence of Aire in 33D1 + DCs led to reduced levels of the chemokine CXCL12 and increased co-stimulatory properties. This resulted in altered activation and recruitment of T-follicular helper cells and germinal centre B cells. The altered balance leads to a change of the early response to a T cell-dependent antigen in Aire −/− mice. These findings add to the understanding of how specific DC subtypes regulate the early responses during T cell-dependent antibody responses within the spleen and further define the role of AIRE in the periphery as regulator of self-antigen expression and lymphocyte migration.

Exosomes, nanosized endogenous vesicles, can induce adaptive immune responses and have been tested in cancer treatment. Similarly, the iNKT cell ligand α-galactosylceramide (αGC) presented on the MHC class I-like molecule CD1d has been used for therapeutic purposes in cancer trials. Here, we investigated if exosomes can be loaded with αGC to activate iNKT cells and potentiate a cancer-specific adaptive immune response. We show that αGC loaded exosomes readily activate iNKT cells both in vitro and in vivo. In addition, exosomes loaded with αGC and the model antigen ovalbumin (OVA) induced a potent NK and γδ T cell innate immune response, followed by synergistically amplified T and B cell responses. Interestingly, αGC/OVA-loaded exosomes did not induce iNKT cell anergy, in contrast to soluble αGC, and were more potent than soluble αGC and OVA in inducing adaptive immunity. Finally, tumor-bearing mice treated with αGC/OVA-loaded exosomes displayed decreased tumor growth and increased T cell tumor infiltration in comparison to mice immunized with OVA-loaded exosomes in an OVA-specific mouse melanoma model. These results show that exosomes loaded with protein antigen and the glycolipid αGC effectively activate both innate and adaptive immunity, important for future cancer immunotherapy design.