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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.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Changes in glycosylation during tumour progression are a key hallmark of cancer. One of the glycan moieties generally overexpressed in cancer are sialic acids, which can induce immunomodulatory ...properties via binding to Siglec receptors. We here show that Pancreatic Ductal Adenocarcinoma (PDAC) tumour cells present an increased sialylation that can be recognized by Siglec-7 and Siglec-9 on myeloid cells. We identified the expression of the α2,3 sialyltransferases ST3GAL1 and ST3GAL4 as main contributor to the synthesis of ligands for Siglec-7 and Siglec-9 in tumour cells. Analysing the myeloid composition in PDAC, using single cell and bulk transcriptomics data, we identified monocyte-derived macrophages as contributors to the poor clinical outcome. Tumour-derived sialic acids dictate monocyte to macrophage differentiation via signalling through Siglec-7 and Siglec-9. Moreover, triggering of Siglec-9 in macrophages reduce inflammatory programmes, while increasing PD-L1 and IL-10 expression, illustrating that sialic acids modulate different myeloid cells. This work highlights a critical role for sialylated glycans in controlling immune suppression and provides new potential targets for cancer immunotherapy in PDAC.
The efficacy of vaccination studies aimed at targeting antigens to human DC-SIGN (hDC-SIGN) have been notoriously difficult to study
, 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
. 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
. 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
.
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-Tn
high
). RNA sequencing and subsequent GO term enrichment analysis of our Tn
high
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-Tn
high
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.
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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
BackgroundPD1 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.MethodsHere, 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.ResultsWe 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.ConclusionThe 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.
What's known on the subject? and What does the study add?
Thermal ablation influences the local tissue microenvironment. Several studies have reported that residual tumour cells may exhibit a more ...aggressive phenotype.
This study shows that incomplete CA and RFA cause an increased proliferation and decreased apptosis of residual renal tumour cells. This may be caused by stimulatory factors such as hypoxia, HSPs and inflammatory cells.
OBJECTIVE
•
To compare the effect of incomplete thermal ablation vs partial nephrectomy (PN) on growth stimulation and cellular survival in renal tumours.
MATERIALS AND METHODS
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Renca renal tumours were transplanted under the renal capsule of mice (four to six mice/group) after which incomplete radiofrequency ablation (RFA), cryoablation (CA) or PN was performed.
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At several time points after treatment, presence of cell proliferation, apoptosis, hypoxic areas, inflammatory factors and the heat‐shock proteins (HSPs) 70 and 90 were evaluated using immunohistochemistry.
RESULTS
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At 2 h after thermal ablation residual tumour cells showed increased proliferation. This hyperproliferation was significantly stronger after RFA than CA (P < 0.05) and not present after PN.
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Residual cells showed increased apoptosis after 2 h and decreased apoptosis from 2 days after thermal ablation. Apoptotic cells were significantly less evident at 3 days after RFA (P < 0.001).
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Hypoxic areas and HSPs were increasingly present from 2 h up to 7 days after thermal ablation (P < 0.001).
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Inflammatory cells infiltrated mainly the necrotic areas after thermal ablation, and their abundance peaked at 1 week after ablation (P < 0.05).
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The increased cell growth was preceded by hypoxia and presence of HSPs.
CONCLUSIONS
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CA and RFA result in an increased proliferation and decreased apoptosis of residual renal tumour cells.
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This hyperproliferation may be caused by stimulatory factors, e.g. hypoxia, HSPs and inflammatory cells, and could facilitate recurrences of renal tumours after thermal ablation.
•
This study highlights the importance of achieving complete tumour destruction.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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.
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 β-lactoglobulin (BLG) and incubated with the oligosaccharides before exposure to BLG to assess direct the effect on degranulation.
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.
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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