P.A. van Veelen

Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines.

Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their α-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis,Escherichia coli, Staphylococcus aureus, andLactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

Rapid Communication: Neuropeptide Expression and Processing as Revealed by Direct Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Single Neurons

Abstract: Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI‐MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI‐ MS study of direct peptide fingerprinting in the single neuron these experients demonstrate that MALDI‐MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single‐cell studies in vertebrates, including humans.

Discovery of T Cell Epitopes Implementing HLA-Peptidomics into a Reverse Immunology Approach

T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry–based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.

Structure, localization and action of a novel inhibitory neuropeptide involved in the feeding of Lymnaea

A neuropeptide that strongly inhibits the spontaneous contractions of the oesophagus in Lymnaea has been characterized as GAPRFVamide. Direct mass spectrometry of nervous tissues and immunocytochemical studies show that the peptide is synthesized by neurones in the buccal ganglia and transported to the oesophagus via the dorso-buccal nerve. In accordance with the function of the peptide, immunoreactive fibres are detected within the muscle layer of the oesophagus. Finally, mass spectrometry reveals the presence of a number of unidentified peptides in the nerves that innervate the oesophagus, which suggests that oesophageal activities may be modified by multiple peptides.

A1.45 Hyperglycosylation of ACPA-IGG variable domains modulates reactivity to citrullinated antigens

Background and Objectives Autoantibodies specific for citrullinated antigens are highly relevant diagnostic and prognostic biomarkers in rheumatoid arthritis and have been considered to be involved in disease pathogenesis. Previous studies have indicated that the ACPA-specific immune response differs from conventional B cell responses by generating polyclonal, cross-reactive antibodies of mostly low-avidity. In addition, ACPA were found to carry aberrant glycosylation patterns at the IgG-Fc tail. The present study was undertaken to further characterise the molecular make-up of ACPA and its potential functional consequences in the context of RA. Materials and Methods Serum components of RA patients were fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. In addition, ACPA-IgG and non-citrulline-specific IgG were affinity purified from RA patient serum and synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised and subsequently analysed by HPLC and mass-spectrometry. Recombinant monoclonal ACPA with variations in ACPA molecular structure were used to study binding affinity by surface plasmon resonance. Results We discovered that ACPA-IgG from RA patients have a higher apparent molecular weight as compared to other IgG molecules including antibodies against recall antigens and other autoantibodies. This higher molecular weight was explained by the overrepresentation of N-linked glycans in the variable domain (Fab region) of ACPA-IgG. Detailed structural analysis of these glycans demonstrated that ACPA-IgG linked Fab glycans are complex-type biantennary N-glycans that differ from the conventional Fc-linked N-glycans by a high degree of sialylation, galactosylation, and fucosylation together with the presence of bisecting N-acetylglucosamine. Using recombinant ACPA-IgG monoclonal antibodies with and without Fab-glycans, we found that Fab-glycans modulate binding affinity of ACPA-IgG for citrullinated antigens. Finally, lectin-immunoblotting showed that ACPA Fab-glycans can bind to sialic acid-binding immunoglobulin-type lectins. Conclusions This study describes an unusual and novel molecular feature of the citrulline-specific immune response in RA. ACPA-IgG, in contrast to non-citrulline-specific IgG, are highly glycosylated in the variable region, which modulates recognition of citrullinated antigens. Moreover, ACPA-IgG linked Fab glycans can be the target of specific lectins, suggesting additional functional features potentially involved in ACPA-mediated pathogenetic effects. This finding points to aberrations in the development of ACPA-specific B cells and further elucidates our understanding of basic disease mechanisms in RA.

Sequence Informative Fragmentation in 252 Cf Plasma Desorption Mass Spectrometry

In the field of peptide chemistry plasma-desorption mass spectrometry (PDMS) is now fully accepted as a useful method for obtaining accurate molecular weight information on peptides up to about 25 kDa molecular weight. Furthermore it has given useful information in C-terminal sequence determination by analyzing the trunk peptide instead of the liberated amino acids [1]. In addition, the method is very useful in peptide mapping by the method introduced by Morris et al. [2] applying fast atom bombardment (FAB) mass spectrometry. In these experiments PDMS has mainly been used as a technique to obtain molecular weight information. Nevertheless in some reports the presence of sequence information has been shown in smaller peptides [3–6]. During peptide mapping of recombinant IL-3 however we obtained a spectrum of a 4600 Da peptide containing considerable sequence informative fragmentation.

A2.05 Carbamylated autoantigens facilitate the break of tolerance: A novel mechanism in the pathogenesis of autoimmune arthritis

Background and objectives Rheumatoid arthritis is characterised by an immune response against posttranslational modified proteins, in particular citrullinated and homo-citrullinated (carbamylated) proteins. Posttranslational protein modification can result in the generation of neo-epitopes that may subsequently trigger auto-immune responses. Antibodies recognising carbamylated proteins (anti-CarP antibodies) are present in sera of RA-patients as well as in different animal models of arthritis. It is currently unknown how responding B cells interact with homo-citrullinated proteins or whether carbamylated self-proteins induce a breach of tolerance. We hypothesise that not only carbamylation of foreign, but also self-proteins can induce anti-carbamylated protein responses both at the T-cell and B-cell level, enabling T-cell dependent antibody production against carbamylated autoantigens. Materials and methods Mice were immunised with carbamylated antigens (ovalbumin/murine albumin/murine fibrinogen) or non-modified antigens. T cell responses were studied by proliferation assays and cytokine ELISAs. Murine anti-CarP hybridomas were sequenced using single cell PCR-based antibody cloning technology. Reactivity of human anti-CarP antibodies towards CaFCS and albumin was determined for by ELISA 160 RA-patients of the Leiden Early Arthritis Cohort and controls. Results After Ca-mAlb immunisation, mice developed high titers of anti-CarP antibodies, recognising different carbamylated foreign- and auto-antigens. In contrast, no anti-CarP antibodies were detected in mAlb immunised mice. Similar responses (i.e. anti-CarP antibody responses against carbamylated human albumin) were observed in RA-patients. Murine monoclonal anti-CarP antibodies show a similar pattern of cross-reactivity towards carbamylated antigens, together indicating that homo-citrulline is seen in a “hapten-like manner” by responding B cells. Analysis of Ig gene sequences revealed high numbers of somatic mutations, indicative of antigen-driven selection for antibody generation. T cell cultures derived from Ca-mAlb immunised mice responded to stimulation with Ca-mAlb, but not to the native protein. Conclusions Carbamylation of both foreign as self-proteins can facilitate a breach of tolerance, resulting in formation of carbamylated protein-specific T-cell and B-cell responses in mice. In mice, homo-citrulline-residues are detected by anti-CarP-antibodies in a “hapten” like manner. Interestingly, carbamylated albumin, which is able to break tolerance in mice, is also recognised by anti-CarP antibodies in RA-patients. These data provide first evidence explaining the abundance of carbamylated self-proteins recognised by anti-CarP antibodies.

S78. Proffered paper: High-affinity CD20-specific TCRs suitable for adoptive immunotherapy can be readily isolated from the allo-repertoire using reverse immunology

Studies using T-cell receptor (TCR) or chimeric antigen receptor (CAR) transduced T-cells have shown the effectiveness of adoptive immunotherapy to treat different malignancies. The efficacy and safety of such interventions greatly depends on good target selection to prevent on-target toxicity. Furthermore, the broad application of TCR-based adoptive immunotherapy is hampered by a lack of an effective immune response against self-antigens. Through self-tolerance, T-cells carrying high-affinity TCRs reactive to self-antigens are deleted during thymic selection. An attractive strategy is to exploit the immunogenicity of foreign human leukocyte antigen (HLA) molecules to generate an effective immune response against these antigens. Here, we describe a protocol to efficiently isolate high-avidity alloHLA-restricted T-cells targeting the B-cell compartment. From a B-cell peptide elution library 15 peptides derived from genes exhibiting B-cell restricted expression patterns were identified and peptide-MHC multimers (pMHC) of HLA-A*0201 were generated. Via MACSorting and FACSorting a plethora of pMHC-multimer binding T-cell clones from HLA-A*0201-negative individuals were isolated. Generated T-cell clones were selected based on peptide-specificity and avidity for further characterization. We successfully isolated two distinct T-cell clones carrying high-affinity TCRs specific for a CD20 peptide presented in HLA-A*0201. CD20 dependent recognition could be demonstrated by genetically engineering CD20-negative K562-A2 cells to express CD20. The isolated T-cell clones efficiently recognized CD20-expressing HLA-A*0201 primary chronic lymphocytic leukaemia (CLL), acute lymphoblastic leukaemia (ALL) and mantle cell lymphoma (MCL), while recognition of CD20-negative hematopoietic and non-hematopoietic cell-subsets was absent. In addition, the CD20-specific T-cell clones were able to more efficiently recognise ALL cell-lines than CD20 specific antibodies. We demonstrated that on ALL cell lines with only very low CD20 surface expression, the CD20-specific T cell clones could still efficiently recognise endogenously processed CD20-derived peptides in the context of HLA-A*0201. In conclusion, we developed a platform for the rapid identification of high-affinity TCRs of therapeutic relevance targeted to self-antigens by combining gene expression data with valuable information on peptide processing from peptide elution studies and exploiting the immunogenicity of foreign HLA. Using this platform we successfully isolated CD20-specific TCRs which could broaden the application of immunotherapies targeted to CD20 in cases were CD20-cell surface expression is low. Based on its general principle the developed platform could easily be adapted to target other malignancies.

OP0177 A High Frequency of N-Glycans in the Acpa-Igg Variable Domain Modulates Reactivity to Citrullinated Antigens

Background Antibodies against citrullinated proteins antigens (ACPA) are the most relevant prognostic and diagnostic biomarker for rheumatoid arthritis (RA). ACPA positive patients are characterized by progressive disease, a high rate of joint destruction, and a low chance to achieve remission. Despite these strong and well-defined associations with clinical phenotype, however, molecular mechanisms underlying the citrulline-specific immune response are incompletely understood. Of note, this immune response is highly polyclonal with considerable cross-reactivity, develops and matures prior to disease onset, and is of remarkably low avidity. These features indicate that the underlying B cell response is different from “conventional” antibody responses, but also different from other autoantibody responses such as the high affinity double-stranded DNA antibody response in SLE. Objectives To study molecular characteristics of ACPA in order to obtain insight into the properties of the underlying B cell response. Methods Serum of ACPA positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. In addition, ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum and synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and subsequently analysed by HPLC and mass-spectrometry. Recombinant monoclonal ACPA were used to study binding affinity for multiple antigens by surface plasmon resonance. Results In almost all donors studied (23 out of 24), ACPA-IgG molecules were found to have a higher molecular weight than non auto-reactive IgG. Structural analysis pinpointed this observation to a high frequency of N-glycans in the ACPA variable regions, which was found on up to 90% of all ACPA. These Fab-glycans were linked to N-glycosylation sites generated by somatic hypermutation, were absent from other disease-specific autoantibodies, were characterized by a high degree of sialylation, and clearly differentiated ACPA from “conventional” IgG molecules. Moreover, ACPA Fab glycans modulated reactivity to citrullinated antigens. Conclusions These data describe a unique and completely novel feature of the citrulline-specific immune response in RA, with important implications for ACPA pathogenicity and disease prognostication. The high frequency of variable domain Fab glycans points to specific developmental abnormalities of the underlying B cell response. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4717

Direct Neuropeptide Profiling of Single Neurons and Target Tissue by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

In the last decade, mass spectrometry has developed into an important analytical tool in the life sciences. This is due to the introduction of softionization techniques such as fast atom bombardment [1], electrospray ionization [2] and matrix-assisted laser desorption ionization [3, 4, 5, 6, 7], which have allowed routine mass measurements of peptides and proteins with high resolution and accuracy. The ability to make precise mass measurements for molecules of interest can solve many problems encountered in the life sciences that are difficult to address by other methods. Mass spectrometry may be used to check correctness of amino acid sequences based on data generated by cDNA cloning studies or Edman degradation of peptides and proteins. It may also serve to confirm the purity of the sample and to quantify the analytes. Mass spectrometry is equally effective in confirming and identifying chemical modifications of peptides with a predicted change in molecular weight, e. g., post-translational modifications such as N-terminal acetylation, C-terminal amidation and disulphide formation [8, 9, 10]. Moreover, the location and nature of carbohydrates, phosphates, lipid moieties, etc., which are important for the bioactivity of proteins, can be determined by mass spectrometry, often in conjunction with other methods such as chemical or enzymatic cleavages. In favourable conditions, noncovalent protein complexes can also be detected, which is important for enzyme-substrate studies, for example.