Martijn H. den Brok

Toll-like receptor 2 controls expansion and function of regulatory T cells

Tregs play a central role in the suppression of immune reactions and prevention of autoimmune responses harmful to the host. During acute infection, however, Tregs might hinder effector T cell activity directed toward the elimination of the pathogenic challenge. Pathogen recognition receptors from the TLR family expressed by innate immune cells are crucial for the generation of effective immunity. We have recently shown the CD4CD25 Treg subset in TLR2 mice to be significantly reduced in number compared with WT littermate control mice, indicating a link between Tregs and TLR2. Here, we report that the TLR2 ligand Pam3Cys, but not LPS (TLR4) or CpG (TLR9), directly acts on purified Tregs in a MyD88-dependent fashion. Moreover, when combined with TCR stimulation, TLR2 triggering augmented Treg proliferation in vitro and in vivo and resulted in a temporal loss of the suppressive Treg phenotype in vitro by directly affecting Tregs. Importantly, WT Tregs adoptively transferred into TLR2 mice were neutralized by systemic administration of TLR2 ligand during the acute phase of a Candida albicans infection, resulting in a 100-fold reduced C. albicans outgrowth. This demonstrates that in vivo TLR2 also controls the function of Tregs and establishes a direct link between TLRs and the control of immune responses through Tregs.

In Situ Tumor Ablation Creates an Antigen Source for the Generation of Antitumor Immunity

Tumor-destructing techniques, like radiofrequency ablation (RFA), allow eradication of large tumors. Potentially, in situ tumor destruction also can provide the immune system with an antigen source for the induction of antitumor immunity. Antigen-presenting cells could take up antigens in the periphery after which they induce specific immune responses. Recent data show that especially antigen-presenting dendritic cells are crucial for the induction of potent immune responses. However, virtually nothing is known regarding the induction of immune responses after in situ tumor destruction in mice or humans. We used the well-defined murine B16-OVA melanoma cell line to develop a novel tumor model to explore: (a) the immunologic consequences of in situ tumor destruction; and (b) the efficacy of a combination approach of tumor destruction and immunostimulation. Applying this model system we demonstrate that following RFA, a weak but detectable immune response develops, directed against OVA, but also against a broader range of B16 antigens. Adoptive transfer experiments further indicate that antitumor reactivity can be transferred to naïve mice by splenocytes. To augment the response observed, we administered a blocking monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 at the time of tumor destruction. Interestingly, this strongly enhanced antitumor immunity, resulting in long-lasting tumor protection. These results illustrate that in situ tumor destruction can provide a useful antigen source for the induction of antitumor immunity, provided that additional immunostimulatory signals are coadministered.

Route of administration of the TLR9 agonist CpG critically determines the efficacy of cancer immunotherapy in mice.

Background The TLR9 agonist CpG is increasingly applied in preclinical and clinical studies as a therapeutic modality to enhance tumor immunity. The clinical application of CpG appears, however, less successful than would be predicted from animal studies. One reason might be the different administration routes applied in most mouse studies and clinical trials. We studied whether the efficacy of CpG as an adjuvant in cancer immunotherapy is dependent on the route of CpG administration, in particular when the tumor is destructed in situ. Methodology/Principal Findings In situ tumor destruction techniques are minimally invasive therapeutic alternatives for the treatment of (nonresectable) solid tumors. In contrast to surgical resection, tumor destruction leads to the induction of weak but tumor-specific immunity that can be enhanced by coapplication of CpG. As in situ tumor destruction by cryosurgery creates an instant local release of antigens, we applied this model to study the efficacy of CpG to enhance antitumor immunity when administrated via different routes: peritumoral, intravenous, and subcutaneous but distant from the tumor. We show that peritumoral administration is superior in the activation of dendritic cells, induction of tumor-specific CTL, and long-lasting tumor protection. Although the intravenous and subcutaneous (at distant site) exposures are commonly used in clinical trials, they only provided partial protection or even failed to enhance antitumor responses as induced by cryosurgery alone. Conclusions/Significance CpG administration greatly enhances the efficacy of in situ tumor destruction techniques, provided that CpG is administered in close proximity of the released antigens. Hence, this study helps to provide directions to fully benefit from CpG as immune stimulant in a clinical setting.

Sweet escape: Sialic acids in tumor immune evasion

Sialic acids represent a family of sugar molecules derived from neuraminic acid that frequently terminate glycan chains and contribute to many biological processes. Already five decades ago, aberrantly high expression of sialic acids has been proposed to protect cancer cells from recognition and eradication by the immune system. Today, increased understanding at the molecular level demonstrates the broad immunomodulatory capacity of tumor-derived sialic acids that is, at least in part, mediated through interactions with immunoinhibitory Siglec receptors. Here we will review current studies from a sialic acid sugar perspective showing that tumor-derived sialic acids disable major killing mechanisms of effector immune cells, trigger production of immune suppressive cytokines and dampen activation of antigen-presenting cells and subsequent induction of anti-tumor immune responses. Furthermore, strategies to modulate sialic acid expression in cancer cells to improve cancer immunotherapy will be discussed.

Mechanical high-intensity focused ultrasound destruction of soft tissue: working mechanisms and physiologic effects

The best known method of high-intensity focused ultrasound is thermal ablation, but interest in non-thermal, mechanical destruction is increasing. The advantages of mechanical ablation are that thermal protein denaturation remains limited and less damage is created to the surrounding tissue by thermal diffusion. The two main techniques for mechanical fragmentation of tissue are histotripsy and boiling histotripsy. These techniques can be used for complete liquefaction of tumor tissue into submicron fragments, after which the fragmented tissue can be easily removed by natural (immunologic) responses. Interestingly it seems that there is a correlation between the degree of destruction and tissue specific characteristics based on the treatment settings used. In this review article, the technical aspects of these two techniques are described, and an overview of the in vivo pathologic and immunologic responses is provided.

Thermal and mechanical high-intensity focused ultrasound: perspectives on tumor ablation, immune effects and combination strategies

Tumor ablation technologies, such as radiofrequency-, cryo- or high-intensity focused ultrasound (HIFU) ablation will destroy tumor tissue in a minimally invasive manner. Ablation generates large volumes of tumor debris in situ, releasing multiple bio-molecules like tumor antigens and damage-associated molecular patterns. To initiate an adaptive antitumor immune response, antigen-presenting cells need to take up tumor antigens and, following activation, present them to immune effector cells. The impact of the type of tumor ablation on the precise nature, availability and suitability of the tumor debris for immune response induction, however, is poorly understood. In this review, we focus on immune effects after HIFU-mediated ablation and compare these to findings using other ablation technologies. HIFU can be used both for thermal and mechanical destruction of tissue, inducing coagulative necrosis or subcellular fragmentation, respectively. Preclinical and clinical results of HIFU tumor ablation show increased infiltration and activation of CD4+ and CD8+ T cells. As previously observed for other types of tumor ablation technologies, however, this ablation-induced enhanced infiltration alone appears insufficient to generate consistent protective antitumor immunity. Therapies combining ablation with immune stimulation are therefore expected to be key to boost HIFU-induced immune effects and to achieve systemic, long-lasting, antitumor immunity.

A transplantable TH-MYCN transgenic tumor model in C57Bl/6 mice for preclinical immunological studies in neuroblastoma

Current multimodal treatments for patients with neuroblastoma (NBL), including anti‐disialoganglioside (GD2) monoclonal antibody (mAb) based immunotherapy, result in a favorable outcome in around only half of the patients with advanced disease. To improve this, novel immunocombinational strategies need to be developed and tested in autologous preclinical NBL models. A genetically well‐explored autologous mouse model for NBL is the TH‐MYCN model. However, the immunobiology of the TH‐MYCN model remains largely unexplored. We developed a mouse model using a transplantable TH‐MYCN cell line in syngeneic C57Bl/6 mice and characterized the immunobiology of this model. In this report, we show the relevance and opportunities of this model to study immunotherapy for human NBL. Similar to human NBL cells, syngeneic TH‐MYCN‐derived 9464D cells endogenously express the tumor antigen GD2 and low levels of MHC Class I. The presence of the adaptive immune system had little or no influence on tumor growth, showing the low immunogenicity of the NBL cells. In contrast, depletion of NK1.1+ cells resulted in enhanced tumor outgrowth in both wild‐type and Rag1−/− mice, showing an important role for NK cells in the natural anti‐NBL immune response. Analysis of the tumor infiltrating leukocytes ex vivo revealed the presence of both tumor associated myeloid cells and T regulatory cells, thus mimicking human NBL tumors. Finally, anti‐GD2 mAb mediated NBL therapy resulted in ADCC in vitro and delayed tumor outgrowth in vivo. We conclude that the transplantable TH‐MYCN model represents a relevant model for the development of novel immunocombinatorial approaches for NBL patients.

Saponin-based adjuvants create a highly effective anti-tumor vaccine when combined with in situ tumor destruction.

Todays most commonly used microbial vaccines are essentially composed of antigenic elements and a non-microbial adjuvant, and induce solid amounts of antibodies. Cancer vaccines mostly aim to induce anti-tumor CTL-responses, which require cross-presentation of tumor-derived antigens by dendritic cells (DCs). Adjuvants that improve DC function and antigen cross-presentation are therefore advantageous for inducing anti-tumor immunity. Previously, we have reported that in situ tumor destruction of established murine tumors by ablation efficiently delivers antigens to DC for the in vivo induction of anti-tumor immunity. Yet, tumor ablation alone resulted in only partial protection against a subsequent tumor-challenge. In this article, the ability of various non-microbial vaccine adjuvants to modulate the immune response following cryo-ablation was tested. The data show that tumor ablation with co-injection of saponin-based adjuvants, but not oil-in-water, water-in-oil or alum-based adjuvants, creates a highly effective in situ vaccine. Draining lymph node CD11c+ DCs acquire antigens more efficiently and become increasingly activated following ablation with saponin adjuvants relative to ablation alone. Moreover, our data reveal that the saponin-based adjuvants facilitate an in this model unprecedented level of antigen cross-presentation, induction of tumor-specific CTL and long-lasting tumor protection. Collectively, combining saponin-based adjuvants with in situ tumor destruction leads to an extremely potent systemic anti-tumor response. This combination approach forms a powerful in situ DC vaccine for which no prior knowledge of tumor antigens is required. As saponin-based adjuvants are currently clinically available, they represent attractive tools for various human and veterinary settings where in situ tumor destruction is applied.

Sialic Acid Glycoengineering Using an Unnatural Sialic Acid for the Detection of Sialoglycan Biosynthesis Defects and On-Cell Synthesis of Siglec Ligands

Sialoglycans play a vital role in physiology, and aberrant sialoglycan expression is associated with a broad spectrum of diseases. Since biosynthesis of sialoglycans is only partially regulated at the genetic level, chemical tools are crucial to study their function. Here, we report the development of propargyloxycarbonyl sialic acid (Ac5NeuNPoc) as a powerful tool for sialic acid glycoengineering. Ac5NeuNPoc showed strongly increased labeling efficiency and exhibited less toxicity compared to those of widely used mannosamine analogues in vitro and was also more efficiently incorporated into sialoglycans in vivo. Unlike mannosamine analogues, Ac5NeuNPoc was exclusively utilized in the sialoglycan biosynthesis pathway, allowing a genetic defect in sialic acid biosynthesis to be specifically detected. Furthermore, Ac5NeuNPoc-based sialic acid glycoengineering enabled the on-cell synthesis of high-affinity Siglec-7 ligands and the identification of a novel Siglec-2 ligand. Thus, Ac5NeuNPoc glycoengineering is a highly efficient, nontoxic, and selective approach to study and modulate sialoglycan interactions on living cells.

Saponin-based adjuvants induce cross-presentation in dendritic cells by intracellular lipid body formation

Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is induced is not clear. Here we show that SBAs uniquely induce intracellular lipid bodies (LBs) in the CD11b+ DC subset in vitro and in vivo. Using genetic and pharmacological interference in models for vaccination and in situ tumour ablation, we demonstrate that LB induction is causally related to the saponin-dependent increase in cross-presentation and T-cell activation. These findings link adjuvant activity to LB formation, aid the application of SBAs as a cancer vaccine component, and will stimulate development of new adjuvants enhancing T-cell-mediated immunity.