Prevention of EAE by tolerogenic vaccination with PEGylated antigenic peptides.

BACKGROUND: Therapeutic treatment options for chronic autoimmune disorders such as multiple sclerosis (MS) rely largely on the use of non-specific immunosuppressive drugs, which are not able to cure the disease. Presently, approaches to induce antigen-specific tolerance as a therapeutic approach; for example, by peptide-based tolerogenic 'inverse' vaccines have regained great interest. We have previously shown that coupling of peptides to carriers can enhance their capacity to induce regulatory T cells in vivo. METHOD: In this present study, we investigated whether the tolerogenic potential of immunodominant myelin T-cell epitopes can be improved by conjugation to the synthetic carrier polyethylene glycol (PEG) in an experimental autoimmune encephalomyelitis (EAE) mouse model for chronic MS (MOG C57BL/6). RESULTS: Preventive administration of the PEGylated antigenic peptide could strongly suppress the development of EAE, accompanied by reduced immune cell infiltration in the central nervous system (CNS). Depletion of regulatory T cells (Tregs) abrogated the protective effect indicating that Tregs play a crucial role in induction of antigen-specific tolerance in EAE. Treatment during the acute phase of disease was safe and did not induce immune activation. However, treatment at the peak of disease did not affect the disease course, suggesting that either induction of Tregs does not occur in the highly inflamed situation, or that the immune system is refractory to regulation in this condition. CONCLUSION: PEGylation of antigenic peptides is an effective and feasible strategy to improve tolerogenic (Treg-inducing) peptide-based vaccines, but application for immunotherapy of overt disease might require modifications or combination therapies that simultaneously suppress effector mechanisms.

Tolerogenic Immunomodulation by PEGylated Antigenic Peptides.

Current treatments for autoimmune disorders rely on non-specific immunomodulatory and global immunosuppressive drugs, which show a variable degree of efficiency and are often accompanied by side effects. In contrast, strategies aiming at inducing antigen-specific tolerance promise an exclusive specificity of the immunomodulation. However, although successful in experimental models, peptide-based tolerogenic "inverse" vaccines have largely failed to show efficacy in clinical trials. Recent studies showed that repetitive T cell epitopes, coupling of peptides to autologous cells, or peptides coupled to nanoparticles can improve the tolerogenic efficacy of peptides, suggesting that size and biophysical properties of antigen constructs affect the induction of tolerance. As these materials bear hurdles with respect to preparation or regulatory aspects, we wondered whether conjugation of peptides to the well-established and clinically proven synthetic material polyethylene glycol (PEG) might also work. We here coupled the T cell epitope OVA323-339 to polyethylene glycols of different size and structure and tested the impact of these nano-sized constructs on regulatory (Treg) and effector T cells in the DO11.10 adoptive transfer mouse model. Systemic vaccination with PEGylated peptides resulted in highly increased frequencies of Foxp3+ Tregs and reduced frequencies of antigen-specific T cells producing pro-inflammatory TNF compared to vaccination with the native peptide. PEGylation was found to extend the bioavailability of the model peptide. Both tolerogenicity and bioavailability were dependent on PEG size and structure. In conclusion, PEGylation of antigenic peptides is an effective and feasible strategy to improve Treg-inducing, peptide-based vaccines with potential use for the treatment of autoimmune diseases, allergies, and transplant rejection.

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1-4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle7.

Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Treg Phenotype.

CD4+ regulatory T cells (Tregs) are key mediators of immunological tolerance and promising effector cells for immuno-suppressive adoptive cellular therapy to fight autoimmunity and chronic inflammation. Their functional stability is critical for their clinical utility and has been correlated to the demethylated state of the TSDR/CNS2 enhancer element in the Treg lineage transcription factor FOXP3. However, proof for a causal contribution of the TSDR de-methylation to FOXP3 stability and Treg induction is so far lacking. We here established a powerful transient-transfection CRISPR-Cas9-based epigenetic editing method for the selective de-methylation of the TSDR within the endogenous chromatin environment of a living cell. The induced de-methylated state was stable over weeks in clonal T cell proliferation cultures even after expression of the editing complex had ceased. Epigenetic editing of the TSDR resulted in FOXP3 expression, even in its physiological isoform distribution, proving a causal role for the de-methylated TSDR in FOXP3 regulation. However, successful FOXP3 induction was not associated with a switch towards a functional Treg phenotype, in contrast to what has been reported from FOXP3 overexpression approaches. Thus, TSDR de-methylation is required, but not sufficient for a stable Treg phenotype induction. Therefore, targeted demethylation of the TSDR may be a critical addition to published in vitro Treg induction protocols which so far lack FOXP3 stability.

Linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo.

Inhibition of influenza A virus infection by multivalent sialic acid inhibitors preventing viral hemagglutinin binding to host cells of the respiratory tract is a promising strategy. However, optimal geometry and optimal ligand presentation on multivalent scaffolds for efficient inhibition both in vitro and in vivo application are still unclear. Here, by comparing linear and dendritic polyglycerol sialosides (LPGSA and dPGSA) we identified architectural requirements and optimal ligand densities for an efficient multivalent inhibitor of influenza virus A/X31/1 (H3N2). Due to its large volume, the LPGSA at optimal ligand density sterically shielded the virus significantly better than the dendritic analog. A statistical mechanics model rationalizes the relevance of ligand density, morphology, and the size of multivalent scaffolds for the potential to inhibit virus-cell binding. Optimized LPGSA inhibited virus infection at IC50 in the low nanomolar nanoparticle concentration range and also showed potent antiviral activity against two avian influenza strains A/Mallard/439/2004 (H3N2) and A/turkey/Italy/472/1999 (H7N1) post infection. In vivo application of inhibitors clearly confirmed the higher inhibition potential of linear multivalent scaffolds to prevent infection. The optimized LPGSA did not show any acute toxicity, and was much more potent than the neuraminidase inhibitor oseltamivir carboxylate in vivo. Combined application of the LPGSA and oseltamivir carboxylate revealed a synergistic inhibitory effect and successfully prevented influenza virus infection in mice.

Multivalent Peptide-Nanoparticle Conjugates for Influenza-Virus Inhibition.

To inhibit binding of the influenza A virus to the host cell glycocalyx, we generate multivalent peptide-polymer nanoparticles binding with nanomolar affinity to the virus via its spike protein hemagglutinin. The chosen dendritic polyglycerol scaffolds are highly biocompatible and well suited for a multivalent presentation. We could demonstrate in vitro that by increasing the size of the polymer scaffold and adjusting the peptide density, viral infection is drastically reduced. Such a peptide-polymer conjugate qualified also in an in vivo infection scenario. With this study we introduce the first non-carbohydrate-based, covalently linked, multivalent virus inhibitor in the nano- to picomolar range by ensuring low peptide-ligand density on a larger dendritic scaffold.

Facilitated Peptide Transport via the Mucosal Epithelium: Impact on Tolerance Induction.

A hallmark of autoimmunity is the breakdown of tolerance and generation of effector responses against self-antigens. Re-establishment of tolerance in autoimmune disorders was always the most desired treatment option; however, despite many efforts, clinical trials have been largely unsuccessful. This also applies to the generation of oral tolerance, which seems to be a default response type of the mucosa-associated lymphoid tissues to harmless antigens. In this study, we report improved efficacy of oral tolerance induction by coupling antigen with the newly identified mucosal carrier peptide 13C. Antigen coupled to 13C is efficiently taken up in the gastrointestinal tract and could be visualized in cells of the lamina propria. Oral, rectal, or nasal treatment effectively induced the proliferation of antigen-specific T cells with some increase in the frequency of regulatory T cells. In a model of delayed-type hypersensitivity, especially intrarectal tolerization treatment resulted in reduced footpad swelling, demonstrating a moderate tolerogenic effect of mucosal treatment with 13C coupled antigen. Coupling of antigens to a transmucosal carrier, therefore, is a promising tool to improve the efficacy of vaccination via mucosal surfaces.

Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene.

E- and P-selectin ligands (E- and P-ligs) guide effector memory T cells into skin and inflamed regions, mediate the inflammatory recruitment of leukocytes, and contribute to the localization of hematopoietic precursor cells. A better understanding of their molecular regulation is therefore of significant interest with regard to therapeutic approaches targeting these pathways. In this study, we examined the transcriptional regulation of fucosyltransferase 7 (FUT7), an enzyme crucial for generation of the glycosylated E- and P-ligs. We found that high expression of the coding gene fut7 in murine CD4+ T cells correlates with DNA demethylation within a minimal promoter in skin/inflammation-seeking effector memory T cells. Retinoic acid, a known inducer of the gut-homing phenotype, abrogated the activation-induced demethylation of this region, which contains a cAMP responsive element. Methylation of the promoter or mutation of the cAMP responsive element abolished promoter activity and the binding of CREB, confirming the importance of this region and of its demethylation for fut7 transcription in T cells. Furthermore, studies on human CD4+ effector memory T cells confirmed demethylation within FUT7 corresponding to high FUT7 expression. Monocytes showed an even more extensive demethylation of the FUT7 gene whereas hepatocytes, which lack selectin ligand expression, exhibited extensive methylation. In conclusion, we show that DNA demethylation within the fut7 gene controls selectin ligand expression in mice and humans, including the inducible topographic commitment of T cells for skin and inflamed sites.

Core 2 ß1,6-N-acetylglucosaminyltransferase-I, crucial for P-selectin ligand expression is controlled by a distal enhancer regulated by STAT4 and T-bet in CD4+ T helper cells 1.

P-selectin ligands (P-ligs) support the recruitment of lymphocytes into inflamed tissues. Binding to P-selectin is mediated by oligosaccharide groups synthesized by means of several glycosyltransferases including core 2 ß1,6-N-acetylglucosaminyltransferase-I (C2GlcNAcT-I), encoded by the gene Gcnt1. Using Gcnt1(-/-) Th1 cells, we show that C2GlcNAcT-I is crucial for inflammatory T cell homing in vivo. To understand the molecular regulation of Gcnt1 in CD4(+) T helper cells, we performed ChIP-on-chip experiments across the Gcnt1 locus assessing the chromatin structure in P-lig-expressing versus non-expressing CD4(+) T cells. This identified a distal region about 20kb upstream of the promoter where the presence of a H3K27me3 mark correlated with Gcnt1 repression. This region possessed IL-12-dependent enhancer activity in reporter assays, in accordance with preferential IL-12-dependent induction of Gcnt1 in vitro. STAT4 and T-bet cooperated in control of the enhancer activity. Deficiency in either one resulted in drastically reduced Gcnt1 mRNA expression in differentiated Th1 cells. While both STAT4 and T-bet were bound to the enhancer early after activation only T-bet binding persisted throughout the expansion phase after TCR signal cessation. This suggests sequential action of STAT4 and T-bet at the enhancer. In summary, we show that Gcnt1 transcription and subsequent P-lig induction in Th1 cells is governed by binding of STAT4 and T-bet to a distal enhancer and further regulated by epigenetic marks such as H3K27me3.

Immune Modulation and Prevention of Autoimmune Disease by Repeated Sequences from Parasites Linked to Self Antigens.

Parasite proteins containing repeats are essential invasion ligands, important for their ability to evade the host immune system and to induce immunosuppression. Here, the intrinsic suppressive potential of repetitive structures within parasite proteins was exploited to induce immunomodulation in order to establish self-tolerance in an animal model of autoimmune neurological disease. We tested the tolerogenic potential of fusion proteins containing repeat sequences of parasites linked to self-antigens. The fusion constructs consist of a recombinant protein containing repeat sequences derived from the S-antigen protein (SAg) of Plasmodium falciparum linked to a CD4 T cell epitope of myelin. They were tested for their efficacy to control the development of experimental autoimmune encephalomyelitis (EAE), In addition, we used the DO11.10 transgenic mouse model to study the immune mechanisms involved in tolerance induced by SAg fusion proteins. We found that repeated sequences of P. falciparum SAg protein linked to self-epitopes markedly protected mice from EAE. These fusion constructs were powerful tolerizing agents not only in a preventive setting but also in the treatment of ongoing disease. The tolerogenic effect was shown to be antigen-specific and strongly dependent on the physical linkage of the T cell epitope to the parasite structure and on the action of anti-inflammatory cytokines like IL-10 and TGF-ß. Other mechanisms include down-regulation of TNF-α accompanied by increased numbers of FoxP3+ cells. This study describes the use of repetitive structures from parasites linked to defined T cell epitopes as an effective method to induce antigen-specific tolerance with potential applicability for the treatment and prevention of autoimmune diseases.

reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4(+) memory T cells.

The combinatorial action of co-localizing chromatin modifications and regulators determines chromatin structure and function. However, identifying co-localizing chromatin features in a high-throughput manner remains a technical challenge. Here we describe a novel reChIP-seq approach and tailored bioinformatic analysis tool, normR that allows for the sequential enrichment and detection of co-localizing DNA-associated proteins in an unbiased and genome-wide manner. We illustrate the utility of the reChIP-seq method and normR by identifying H3K4me3 or H3K27me3 bivalently modified nucleosomes in primary human CD4(+) memory T cells. We unravel widespread bivalency at hypomethylated CpG-islands coinciding with inactive promoters of developmental regulators. reChIP-seq additionally uncovered heterogeneous bivalency in the population, which was undetectable by intersecting H3K4me3 and H3K27me3 ChIP-seq tracks. Finally, we provide evidence that bivalency is established and stabilized by an interplay between the genome and epigenome. Our reChIP-seq approach augments conventional ChIP-seq and is broadly applicable to unravel combinatorial modes of action.

IL-10+ Innate-like B Cells Are Part of the Skin Immune System and Require α4ß1 Integrin To Migrate between the Peritoneum and Inflamed Skin.

The skin is an important barrier organ and frequent target of autoimmunity and allergy. In this study, we found innate-like B cells that expressed the anti-inflammatory cytokine IL-10 in the skin of humans and mice. Unexpectedly, innate-like B1 and conventional B2 cells showed differential homing capacities with peritoneal B1 cells preferentially migrating into the inflamed skin of mice. Importantly, the skin-homing B1 cells included IL-10-secreting cells. B1 cell homing into the skin was independent of typical skin-homing trafficking receptors and instead required α4ß1-integrin. Moreover, B1 cells constitutively expressed activated ß1 integrin and relocated from the peritoneum to the inflamed skin and intestine upon innate stimulation, indicating an inherent propensity to extravasate into inflamed and barrier sites. We conclude that innate-like B cells migrate from central reservoirs into skin, adding an important cell type with regulatory and protective functions to the skin immune system.

Surface-modified yeast cells: A novel eukaryotic carrier for oral application.

The effective targeting and subsequent binding of particulate carriers to M cells in Peyer's patches of the gut is a prerequisite for the development of oral delivery systems. We have established a novel carrier system based on cell surface expression of the ß1-integrin binding domain of invasins derived from Yersinia enterocolitica and Yersinia pseudotuberculosis on the yeast Saccharomyces cerevisiae. All invasin derivatives were shown to be effectively expressed on the cell surface and recombinant yeast cells showed improved binding to both human HEp-2 cells and M-like cells in vitro. Among the different derivatives tested, the integrin-binding domain of Y. enterocolitica invasin proved to be the most effective and was able to target Peyer's patches in vivo. In conclusion, cell surface-modified yeasts might provide a novel bioadhesive, eukaryotic carrier system for efficient and targeted delivery of either antigens or drugs via the oral route.

Depletion of regulatory T cells leads to an exacerbation of delayed-type hypersensitivity arthritis in C57BL/6 mice that can be counteracted by IL-17 blockade.

Rodent models of arthritis have been extensively used in the elucidation of rheumatoid arthritis (RA) pathogenesis and are instrumental in the development of therapeutic strategies. Here we utilise delayed-type hypersensitivity arthritis (DTHA), a model in C57BL/6 mice affecting one paw with synchronised onset, 100% penetrance and low variation. We investigate the role of regulatory T cells (Tregs) in DTHA through selective depletion of Tregsand the role of IL-17 in connection with Tregdepletion. Given the relevance of Tregsin RA, and the possibility of developing Treg-directed therapies, this approach could be relevant for advancing the understanding of Tregsin inflammatory arthritis. Selective depletion of Tregswas achieved using aFoxp3-DTR-eGFPmouse, which expresses the diphtheria toxin receptor (DTR) and enhanced green fluorescent protein (eGFP) under control of theFoxp3gene. Anti-IL-17 monoclonal antibody (mAb) was used for IL-17 blockade. Numbers and activation of Tregsincreased in the paw and its draining lymph node in DTHA, and depletion of Tregsresulted in exacerbation of disease as shown by increased paw swelling, increased infiltration of inflammatory cells, increased bone remodelling and increased production of inflammatory mediators, as well as increased production of anti-citrullinated protein antibodies. Anti-IL-17 mAb treatment demonstrated that IL-17 is important for disease severity in both the presence and absence of Tregs, and that IL-17 blockade is able to rescue mice from the exacerbated disease caused by Tregdepletion and caused a reduction in RANKL, IL-6 and the number of neutrophils. We show that Tregsare important for the containment of inflammation and bone remodelling in DTHA. To our knowledge, this is the first study using theFoxp3-DTR-eGFPmouse on a C57BL/6 background for Tregdepletion in an arthritis model, and we here demonstrate the usefulness of the approach to study the role of Tregsand IL-17 in arthritis.

Identification of Targeting Peptides for Mucosal Delivery in Sheep and Mice.

In this study we identified and characterized a novel cyclic peptide that facilitates the rapid transportation of conjugated molecules across the epithelial layer of the small intestine. The peptide was initially selected from phage display libraries using a large animal experimental model, which employed consecutive in vitro and in vivo panning. The procedure was designed to enrich for peptides that facilitated transcytosis across the intestinal epithelium into the intestinal afferent lymphatic system. A small set of peptides was repeatedly isolated using this selection method; however, the cyclic nonamer CTANSSAQC, 13C, dominated. The activity of the putative targeting peptide 13C was then verified using a mouse model. These experiments showed that the 13C peptide as well as macromolecules conjugated to it were rapidly transported across the intestinal mucosa into distinct subsets of epithelial cells and CD11c+ cells located in the lamina propria and Peyer's Patches. Significant amounts of intact protein could be delivered into the systemic circulation after rectal and nasal application. Thus, peptide 13C is regarded as an attractive carrier candidate for mucosal delivery of large molecules. The preferential targeting to distinct intestinal cells may be utilized to deliver active biological drugs for the effective control of diseases of the gut.

Anti-Inflammatory Effects of IL-27 in Zymosan-Induced Peritonitis: Inhibition of Neutrophil Recruitment Partially Explained by Impaired Mobilization from Bone Marrow and Reduced Chemokine Levels.

Rapid activation of the innate immune system is critical for an efficient host response to invading pathogens. However, the inflammatory reaction has to be strictly controlled to minimize harmful immunopathology. A number of mediators including the cytokine interleukin-27 (IL-27) appear to be responsible for limitation and resolution of inflammation. Despite increasing knowledge of its suppressive effects on T cells, the influence on neutrophils and macrophages is poorly understood. To determine the role of IL-27 in innate immune responses we analysed the effect of IL-27 in a T cell independent model of zymosan-induced peritonitis. Early administration of recombinant IL-27 strongly reduced the number of neutrophils recruited to the peritoneal cavity after zymosan application as well as the neutrophil frequency in the blood. Simultaneously, IL-27 reduced the release of neutrophils from the bone marrow upon inflammation. Although cytokine levels were not affected by IL-27 treatment, the levels of the chemokines KC, MCP-1 and MIP-1α in the peritoneal fluid were strongly decreased. These findings demonstrate that IL-27 is able to control mobilisation and recruitment of neutrophils into the peritoneal cavity and identify a novel mechanism to limit inflammation caused by innate immune cells.

Integrin αE(CD103) Is Involved in Regulatory T-Cell Function in Allergic Contact Hypersensitivity.

Murine contact hypersensitivity (CHS) is a dendritic cell (DC)-dependent T-cell-mediated inflammation with CD8+ T cells as effectors and CD4+ T cells as regulators (Treg cells) that models human allergic contact dermatitis. The integrin αE(CD103) is expressed by some T-cell and DC subsets and has been implicated in epithelial lymphocyte localization, but its role in immune regulation remains enigmatic. We have identified a function for CD103 in the development of cutaneous allergic immune responses. CHS responses, but not irritant contact dermatitis, were significantly augmented in CD103-deficient mice in hapten-challenged skin. Phenotype and function of skin DCs during sensitization were normal, whereas adoptive transfer experiments revealed that the elevated CHS response in CD103-deficient mice is transferred by primed T cells and is independent of resident cells in recipient mice. While T-cell counts were elevated in challenged skin of CD103-deficient mice, the FoxP3 expression level of CD4+CD25+ Treg cells was significantly reduced, indicating impaired functionality. Indeed, Treg cells from CD103-deficient mice were not able to suppress CHS reactions during the elicitation phase. Further, CD103 on FoxP3+ Treg cells was involved in Treg retention to inflamed skin. These findings indicate an unexpected dichotomous functional role for CD103 on Treg cells by modulating FoxP3 expression.

Tolerogenic modulation of the immune response by oligoglycerol- and polyglycerol-peptide conjugates.

Peptide-based therapy is a promising strategy for antigen-specific immunosuppression to treat or even heal autoimmune diseases with significantly reduced adverse effects compared to conventional therapies. However, there has been no major success due to the drawbacks of native peptides, i.e., limited bioavailability. Considering the importance and limitations of peptide-based therapies for treatment of autoimmune diseases, we designed and constructed oligoglycerol (OG)- and polyglycerol (PG)-based peptide conjugates. They were evaluated for their biological activity (in vitro and in vivo), bioavailability, and tolerogenic potential. Among the OG- and PG-peptide constructs, PG-peptide constructs exhibited an extended bioavailability compared to OG-peptide constructs and unconjugated peptide. Interestingly, size, structure, and linker chemistry played a critical role for the tolerogenic capacity of the constructs. The PG-peptide construct bound via an ester linkage was the most tolerogenic conjugate, while the PG-peptide construct bound via an amide induced stronger proliferation, but also higher TNF production and lower frequencies of Foxp3(+) regulatory T-cells. Therefore, we conclude that PG-peptide conjugates bound via an ester linkage are not only promising candidates for tolerogenic vaccination, but also open a new avenue toward the application of peptides for the treatment of autoimmune diseases.

Human cytomegalovirus drives epigenetic imprinting of the IFNG locus in NKG2Chi natural killer cells.

Memory type 1 T helper (T(H)1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8(+) T cells or T(H)1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C(hi) NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.