GILT Expression in Human Melanoma Cells Enhances Generation of Antigenic Peptides for HLA Class II-Mediated Immune Recognition

Melanoma is an aggressive skin cancer that has become increasingly prevalent in western populations. Current treatments such as surgery, chemotherapy, and high-dose radiation have had limited success, often failing to treat late stage, metastatic melanoma. Alternative strategies such as immunotherapies have been successful in treating a small percentage of patients with metastatic disease, although these treatments to date have not been proven to enhance overall survival. Several melanoma antigens (Ags) proposed as targets for immunotherapeutics include tyrosinase, NY-ESO-1, gp-100, and Mart-1, all of which contain both human leukocyte antigen (HLA) class I and class II-restricted epitopes necessary for immune recognition. We have previously shown that an enzyme, gamma-IFN-inducible lysosomal thiol-reductase (GILT), is abundantly expressed in professional Ag presenting cells (APCs), but absent or expressed at greatly reduced levels in many human melanomas. In the current study, we report that increased GILT expression generates a greater pool of antigenic peptides in melanoma cells for enhanced CD4+ T cell recognition. Our results suggest that the induction of GILT in human melanoma cells could aid in the development of a novel whole-cell vaccine for the enhancement of immune recognition of metastatic melanoma.     

The ABCs of Antigen Presentation by Stromal Non-Professional Antigen-Presenting Cells

Professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, are known for their ability to present exogenous antigens to T cells. However, many other cell types, including endothelial cells, fibroblasts, and lymph node stromal cells, are also capable of presenting exogenous antigens to either CD8+ or CD4+ T cells via cross-presentation or major histocompatibility complex (MHC) class II-mediated presentation, respectively. Antigen presentation by these stromal nonprofessional APCs differentially affect T cell function, depending on the type of cells that present the antigen, as well as the local (inflammatory) micro-environment. It has been recently appreciated that nonprofessional APCs can, as such, orchestrate immunity against pathogens, tumor survival, or rejection, and aid in the progression of various auto-immune pathologies. Therefore, the interest for these nonprofessional APCs is growing as they might be an important target for enhancing various immunotherapies. In this review, the different nonprofessional APCs are discussed, as well as their functional consequences on the T cell response, with a focus on immuno-oncology.     

Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.     

Cyclic Peptide Mimotopes for the Detection of Serum Anti–ATIC Autoantibody Biomarker in Hepato-Cellular Carcinoma

Tumor-associated (TA) autoantibodies have been identified at the early tumor stage before developing clinical symptoms, which holds hope for early cancer diagnosis. We identified a TA autoantibody from HBx-transgenic (HBx-tg) hepatocellular carcinoma (HCC) model mouse, characterized its target antigen, and examined its relationship to human HCC. The mimotopes corresponding to the antigenic epitope of TA autoantibody were screened from a random cyclic peptide library and used for the detection of serum TA autoantibody. The target antigen of the TA autoantibody was identified as an oncogenic bi-functional purine biosynthesis protein, ATIC. It was upregulated in liver cancer tissues of HBx-tg mouse as well as human HCC tissues. Over-expressed ATIC was also secreted extracellularly via the cancer-derived exosomes, which might cause auto-immune responses. The cyclic peptide mimotope with a high affinity to anti-ATIC autoantibody, CLPSWFHRC, distinguishes between serum samples from HCC patients and healthy subjects with 70.83% sensitivity, 90.68% specificity (AUC = 0.87). However, the recombinant human ATIC protein showed a low affinity to anti-ATIC autoantibody, which may be incompatible as a capture antigen for serum TA autoantibody. This study indicates that anti-ATIC autoantibody can be a potential HCC-associated serum biomarker and suggests that autoantibody biomarker’s efficiency can be improved by using antigenic mimicry to native antigens present in vivo.     

Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides

Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high-affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine- and threonine-phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well-defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol can be modified and enriched from a mixture of peptides. During the development of the beta-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphorylated residues.     

Multiple Roles for Cytokines in Atopic Dermatitis: From Pathogenic Mediators to Endotype-Specific Biomarkers to Therapeutic Targets

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, which generally presents with intense itching and recurrent eczematous lesions. AD affects up to 20% of children and 10% of adults in high-income countries. The prevalence and incidence of AD have increased in recent years. The onset of AD mostly occurs in childhood, although in some cases AD may persist in adult life or even manifest in middle age (adult-onset AD). AD pathophysiology is made of a complex net, in which genetic background, skin barrier dysfunction, innate and adaptive immune responses, as well as itch contribute to disease development, progression, and chronicization. One of the most important features of AD is skin dehydration, which is mainly caused by filaggrin mutations that determine trans-epidermal water loss, pH alterations, and antigen penetration. In accordance with the “outside-inside” theory of AD pathogenesis, in a context of an altered epidermal barrier, antigens encounter epidermal antigen presentation cells (APCs), such as epidermal Langerhans cells and inflammatory epidermal dendritic cells, leading to their maturation and Th-2 cell-mediated inflammation. APCs also bear trimeric high-affinity receptors for immunoglobulin E (IgE), which induce IgE-mediated sensitizations as part of pathogenic mechanisms leading to AD. In this review, we discuss the role of cytokines in the pathogenesis of AD, considering patients with various clinical AD phenotypes. Moreover, we describe the cytokine patterns in patients with AD at different phases of the disease evolution, as well as in relation to different phenotypes/endotypes, including age, race, and intrinsic/extrinsic subtypes. We also discuss the outcomes of current biologics for AD, which corroborate the presence of multiple cytokine axes involved in the background of AD. A deep insight into the correlation between cytokine patterns and the related clinical forms of AD is a crucial step towards increasingly personalized, and therefore more efficient therapy.     

Surface engineering on mesoporous silica chips for enriching low molecular weight phosphorylated proteins

Phosphorylated peptides and proteins play an important role in normal cellular activities, e.g., gene expression, mitosis, differentiation, proliferation, and apoptosis, as well as tumor initiation, progression and metastasis. However, technical hurdles hinder the use of common fractionation methods to capture phosphopeptides from complex biological fluids such as human sera. Herein, we present the development of a dual strategy material that offers enhanced capture of low molecular weight phosphoproteins: mesoporous silica thin films with precisely engineered pore sizes that sterically select for molecular size combined with chemically selective surface modifications (i.e. Ga3+, Ti4+ and Zr4+) that target phosphoroproteins. These materials provide high reproducibility (CV=18%) and increase the stability of the captured proteins by excluding degrading enzymes, such as trypsin. The chemical and physical properties of the composite mesoporous thin films were characterized by X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy and ellipsometry. Using mass spectroscopy and biostatistics analysis, the enrichment efficiency of different metal ions immobilized on mesoporous silica chips was investigated. The novel technology reported provides a platform capable of efficiently profiling the serum proteome for biomarker discovery, forensic sampling, and routine diagnostic applications.     

Carbohydrate-mediated targeting of antigen to dendritic cells leads to enhanced presentation of antigen to T cells

The unique therapeutic value of dendritic cells (DCs) for the treatment of allergy, autoimmunity and transplant rejection is predicated upon our ability to selectively deliver antigens, drugs or nucleic acids to DCs in vivo. Here we describe a method for delivering whole protein antigens to DCs based on carbohydrate-mediated targeting of DC-expressed lectins. A series of synthetic carbohydrates was chemically-coupled to a model antigen, ovalbumin (OVA), and each conjugate was evaluated for its ability to increase the efficiency of antigen presentation by murine DCs to OVA-specific T cells (CD4(+) and CD8(+)). In vitro data are presented that demonstrate that carbohydrate modification of OVA leads to a 50-fold enhancement of presentation of antigenic peptide to CD4(+) T cells. A tenfold enhancement is observed for CD8(+) T cells; this indicates that the targeted lectin(s) can mediate cross-presentation of antigens on MHC class I. Our data indicate that the observed enhancements in antigen presentation are unique to OVA that is conjugated to complex oligosaccharides, such as a high-mannose nonasaccharide, but not to monosaccharides. Taken together, our data suggest that a DC targeting strategy that is based upon carbohydrate-lectin interactions is a promising approach for enhancing antigen presentation via class I and class II molecules.     

CD40/APC-specific antibodies with three T-cell epitopes loaded in the constant domains induce CD4+ T-cell responses

CD4+ T lymphocytes play a central role in the orchestration and maintenance of the adaptive immune response. Targeting of antigen to antigen presenting cells (APCs) increases peptide loading of major histocompatibility complex (MHC) class II molecules and CD4+ T-cell activation. APCs have been targeted by APC-specific recombinant antibodies (rAbs) with single T-cell epitopes integrated in the constant region of the heavy chain (C(H)). However, the strategy may be improved if several T-cell epitopes could be delivered simultaneously by one rAb. We here demonstrate that a single rAb can be loaded with multiple identical or different T-cell epitopes, integrated as loops between β-strands in C(H) domains. One epitope was inserted in C(H)1, while two were placed in C(H)2 of IgG. T-cell proliferation assays showed that all three peptides were excised from loops and presented on MHC class II to T-cells. Induction of T-cell activation by each epitope in the multi-peptide rAb was as good, or even better, than that elicited by corresponding single-peptide rAbs. Furthermore, following DNA vaccination of mice with plasmids that encode CD40-specific rAbs loaded with either one or three peptides, T-cell responses were induced. Thus, integration of multiple epitopes in C(H) region loops of APC-specific rAbs is feasible and may be utilized in design of multi-vaccines.     

Traceless Cleavage of Protein–Biotin Conjugates under Biologically Compatible Conditions

Biotinylation of amines is widely used to conjugate biomolecules, but either the resulting label is non‐removable or its removal leaves a tag on the molecule of interest, thus affecting downstream processes. We present here a set of reagents (RevAmines) that allow traceless, reversible biotinylation under biologically compatible, mild conditions. Release following avidin‐based capture is achieved through the cleavage of a (2‐(alkylsulfonyl)ethyl) carbamate linker under mild conditions (200 mm ammonium bicarbonate, pH 8, 16–24 h, room temperature) that regenerates the unmodified amine. The capture and release of biotinylated proteins and peptides from neutravidin, fluorescent labelling through reversible biotinylation at the cell surface and the selective enrichment of proteins from bacterial periplasm are demonstrated. The tags are easily prepared, stable and offer the potential for future application in proteomics, activity‐based protein profiling, affinity chromatography and bio‐molecule tagging and purification.     

Effects of Hapten Density on the Induced Antibody Repertoire

Small peptides and oligosaccharides are important antigens for the development of vaccines and the production of monoclonal antibodies. Because of their small size, peptides and oligosaccharides are non‐immunogenic on their own and typically must be conjugated to a larger carrier protein to elicit an immune response. Selection of a suitable carrier protein, conjugation method, and hapten density are critical for generating an optimal immune response. We used a glycan array to compare the repertoire of antibodies induced after immunizing with either low or high‐density conjugates of the tumor‐associated Tn antigen. At high hapten density, a broader range of antibodies was induced, and reactivity to the clustered Tn antigen was observed. In contrast, antibodies induced by the low‐density conjugate had narrower reactivity and did not bind the clustered Tn antigen.     

应用免疫金银法检测病毒抗原

应用兔疫金银染色法(IGSS)、使用不同的第Ⅰ抗体,自制胶体金标记的第Ⅱ抗体,如金标羊抗兔IgG(SARG)、金标羊抗鼠IgG(SAMG)及金标蛋白A(PAG)等,检测石蜡切片中的流行性出血热病毒(EHFV)、乙型脑炎病毒(JBEV)和乙肝病毒的表面抗原(HBs Ag)和核心抗原(HBc Ag)等,均获得满意的结果。该法特异性强、敏感度高、制备简单、无毒性、经济实用,仅用普通光学显微镜即可判定结果。     

Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma

We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody–peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody–peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody–peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed.     

Selection of an active enzyme by phage display on the basis of the enzyme's catalytic activity in vivo

We have developed a novel phage display method based on catalytic activity for the in vivo selection of an enzyme. To confirm the validity of our method and to demonstrate its potential utility, we used biotin protein ligase (BPL) from Escherichia coli as a model enzyme. We were able to demonstrate the potential value of our method by selective enrichment for the birA gene, which encodes BPL, in a mixed library. The presented method for in vivo selection should allow selection of various enzymes that catalyze modification of peptides or proteins, such as protein ligase, acetylase, kinase, phosphatase, ubiquitinase, and protease (including caspase). The method should be useful in efforts to analyze mechanisms of signal transduction, to find unidentified enzymes encoded by cDNA libraries, and to exploit artificial enzymes.     

Ligand-Directed Chemistry on Glycoside Hydrolases – A Proof of Concept Study

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near – but not in – the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model β-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.     

The Antirheumatic Drug Gold, a Coin With Two Faces: AU(I) and AU(III). Desired and Undesired Effects on the Immune System

Three new findings are reviewed that help to understand the mechanisms of action of antirheumatic Au(I) drugs, such as disodium aurothiomalate (Na(2)Au(I)TM): (i) We found that Na(2)Au(I)TM selectively inhibits T cell receptor (TCR)-mediated antigen recognition by murine CD(4+) T cell hybridomas specific for antigenic peptides containing at least two cysteine residues. Presumably, Au(I) acts as a chelating agent forming linear complexes (Cys-Au(I)-Cys) which prevent correct antigen-processing and/or peptide recognition by the TCR. (ii) We were able to show that Au(I) is oxidized to Au(III) in phagocytic cells, such as macrophages. Because Au(III) is re-reduced to Au(I) this may introduce an Au(I)/Au(III) redox system into phagocytes which scavenges reactive oxygen species, such as OCl(-) and inactivates lysosomal enzymes. (iii) Pretreatment with Au(III) of a model protein antigen, bovine ribonuclease A (RNase A), induced novel antigenic determinants recognized by CD(4+) T lymphocytes. Analysis of the fine specificity of these 'Au(III)-specific' T cells revealed that they react to RNase peptides that are not presented to T cells when the native protein, i.e., not treated with Au(III), is used as antigen. The T cell recognition of these cryptic peptides did not require the presence of gold. This finding has important implications for understanding the pathogenesis of allergic and autoimmune responses induced by Au(I) drugs. Taken together, our findings indicate that Au(I) and Au(III) each exert specific effects on several distinct components of macrophages and the subsequent activation of T cells; these effects may explain both the desired anti-inflammatory and the adverse effects of antirheumatic gold drugs.     

Development of a novel strategy for engineering high-affinity proteins by yeast display

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.     

Trifunctional Saxitoxin Conjugates for Covalent Labeling of Voltage-Gated Sodium Channels**

Voltage-gated sodium ion channels (Na_Vs) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of Na_Vs hold potential value for understanding how channel regulation and post-translational modification are influenced during development and in response to diseases and disorders of the nervous system. We have developed chemical reagents patterned after (+)-saxitoxin (STX) – a potent and reversible inhibitor of multiple Na_V isoforms – and affixed with a reactive electrophile and either a biotin cofactor, fluorophore, or ‘click’ functional group for labeling wild-type channels. Our studies reveal enigmatic structural effects of the probes on the potency and efficiency of covalent protein modification. Among the compounds analyzed, a STX-maleimide-coumarin derivative is most effective at irreversibly blocking Na⁺ conductance when applied to recombinant Na_Vs and endogenous channels expressed in hippocampal neurons. Mechanistic analysis supports the conclusion that high-affinity toxin binding is a prerequisite for covalent protein modification. Results from these studies are guiding the development of next-generation tool compounds for selective modification of Na_Vs expressed in the plasma membranes of cells.