Immunological dynamics after subcutaneous immunization with a squalene-based oil-in-water adjuvant.

The clinically successful adjuvant MF59 is used in seasonal influenza vaccines, which is proposed to enhance immunity by creating an immune-competent microenvironment in the muscle that allows recruitment of immune cells that drive adaptive immune responses. Here, we examined whether the clinically successful adjuvants MF59/AddaVax could be used for subcutaneous use and how antigen delivery can be synergized with cellular dynamics at the vaccination site. Subcutaneous injection of AddaVax leads to thickening of the skin, characterized by a neutrophil-monocyte recruitment sequence. Skin-infiltrating CCR2+Ly6Chigh monocytes showed differentiation to CD11b+Ly6C+MHCII+CD11c+CD64+ monocyte-derived DCs over time in the hypodermal layers of the skin, expressing high levels of CD209a/mDC-SIGN. Surprisingly, skin thickening was accompanied with increased white adipose tissue highly enriched with monocytes. Analysis of the skin-draining lymph nodes revealed early increases in neutrophils and moDCs at 12 hours after injection and later increases in migratory cDC2s. Subcutaneous vaccination with AddaVax enhanced antigen-specific CD8+ and CD4+ T cell responses, while moDC targeting using antigen-coupled CD209a antibody additionally boosted humoral responses. Hence, oil-in-water emulsions provide an attractive immune modulatory adjuvants aimed at increasing cellular responses, as well as antibody responses when combined with moDC targeting.

Non-digestible oligosaccharides scFOS/lcFOS facilitate safe subcutaneous immunotherapy for peanut allergy.

BACKGROUND: Improving the safety of subcutaneous immunotherapy (SCIT) for food allergy is necessary to reduce side effects and achieve long-term tolerance. We determined the effect of dietary supplementation with 1% non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) on safety and efficacy of SCIT using a peanut allergy mouse model. METHODS: After sensitization, mice received a scFOS/lcFOS or control diet for the rest of the study. To study safety of SCIT, mice were dosed with a single subcutaneous injection of peanut extract (PE) or PBS. To study efficacy, mice were dosed subcutaneously (SCIT, 3 times/week) with PE or PBS for 3 weeks. Hereafter, acute allergic skin responses, anaphylactic shock symptoms and body temperature were assessed. To study the mechanism in vitro, the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) line was sensitized with an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (BLG) and incubated with the oligosaccharides before exposure to BLG to assess direct the effect on degranulation. RESULTS: scFOS/lcFOS reduced anaphylaxis caused by a single PE SCIT dose. scFOS/lcFOS alone also reduced the acute allergic skin response. Moreover, scFOS/lcFOS supplementation resulted in lower MMCP-1 levels in serum after PE SCIT dose compared to control diet, while antibody levels were not affected by the diet. In vitro incubation with scFOS/lcFOS at 0.5% suppressed the degranulation of IgE-sensitized RBL cells. However, dietary supplementation with scFOS/lcFOS did not improve the efficacy of SCIT. CONCLUSIONS: We show that scFOS/lcFOS diet improves the safety of SCIT, as evidenced by lower anaphylactic responses without compromising the efficacy in a mouse model for peanut allergy. This effect is likely to result from the suppression of mast cell effector function.

Palmitoylated antigens for the induction of anti-tumor CD8+ T cells and enhanced tumor recognition.

Induction of tumor-specific cytotoxic CD8+ T cells (CTLs) via immunization relies on the presentation of tumor-associated peptides in major histocompatibility complex (MHC) class I molecules by dendritic cells (DCs). To achieve presentation of exogenous peptides into MHC class I, cytosolic processing and cross-presentation are required. Vaccination strategies aiming to induce tumor-specific CD8+ T cells via this exogenous route therefore pose a challenge. In this study, we describe improved CD8+ T cell induction and in vivo tumor suppression of mono-palmitic acid-modified (C16:0) antigenic peptides, which can be attributed to their unique processing route, efficient receptor-independent integration within lipid bilayers, and continuous intracellular accumulation and presentation through MHC class I. We propose that this membrane-integrating feature of palmitoylated peptides can be exploited as a tool for quick and efficient antigen enrichment and MHC class I loading. Importantly, both DCs and non-professional antigen-presenting cells (APCs), similar to tumor cells, facilitate anti-tumor immunity by efficient CTL priming via DCs and effective recognition of tumors through enhanced presentation of antigens.

Adaptable antigen matrix platforms for peptide vaccination strategies and T cell-mediated anti-tumor immunity.

Injection of antigenic peptides has been widely used as a vaccine strategy to boost T cell immunity. However, the poor immunogenicity of single peptides can potentially be strengthened through modification of the tertiary structure and the selection of the accompanying adjuvant. Here, we generated antigenic peptides into non-linear trimers by solid phase peptide synthesis, thereby enhancing antigen presentation by dendritic cells to CD8+ T cells in vitro and in vivo. CD8+ T cells from mice vaccinated with trimers showed an KLRG1+ effector phenotype and were able to recognize and kill antigen-expressing tumor cells ex vivo. Importantly, trimers outperformed synthetic long peptide in terms of T cell response even when equal number of epitopes were used for immunization. To improve the synthesis of trimers containing difficult peptide sequences, we developed a novel small molecule that functions as conjugation platform for synthetic long peptides. This platform , termed Antigen MAtriX (AMAX) improved yield, purity and solubility of trimers over conventional solid phase synthesis strategies. AMAX outperformed synthetic long peptides in terms of both CD8+ and CD4+ T cell responses and allowed functionalization with DC-SIGN-binding carbohydrates for in vivo dendritic cell targeting strategies, boosting T cell responses even further. Moreover, we show that agonistic CD40 antibody combined with MF59 (AddaVax) emulsion synergistically improves the antigen-specific T cell response of the AMAX in vivo. Also, tumor-associated antigens and neo-antigens could be incorporated in AMAX for tumor-specific CD8+ T cell responses. Importantly, immunization with a mix of neoantigen AMAX could reduce tumor growth in a pre-clinical syngeneic mouse model. Hence, we provide pre-clinical support for the induction of effector CD8+ T cells through the adaptable AMAX platform as easy implementable peptidic vaccination strategy against any antigen of choice, including neoantigens for anti-tumor immunity.

Tn Antigen Expression Contributes to an Immune Suppressive Microenvironment and Drives Tumor Growth in Colorectal Cancer.

Expression of the tumor-associated glycan Tn antigen (αGalNAc-Ser/Thr) has been correlated to poor prognosis and metastasis in multiple cancer types. However, the exact mechanisms exerted by Tn antigen to support tumor growth are still lacking. One emerging hallmark of cancer is evasion of immune destruction. Although tumor cells often exploit the glycosylation machinery to interact with the immune system, the contribution of Tn antigen to an immunosuppressive tumor microenvironment has scarcely been studied. Here, we explored how Tn antigen influences the tumor immune cell composition in a colorectal cancer (CRC) mouse model. CRISPR/Cas9-mediated knock out of the C1galt1c1 gene resulted in elevated Tn antigen levels on the cell surface of the CRC cell line MC38 (MC38-Tnhigh). RNA sequencing and subsequent GO term enrichment analysis of our Tnhigh glycovariant not only revealed differences in MAPK signaling and cell migration, but also in antigen processing and presentation as well as in cytotoxic T cell responses. Indeed, MC38-Tnhigh tumors displayed increased tumor growth in vivo, which was correlated with an altered tumor immune cell infiltration, characterized by reduced levels of cytotoxic CD8+ T cells and enhanced accumulation of myeloid-derived suppressor cells. Interestingly, no systemic differences in T cell subsets were observed. Together, our data demonstrate for the first time that Tn antigen expression in the CRC tumor microenvironment affects the tumor-associated immune cell repertoire.

Monocyte-derived APCs are central to the response of PD1 checkpoint blockade and provide a therapeutic target for combination therapy.

BACKGROUND: PD1 immune checkpoint blockade (αPD1 ICB) has shown unparalleled success in treating many types of cancer. However, response to treatment does not always lead to tumor rejection. While αPD1 ICB relies on cytotoxic CD8+ T cells, antigen-presenting cells (APCs) at the tumor site are also needed for costimulation of tumor-infiltrating lymphocytes (TILs). It is still unclear how these APCs develop and function before and during αPD1 ICB or how they are associated with tumor rejection. METHODS: Here, we used B16 mouse melanoma and MC38 colorectal carcinoma tumor models, which show differential responses to αPD1 ICB. The immune composition of ICB insensitive B16 and sensitive MC38 were extensively investigated using multi-parameter flow cytometry and unsupervised clustering and trajectory analyses. We additionally analyzed existing single cell RNA sequencing data of the myeloid compartment of patients with melanoma undergoing αPD1 ICB. Lastly, we investigated the effect of CD40 agonistic antibody on the tumor-infiltrating monocyte-derived cells during αPD1 ICB. RESULTS: We show that monocyte-derived dendritic cells (moDCs) express high levels of costimulatory molecules and are correlated with effector TILs in the tumor microenvironment (TME) after αPD1 ICB only in responding mouse tumor models. Tumor-resident moDCs showed distinct differentiation from monocytes in both mouse and human tumors. We further confirmed significant enrichment of tumor-resident differentiated moDCs in patients with melanoma responding to αPD1 ICB therapy compared with non-responding patients. Moreover, moDCs could be targeted by agonistic anti-CD40 antibody, supporting moDC differentiation, effector T-cell expansion and anti-tumor immunity. CONCLUSION: The combined analysis of myeloid and lymphoid populations in the TME during successful and non-successful PD1 ICB led to the discovery of monocyte-to-DC differentiation linked to expanding T-cell populations. This differentiation was found in patients during ICB, which was significantly higher during successful ICB. The finding of tumor-infiltrating monocytes and differentiating moDCs as druggable target for rational combination therapy opens new avenues of anti-tumor therapy design.

Immune involvement of the contralateral hemisphere in a glioblastoma mouse model.

BACKGROUND: Glioblastoma (GBM) is the most common and deadliest form of brain cancer in adults. Standard treatment, consisting of surgery and radiochemotherapy, only provides a modest survival benefit and is incapable of combating infiltrating GBM cells in other parts of the brain. New therapies in clinical trials, such as anti-programmed cell death 1 immunotherapy, have so far shown limited success in GBM. Moreover, it is unclear how the growth of GBM suppresses the immune system locally at the site of the brain tumor or if distant sites of tumor cell migration are also involved. Invasive GBM cells in brain tissue beyond the primary tumor limit the use of surgery, thus immunotherapy could be beneficial if activated/suppressed immune cells are present in the contralateral hemisphere. METHODS: Here, we used a syngeneic orthotopic GL26 GBM mouse model and multiparameter fluorescence-activated cell sorting analysis to study the phenotype of resident and infiltrating immune cells in both the brain tumor hemisphere and contralateral hemisphere. RESULTS: We show that lymphoid cells, including tumor antigen-specific CD8+ tumor-infiltrating lymphocytes (TILs) are present in the tumor and are characterized by a tolerogenic phenotype based on high immune checkpoint expression. Massive infiltration of myeloid cells is observed, expressing immune checkpoint ligands, suggesting an immune-dependent coinhibitory axis limiting TIL responses. Surprisingly, these phenotypes are paralleled in the contralateral hemisphere, showing that infiltrating immune cells are also present at distant sites, expressing key immune checkpoints and immune checkpoint ligands. CONCLUSION: Whole-brain analysis indicates active immune involvement throughout the brain, both at the site of the primary tumor and in the contralateral hemisphere. Using the right combination and timing, immune checkpoint blockade could have the potential to activate immune cells at the site of the brain tumor and at distant sites, thereby also targeting diffusely infiltrating GBM cells.

Outer membrane vesicles engineered to express membrane-bound antigen program dendritic cells for cross-presentation to CD8+ T cells.

Outer membrane vesicles (OMVs) are vesicular nano-particles produced by Gram-negative bacteria that are recently being explored as vaccine vector. The fact that OMVs can be efficiently produced by a hypervesiculating Salmonella typhimurium strain, are packed with naturally-occurring adjuvants like lipopolysaccharides (LPS), and can be engineered to express any antigen of choice, makes them ideal candidates for vaccinology. However, it is unclear whether OMVs induce dendritic cell (DC)-mediated antigen-specific T cell responses and how immune activation is coordinated. Here, we show that OMVs induce maturation of human monocyte-derived DCs, murine bone marrow-derived DCs and CD11c+ splenic DCs. OMV-induced DC maturation was dependent on the presence of LPS and the myeloid differentiation primary response 88 (MyD88) adapter protein downstream of toll-like receptor signaling. Importantly, OMVs did not induce pyroptosis/cell death, but instead provided a significant survival benefit in DCs over non-stimulated DCs. OMVs displaying a sizeable ovalbumin fragment at the vesicle surface induce potent cross-presentation in BMDCs and splenic CD11c+ DCs to OTI CD8+ T cells, dependent on MyD88. Interestingly, the OMV-induced preference to cross-presentation was only partly dependent on the BATF3-dependent CD8a+ professional cross-presenting DC subset. Hence, an OMV-specific programming of DCs that induces maturation and provides a survival benefit for antigen presentation to T cells is identified. Additionally, for the first time, antigen-specific and potent cross-presentation of antigen-loaded OMVs to CD8+ T cells is demonstrated. These data provide mechanistical insight into the processes needed for the DC-mediated cross-presentation of OMV-derived antigens to CD8+ T cells with implications for therapeutic strategies. STATEMENT OF SIGNIFICANCE: Bacteria are primarily known to cause disease. However, recent research has focused on using engineered bacteria and its byproducts as vaccine agents. In particular, outer membrane vesicles (OMVs) have shown promise in eliciting potent immunity against a variety of pathogens. While most vaccines rely on the generation of antibodies, the control of viral replication and tumor growth is driven by cytotoxic CD8+ T cells induced by dendritic cells (DCs). As such, there is a dire need for vaccines that use DCs to elicit CD8+ T cell responses. Studying OMVs as engineered biomaterial and its interaction with DCs allows tailored induction of immunity. This study includes important findings on OMV-dendritic cell interactions and for the first time supports OMVs as vehicles for the induction of antigen-specific CD8+ T cell responses. Additionally, important mechanistical insight into the molecular pathways needed for the cross-presentation of OMV-derived antigens to CD8+ T cells is provided.

Mouse DC-SIGN/CD209a as Target for Antigen Delivery and Adaptive Immunity.

The efficacy of vaccination studies aimed at targeting antigens to human DC-SIGN (hDC-SIGN) have been notoriously difficult to study in vivo, as eight dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) homologs have been described in mice. CD209a/SIGNR5 has been coined as the mouse DC-SIGN (mDC-SIGN) ortholog, based on its expression and location in the genome. Nonetheless, which properties of hDC-SIGN are covered by mDC-SIGN is poorly investigated. One of the most important functions of DC-SIGN is the induction of adaptive immunity. As such, the aim of this study is to determine the capability of mDC-SIGN to induce adaptive immune responses. Here, we show that mDC-SIGN is expressed on GM-CSF cultured bone marrow-derived dendritic cells (BMDCs) and macrophages. However, mDC-SIGN is an internalizing receptor which, unlike hDC-SIGN, quickly resurfaces after internalization. Binding of OVA-coupled anti-mDC-SIGN antibody by BMDCs leads to quick internalization, processing, and presentation to antigen-specific CD8+ and CD4+ T cells, which can be boosted using the TLR4 ligand, monophosphoryl lipid A. In the homeostatic condition, mDC-SIGN is mostly expressed on myeloid cells in the skin and spleen. A subcutaneous injection of fluorescent anti-mDC-SIGN reveals specific targeting to mDC-SIGN+ skin dendritic cells (DCs) and monocyte-derived DCs in situ. A subcutaneous vaccination strategy containing OVA-coupled anti-mDC-SIGN antibody generated antigen-specific polyfunctional CD8+ T cell and CD4+ T cell responses and a strong isotype-switched OVA-specific antibody response in vivo. We conclude that mDC-SIGN shows partly overlapping similarities to hDC-SIGN and that targeting mDC-SIGN provides a valuable approach to investigate the immunological function of DC-SIGN in vivo.

Non-dioxin-like AhR ligands in a mouse peanut allergy model.

Recently, we have shown that AhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses sensitization to peanut at least in part by inducing a functional shift toward CD4(+)CD25(+)Foxp3(+) T cells. Next to TCDD, numerous other AhR ligands have been described. In this study, we investigated the effect of three structurally different non-dioxin-like AhR ligands, e.g., 6-formylindolo[3,2-b]carbazole (FICZ), ß-naphthoflavone (ß-NF), and 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), on peanut sensitization. Female C57BL/6 mice were sensitized by administering peanut extract (PE) by gavage in the presence of cholera toxin. Before and during peanut sensitization, mice were treated with FICZ, ß-NF, or 6-MCDF. AhR gene transcription in duodenum and liver was investigated on day 5, even as the effect of these AhR ligands on CD4(+)CD25(+)Foxp3(+) T(reg) cells in spleen and mesenteric lymph nodes (MLNs). Mice treated with TCDD were included as a positive control. Furthermore, the murine reporter cell line H1G1.1c3 (CAFLUX) was used to investigate the possible role of metabolism of TCDD, FICZ, ß-NF, and 6-MCDF on AhR activation in vitro. TCDD, but not FICZ, ß-NF, and 6-MCDF, suppressed sensitization to peanut (measured by PE-specific IgE, IgG1, IgG2a and PE-induced interleukin (IL)-5, IL-10, IL-13, IL-17a, IL-22, and interferon-γ). In addition, FICZ, ß-NF, and 6-MCDF treatments less effectively induced AhR gene transcription (measured by gene expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1) compared with TCDD-treated mice. Furthermore, FICZ, ß-NF and 6-MCDF did not increase the percentage of CD4(+)CD25(+)Foxp3(+) T(reg) cells in spleen and mesenteric lymph nodes compared with PE-sensitized mice, in contrast to TCDD. Inhibition of metabolism in vitro increased AhR activation. Together, these data shows that TCDD, but not FICZ, ß-NF, and 6-MCDF suppresses sensitization to peanut. Differences in metabolism, AhR binding and subsequent gene transcription might explain these findings and warrant further studies to investigate the role of the AhR in food allergic responses.