Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells

The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created "sialic acid low" tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing "sialic acid low" tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumorspecific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attack

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 ag

Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells

Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4+ and CD8+ T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen–loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E–mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro–established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance

Controlled release of a model vaccine by nanoporous ceramic microneedle arrays

\u3cp\u3eAbstract Current vaccination technology can advance from the use of novel ceramic nanoporous microneedle arrays (npMNA), where the material serves as a storage reservoir for vaccines. Moreover, npMNA will enhance vaccine efficacy by more precisely reaching skin dendritic cells, the kickstarters of T and B cell immunity. In the present study we assessed the efficacy of vaccination using npMNAs by in vivo application of OVA\u3csub\u3e257-264\u3c/sub\u3e peptides mixed with agonistic anti-CD40 antibodies as adjuvant. The induction of OVA-specific CD8\u3csup\u3e+\u3c/sup\u3e T cells via npMNA was comparable with the frequency induced via intradermal injection using needle-syringe. However, only when expanding the vaccination area by using two npMNAs the frequencies of induced IFN-γ-specific effector CD8\u3csup\u3e+\u3c/sup\u3e T cells were comparable with those induced via needle-syringe injection. Analysis of vaccine release from npMNA in a human ex vivo skin explant model revealed that OVA\u3csub\u3e257-264\u3c/sub\u3e peptides were indeed delivered intradermal, and release also increased by prolonging the npMNA application time on the human skin. Together, our studies demonstrate the potential of npMNA for vaccine delivery in human skin and in vivo induction of CD8\u3csup\u3e+\u3c/sup\u3e effector T cell responses.\u3c/p\u3

The stress system in Solea senegalensis: molecular and physiological approaches

Trabajo presentado en el V Workshop The cultivation of the Soles, celebrado en Faro (Portugal) del 5 al 7 de abril de 2011.The hypothalamus-pituitary-interrenal (HPI) axis is pivotal in the adaptive stress response of fish. Hypothalamic corticotropin-releasing hormone (CRH) initiates the endocrine stress response and stimulates the release of adrenocorticotropic hormone (ACTH) from the pituitary pars distalis. ACTH activates the interrenal cells of the head kidney to produce and release cortisol, which is the main stress steroid. Together with other peptides like MSH and ß-endorphin, ACTH derives from the precursor protein proopiomelanocortin (POMC) by post-translational cleavage. CRH activity is regulated by a specific CRH binding protein (CRH-BP), which is generally considered to be a potent antagonist of CRH. Chronic exposure to stressors can lead to allostatic overload, which negatively affects reproduction, growth and immune functions leading to diseases and reduced animal welfare. Considering plasma cortisol levels in fish as readout for stress, increased stocking density (a typical situation in aquaculture activity) is known to evoke crowding stress. However, the precise role of the central HPI axis has not yet been established. Furthermore, fish in aquaculture must cope with exposure to a series of acute stressors such as transport, weighing, sorting/grading and sudden environmental changes e.g. in food availability, water temperature and salinity. Proceeding from this notion, we developed new tools at molecular level (characterization of the cDNAs coding for S. senegalensis CRH, CRH-BP and two forms of POMC) and investigated their roles in relation to a chronic stressor (viz. increased stocking density) and the ability to cope with a subsequent acute stressor (viz. transfer to increased ambient salinity). Therefore, juveniles were kept at three densities (1.9, 4.7 and 9.8 kg/m2) for 33 days, and then transferred from seawater (SW, 39¿ salinity) to high salinity seawater (HSW, 55¿). The highest density imposed stress as indicated by elevated cortisol levels and CRH mRNA expression, compared to fish kept at low density. Fish kept at high density responded differently to a posterior transfer to HSW showing no cortisol or CRH response, but osmoregulatory and metabolic parameters were affected. These results indicate a compromised capacity to cope with a subsequent acute stressor, which might implicate that S. senegalensis does not adapt successfully to high stocking density. No differences in CRH-BP mRNA expression levels were found at different stocking densities but transfer to HSW enhanced expression in both low and high density stocked fish, suggesting that CRH-BP acts as a modulator of the acute stress response under our experimental conditions. As for POMC, two transcripts were found, most likely presenting subfunction partitioning between the paralogues: S. senegalensis POMC ¿A¿ shows a high homology of 98% identity with S. solea POMC, but cannot cleave to ACTH and completely lacks the opioid consensus in the ß-endorphin part. POMC ¿B¿, however, lacks the N-terminal cleavage site for ß-MSH. Furthermore, ß-endorphin derived from POMC A seems to be under neutral evolution, whereas POMC B possesses well-conserved peptide regions under strong purifying selection. Also, differential mRNA expression was seen between POMC A and B, suggesting different physiological roles, which will be further investigated. When investigating POMC mRNA expression in the Solea stress system, we recommend keeping in mind POMC¿s structure in order for correct interpretation.Peer Reviewe

Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells

The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created "sialic acid low" tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing "sialic acid low" tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumor-specific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attac

Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells

The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created "sialic acid low" tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing "sialic acid low" tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumor-specific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attack

Antigen targeting to dendritic cells combined with transient regulatory T cell inhibition results in long-term tumor regression.

Therapeutic vaccinations against cancer are still largely ineffective. Major caveats are inefficient delivery of tumor antigens to dendritic cells (DCs) and excessive immune suppression by Foxp3(+) regulatory T cells (Tregs), resulting in defective T cell priming and failure to induce tumor regression. To circumvent these problems we evaluated a novel combinatorial therapeutic strategy. We show that tumor antigen targeting to DC-SIGN in humanized hSIGN mice via glycans or specific antibodies induces superior T cell priming. Next, this targeted therapy was combined with transient Foxp3(+) Treg depletion employing hSIGNxDEREG mice. While Treg depletion alone slightly delayed B16-OVA melanoma growth, only the combination therapy instigated long-term tumor regression in a substantial fraction of mice. This novel strategy resulted in optimal generation of antigen-specific activated CD8(+) T cells which accumulated in regressing tumors. Notably, Treg depletion also allowed the local appearance of effector T cells specific for endogenous B16 antigens. This indicates that antitumor immune responses can be broadened by therapies aimed at controlling Tregs in tumor environments. Thus, transient inhibition of Treg-mediated immune suppression potentiates DC targeted antigen vaccination and tumor-specific immunity