Natascha Stergiou

Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo

The quality of the adaptive immune response depends on the differentiation of distinct CD4+ helper T cell subsets, and the magnitude of an immune response is controlled by CD4+Foxp3+ regulatory T cells (Treg cells). However, how a tissue- and cell type–specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell–specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the β-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3+ Treg cell subpopulation that was unable to control the maturation of IRF4+PD-L2+ dendritic cells required for the development of TH2 responses in vivo.

CpG-Loaded Multifunctional Cationic Nanohydrogel Particles as Self-Adjuvanting Glycopeptide Antitumor Vaccines

Self-adjuvanting antitumor vaccines by multifunctional cationic nanohydrogels loaded with CpG. A conjugate consisting of tumor-associated MUC1-glycopeptide B-cell epitope and tetanus toxin T-cell epitope P2 is linked to cationic nanogels. Oligonucleotide CpG complexation enhances toll-like receptor (TLR) stimulated T-cell proliferation and rapid immune activation. This co-delivery promotes induction of specific MUC1-antibodies binding to human breast tumor cells without external adjuvant.

A Fully Synthetic Four-Component Antitumor Vaccine Consisting of a Mucin Glycopeptide Antigen Combined with Three Different T-Helper-Cell Epitopes†

In a new concept of fully synthetic vaccines, the role of T-helper cells is emphasized. Here, a synthetic antitumor vaccine consisting of a diglycosylated tumor-associated MUC1 glycopeptide as the B-cell epitope was covalently cross-linked with three different T-helper-cell epitopes via squaric acid ligation of two linear (glyco)peptides. In mice this four-component vaccine administered without external immune-stimulating promoters elicit titers of MUC1-specific antibodies that were about eight times higher than those induced by a vaccine containing only one T-helper-cell epitope. The promising results indicate that multiple activation of different T-helper cells is useful for applications in which increased immunogenicity is required. In personalized medicine, in particular, this flexible construction of a vaccine can serve as a role model, for example, when T-helper-cell epitopes are needed that match human leukocyte antigens (HLA) in different patients.

A Synthetic Glycopeptide Vaccine for the Induction of a Monoclonal Antibody that Differentiates between Normal and Tumor Mammary Cells and Enables the Diagnosis of Human Pancreatic Cancer

In studies within the realm of cancer immunotherapy, the synthesis of exactly specified tumor-associated glycopeptide antigens is shown to be a key strategy for obtaining a highly selective biological reagent, that is, a monoclonal antibody that completely differentiates between tumor and normal epithelial cells and specifically marks the tumor cells in pancreas tumors. Mucin MUC1, which is overexpressed in many prevalent cancers, was identified as a promising target for this strategy. Tumor-associated MUC1 differs significantly from that expressed by normal cells, in particular by altered glycosylation. Structurally defined tumor-associated MUC1 cannot be isolated from tumor cells. We synthesized MUC1-glycopeptide vaccines and analyzed their structure-activity relationships in immunizations; a monoclonal antibody that specifically distinguishes between human normal and tumor epithelial cells was thus generated.

Protein kinase CK2 governs the molecular decision between encephalitogenic TH17 cell and Treg cell development

Significance Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system in the western world and leads to devastating disability in young adults, with only limited treatment options currently available. Our recent work demonstrates that pharmacological inhibition of the protein kinase CK2 (CK2) results in inhibition of encephalitogenic human and mouse T helper 17 (TH17) cell development and effector function while at the same time promoting development of induced regulatory T (iTreg) cells. Hence, modulation of CK2 activity might represent a promising approach for the treatment of MS and other TH17 cell-driven inflammatory diseases. T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance. Thus, the balance between Treg cells and TH17 cells determines the severity of a TH17 cell-driven disease and therefore is a promising target for treating autoimmune diseases. However, the molecular mechanisms controlling this balance are still unclear. Here, we report that pharmacological inhibition as well as genetic ablation of the protein kinase CK2 (CK2) ameliorates experimental autoimmune encephalomyelitis (EAE) severity and relapse incidence. Furthermore, CK2 inhibition or genetic ablation prevents TH17 cell development and promotes the generation of Treg cells. Molecularly, inhibition of CK2 leads to reduced STAT3 phosphorylation and strongly attenuated expression of the IL-23 receptor, IL-17, and GM-CSF. Thus, these results identify CK2 as a nodal point in TH17 cell development and suggest this kinase as a potential therapeutic target to treat TH17 cell-driven autoimmune responses.

Tick Salivary Sialostatin L Represses the Initiation of Immune Responses by Targeting IRF4-Dependent Transcription in Murine Mast Cells

Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1β and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R–deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell–derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell–specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.

Secondary-Structure-Driven Self-Assembly of Reactive Polypept(o)ides: Controlling Size, Shape, and Function of Core Cross-Linked Nanostructures

Achieving precise control over the morphology and function of polymeric nanostructures during self-assembly remains a challenge in materials as well as biomedical science, especially when independent control over particle properties is desired. Herein, we report on nanostructures derived from amphiphilic block copolypept(o)ides by secondary-structure-directed self-assembly, presenting a strategy to adjust core polarity and function separately from particle preparation in a bioreversible manner. The peptide-inherent process of secondary-structure formation allows for the synthesis of spherical and worm-like core-cross-linked architectures from the same block copolymer, introducing a simple yet powerful approach to versatile peptide-based core-shell nanostructures.

Antibody Induction Directed against the Tumor‐Associated MUC4 Glycoprotein

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti‐tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor‐associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor‐associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem‐repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4‐based vaccines induced very strong antigen‐specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies.

Immunogenicity of a Fully Synthetic MUC1 Glycopeptide Antitumor Vaccine Enhanced by Poly(I:C) as a TLR3-Activating Adjuvant

Fully synthetic MUC1 glycopeptide antitumor vaccines have a precisely specified structure and induce a targeted immune response without suppression of the immune response when using an immunogenic carrier protein. However, tumor‐associated aberrantly glycosylated MUC1 glycopeptides are endogenous structures, “self‐antigens”, that exhibit only low immunogenicity. To overcome this obstacle, a fully synthetic MUC1 glycopeptide antitumor vaccine was combined with poly(inosinic acid:cytidylic acid), poly(I:C), as a structurally defined Toll‐like receptor 3 (TLR3)‐activating adjuvant. This vaccine preparation elicited extraordinary titers of IgG antibodies which strongly bound human breast cancer cells expressing tumor‐associated MUC1. Beside the humoral response, the poly(I:C) glycopeptide vaccine induced a pro‐inflammatory environment, very important to overcome the immune‐suppressive mechanisms, and elicited a strong cellular immune response crucial for tumor elimination.

Immunization with a Synthetic Human MUC1 Glycopeptide Vaccine against Tumor-Associated MUC1 Breaks Tolerance in Human MUC1 Transgenic Mice

Breaking tolerance is crucial for effective tumor immunotherapy. We showed that vaccines containing tumor‐associated human MUC1 glycopeptides induce strong humoral antitumor responses in mice. The question remained whether such vaccines work in humans, in systems where huMUC1 is a self‐antigen. To clarify the question, mice transgenic in expressing huMUC1, mimicking the self‐tolerant environment, and wild‐type mice were vaccinated with a synthetic vaccine. This vaccine comprised STn and Tn antigens bound to a MUC1 tandem repeat peptide coupled to tetanus toxoid. The vaccine induced strong immune responses in wild‐type and huMUC1‐transgenic mice without auto‐aggressive side effects. All antisera exhibited almost equivalent binding to human breast tumor cells. Similar increases of activated B‐, CD4+ T‐, and dendritic cells was found in the lymph nodes. The results demonstrate that tumor‐associated huMUC1 glycopeptides coupled to tetanus toxoid are promising antitumor vaccines.