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IgG4-Treat consortium is looking for highly motivated and talented PhD students to contribute to groundbreaking research in the field of IgG4-autoimmune diseases (IgG4-AID). 

The PhD researchers will have the opportunity to participate in a comprehensive training programme in academic and industry setup. Next to their transferable skills training, the IgG4-Treat PhD students will pursue 10 individual, multidisciplinary research projects (organised into four scientific work packages) aimed to advance our understanding of IgG4-AID. 

PhD candidates will become part of a network of 9 host laboratories and 7 associated partners from 7 different European countries.  Funding for these projects has been provided by the EU under the competitive MSCA Doctoral Networks 2022 program (Grant Agreement No. 101119457).

Eligibility criteria

The MSCA Doctoral Networks program requirements:

• MSCA Mobility Rule: Applicants must not have resided or carried out their main activity (work, studies, etc.) in the host country for more than 12 months in the 36 months immediately preceding the starting date of the PhD (earliest starting date: 31/03/2024).

• Doctoral Candidate Definition: At the time of recruitment, the applicants must not be in possession of a doctoral degree or have successfully defended a doctoral thesis awaiting formal notification.
• In addition, applicants must be eligible for enrolment for PhD studies at the hosting institution.

Benefits:

The funding is guaranteed for 36 months* and will include:

• a monthly living allowance of 3,400 EUR/month (gross salary will be adjusted through the application of a country correction coefficient for the country in which the researcher is recruited; as indicated in Table 1 of the MSCA Work Programme)
• a monthly mobility allowance of 600 EUR/month;
• a monthly family allowance (if applicable), of 660 EUR/month.

Allowances are gross amounts, which means that all compulsory social security contributions, direct taxes, and any other compulsory deductions under national legislation will be deducted from these amounts.

*As PhD projects in the Netherlands (NL) are required to last 4 years, research groups in the NL guarantee funding for 48 months.

R1: Serological, histological, cellular and genetic characteristics of the IgG4 immune response (WP1 and WP2)

Research project description:

IgG4-AID are emerging as a distinct disease group with pathogenic IgG4 antibodies targeting antigens in the central and peripheral nervous system. IgG4 is produced in response to chronic or strong antigen stimulation and is thought to play a role as an anti-inflammatory or tolerogenic antibody. IgG4 competes with antibodies of other classes and subclasses for antigen and blocks the epitope, so that the effector function of the competing antibody is abolished. Despite their protective role, in IgG4-AID neural cell–specific antibodies of the IgG4 subclass exert pathogenic effects by inhibiting cellular adhesion, blocking enzymatic activity, or disrupting protein–protein interactions affecting signal transduction pathways, in contrast to IgG1-3 antibodies, which cause inflammatory-mediated tissue destruction. The IgG4 and IgG1-3 antibodies are thought to be produced by distinct subsets of plasma cells, and patients with IgG4 autoantibodies often require distinct treatment from patients with IgG1-3 antibodies, and patients also often present with a mix of different IgG subclasses.

For targeted therapies, it is important to understand which IgG subsets predominate and which immune cells play a role in the production of pathogenic autoantibodies. To date, our understanding of the relevant B and T cell subsets that drive the IgG4 antibody production is limited. We observed tissue-resident immune cells in the brain of patients with antineuronal IgG4-AID that may be relevant for disease, and characterization of these cells may identify dysregulated or dysfunctional IgG4 regulators, and constitute novel biomarkers.

Main objectives:

• A thorough and comparative clinicopathological characterization of patients with very well-defined neurological IgG4-AID, and comparison to samples from healthy and disease control individuals from all centers. Comparative analysis of the clinical and immunological aspects of IgG4-AID, including the serological characterization of IgG4-AID by cell- and tissue-based assays, histopathological characterization of patient biopsy and autopsy material, and correlation to the clinical characteristics.
• Characterization of tissue-resident immune cells to determine which cells contribute to the production of pathogenic antibodies by analysis of IgG4-AID patient autopsy and biopsy material via immunohistochemistry and imaging mass cytometry.
• Measurement of anti-drug antibodies and HLA genotype in ADA patients and correlation of IgG1- and IgG4 response to the HLA genotype.

Methods used in the project:

Cell-/tissue-based assays, Immunohistochemistry, Imaging mass cytometry, Flow cytometry, ELISA, Biobanking, Western blot, PBMC isolation, HLA genotyping + Animal work during a secondment 

Required project-specific qualifications and skills:

• Previous experience in immunology
• English language proficiency is required
• Hands-on experience with flow cytometry, immunohistochemistry, imaging mass cytometry and cell culture
• Medical degree (preferred)

Project duration: 36 months (40h/week)  

Foreseen mobility: Secondments are planned in:

• INSERM (supervised by R. Le Panse, France): Validation of dysregulated tissue immune cells by flow or spectral cytometry
• TND (Tzartos NeuroDiagnostics, supervised by S. Tzartos, Greece): Validation of diagnostic findings by complementary methodology (rodent model).

Hosting lab description:

PhD student will work under the supervision of Dr. Inga Koneczny and Prof. Romana Höftberger in the Division of Neuropathology and Neurochemistry, Department of Neurology, Medical University of Vienna (MUW). Division of Neuropathology and Neurochemistry at MUW is the Austrian reference center for antineuronal autoantibodies. Further, the lab is hosting the IgG4 Biobank, including a large collection of longitudinal patient serum and CSF samples and patient autopsy and biopsy material. All required methods, including cell- and tissue-based assays, flow cytometry, ELISA, PBMC isolation, and imaging mass cytometry are established at MUW.

R2: Identification of specific serum biomarkers and immune cell dysregulation commonly associated with IgG4 autoimmune diseases (WP2)

Research project description:

IgG4 autoimmune diseases (IgG4-AID) are a group of severe autoimmunopathies that are characterized by pathogenic IgG4 autoantibodies against defined target antigens. To better understand IgG4-AID, we plan to identify specific serum biomarkers and immune cell dysregulation commonly associated with IgG4 AID.

Our team has a long-time expertise in autoimmune Myasthenia Gravis (MG) including MuSK MG mediated by IgG4 autoantibodies. The PhD project proposed in our laboratory will investigate serum biomarkers associated with IgG4-AID using the Olink technology that allows the analysis of at least 384 serum proteins per sample. In parallel, immune cell dysregulation in IgG4-AID will also be investigated by mass cytometry (CyTOF). These multiparametric analyses will be performed by comparing healthy donors with anti-MuSK-MG patients, but also anti-LGI1 encephalitis patients obtained from partners of the IgG4-AID network. Specific serum biomarkers and immune cell dysregulation will be further validated in other IgG4-AID by the PhD student during a secondment at TND (Tzartos NeuroDiagnostics, Greece) (ELISA and SIMOA analyses) or by different partners. In addition, the implication of defined immune dysregulation will be investigated by flow cytometry in the experimental MuSK-MG mouse models in a secondment at Istanbul University (supervised by V. Yilmaz and E. Tüzün, Turkey).

Methods used in the project:

Mass and flow cytometry, Protein analyses (ELISA, SIMOA…), Bioinformatics skills for multiparametric analyses,  Human blood sample preparation, Animal work during a secondment 

Required project-specific qualifications and skills:

• Qualification in immunology
• Good skills in flow cytometry, data analysis with an affinity for bioinformatic
• Good level in spoken and written English

Project duration: 36 months, 35h/week 

Foreseen mobility: Secondments are planned in:

• TND (Tzartos NeuroDiagnostics, supervised by S.Tzartos, Greece): Validate Olink findings using SIMOA technology.
• Istanbul University (supervised by V. Yilmaz and E. Tüzün, Turkey): Analyze immune cell dysregulation in IgG4-AID active immunization mouse model (MuSK MG).

Hosting lab description:

Dr Rozen Le Panse's team is part of the Center of Research in Myology located in Paris (hospital Pitié-Salpêtrière). She has been working on Myasthenia Gravis (MG) for more than 20 years. The team is investigating the etiological and pathophysiological mechanisms involved in MG. The team is also involved in the search for new therapeutic approaches using different experimental models. In collaboration with clinicians working on MG, the team is also looking for new cellular and molecular biomarkers for the follow up of the patients using new technological approaches. More information on Le Panse's team.

R3: Identification of genetic predisposition and transcriptional factors involved in isotype-switching of IgG4-AID B-cell clones (WP2)

Research project description:

The autoimmune encephalitis (AE) subtype with autoantibodies targeting the protein Leucin-rich glioma inactivated 1 (LGI1) is one of the most common AE variants and its autoantibodies are dominantly of the IgG4 isotype. Similar to other IgG4 autoimmunopathies, the reason for the IgG4 fingerprint and underlying transcriptional changes of the respective plasma cells remains elusive. LGI1-AE is an intriguing model disease because we and others have already shown that there is a highly clonally expanded B-cell repertoire in CSF, whose large clonotypes are mostly autoantigen specific. Together with colleagues, we have prospectively recruited seven untreated patients with LGI1-AE, whose CSF and PBMCs underwent single-cell sequencing (10x, 5', TCR, BCR). In several of these patients, we have also obtained longitudinal PBMC samples and sometimes CSF cells. We hypothesize, that analyzing CSF-restricted, LGI1-specific B-cell clones producing IgG4 antibodies in comparison to ones producing LGI1-IgG1/2 antibodies will provide an answer to the question of what differentiates these two plasma cell variants on a transcriptional level. We will compare these transcriptional phenotypes to longitudinally acquired samples of the same patients and identify the temporal stability of IgG1/IgG4 clonotypes over time, e.g. do we observe an isotype switch in the course of disease from IgG1/2 to igG4? We will also analyze together with consortium partners the relative pattern of IgG1/2 vs IgG4 in serum and CSF of patients and correlate these to available GWAS/whole genome data from these patients. Finally, the project will try to enrich LGI1-specific B-cells from the blood of affected patients using labeled, recombinant LGI1 domains, cultivate B-cells in vitro and characterize their functional effects.

Objectives:

• To identify genetic predisposition for IgG4-mediated AID within and between subgroups.
• To examine transcriptional changes associated with IgG4-switching B-cells in CSF from patients with IgG4-mediated-autoimmune-encephalitis.

Expected Results:

We will analyze DNA samples recruited by the consortium of patients with IgG4- AID by genotyping using Illumina GSAv2 Chip. SNPs associated with specific subtypes or IgG4-mediated autoimmunity in general will be identified and function effects examined using available and future single-cell transcriptomic data from patients. We will also analyze available data from whole genome sequencing of 150 LGI1 patients and healthy controls for confirmation and true genome-wide associations. We will analyze antigen-specific B-cells in CSF at two timepoints in patients by combining single-cell transcriptomics (10xGenomics) including single single-cell and B-cell receptor sequencing (CSF), direct labeling of antigen-specific B-cells by antigen-tetramers (CITE-seq) and recombinant cloning (all established, unpublished). We will track identical or affinity-matured daughter clones in CSF at the second timepoint 3-6 months later and identify transcriptional changes in clones which have undergone a class-switch from IgG1 to IgG4. This will shed light on genetic and transcriptional mechanisms relevant for the initiation and propagation of IgG4-mediated autoimmunity and help in designing targeted therapies.

Methods used in the project:

Serology, Immunofluorescence (cell-based assays), Cell culture, ex vivo handling of CSF and PBMCs, Single-cell sequencing, Transfections and viral transduction, Recombinant T-cell receptor and B-cell receptor cloning, Flow cytometry, Functional studies of recombinant BCR/TCR, Genome-wide association studies (Cooperation Prof. Gregor Kuhlenbäumer Neurogenetics).

Required project-specific qualifications and skills:

• M.Sc. degree in biology, biotechnology, molecular medicine or related fields
• English proficiency in spoken and written form is required 
  
• Previous knowledge in neuroimmunology could be an advantage
• Laboratory experience with cell culture, transfection and single-cell sequencing analysis would be appreciated

Project duration: 36 months (38.5h/week)

Foreseen mobility: Secondments are planned in:

• INSERM (supervised by R. Le Panse, France): Analyze LGI1-AE samples by flow cytometry to confirm scRNAseq and CytOF observations in switched B-cells from patients with LGI1-AE.

• Sanquin (supervised by T. Rispens, the Netherlands): Pilot experiments analyzing feasibility of blocking pathogenic IgG4 binding to LGI1 by using a hexamerized decoy LGI1-Fc fusion protein in vitro.

Hosting lab description:

The PhD candidate will work in the neuroimmunology group (Headed by Dr. Frank Leypoldt and Prof. Klaus-Peter Wandinger) at the UKSH Kiel and with extensive interaction with the clinical neuroimmunology department and neurogenetics (Prof. Kuhlenbäumer) and bioinformatics (Dr. Daniela Esser). Our group is focused on autoantibody-associated diseases in neurology and has extensive experience in genetic predisposition, single-cell sequencing, recombinant cloning, CSF analysis, function characterization of autoantibodies. All procedures are established and instruments are available in the group. Our group has extensive national and international collaborations in the field of autoimmune encephalitis. It is a key contributor to the German Network for Research on autoimmune encephalitis. The PhD will be awarded by Kiel University.

R4: New animal models for studying IgG4 and IgG4-AID (WP4)

Research project description:

In vivo studies toward IgG4 function and its role in disease are limited because of the lack of a good homolog in mice. In this project, we aim to develop a novel mouse model expressing a humanized version of IgG4. We will base the design of this model on in vitro experiments testing mouse IgG and modifying it with human IgG4 features. The mouse model will next be used to study the effect of immunizing with IgG4-autoimmune eliciting antigens and the ability of an IgG4-specific treatment to alleviate disease development in these animals. The ultimate ambition of this project is to develop a novel humanized IgG4 mouse model and use it to provide proof of concept for this novel IgG4-targeted therapy.

Methods used in the project:

Animal work, ELISA, CRISPR-Cas genetic modification, surface plasmon resonance (SPR), Passive transfer mouse model, Western blotting, cell culture, Antibody design, Affinity chromatography, recombinant antibody production.

Required project-specific qualifications and skills:

• M.Sc. degree or equivalent in Biochemistry/Molecular Biology/Immunology or related fields
• English language proficiency is required
  
• Animal experimental work experience and a valid certificate for working with experimental animals are favored

Project duration: 48 months* (36h/week)

*In The Netherlands a PhD project takes at least 4 years, therefore the project will continue one year beyond the duration of the consortium.

Foreseen mobility: Secondments are planned in:

• Maastricht University (supervised by P. Martinez Martinez, the Netherlands): Perform active immunization experiments in the humanized IgG4 model.

• TND (Tzartos NeuroDiagnostics, supervised by S.Tzartos, Greece): Test of in vitro apheresis with serum IgG4 isolated from the humanized IgG4 mouse model.

• Hellenic Pasteur Institute (supervised by K. Lazaridis, Greece): Test therapeutic effect of IgG4 apheresis in the humanized IgG4 mouse model.

Hosting lab description:

The project will be executed in the Translational Neuroimmunology lab led by Dr. Maartje G. Huijbers at Leiden University Medical Center, The Netherlands. The lab of Dr. Huijbers is an expert in neuromuscular autoimmune disease with a special interest in the IgG4-AID MuSK myasthenia gravis. They have several in vitro and in vivo models developed to study these diseases including myotube cultures, iPSC-based NMJs, immune cell cultures, recombinant antibody technology and passive transfer models. Please check more details on Huijbers lab website.

R5: Investigation of potential IgG4 regulators in vivo and in vitro (WP2 and WP3) 

Research project description:

We aim to investigate the significance of the HLA genotype and identify key immunological molecules involved in IgG4 production and class switching. The main questions of the project:

1) Which HLA genotypes demonstrate associations with IgG4 production and isotype switching?

T cells and antigen-presenting cells derived from MuSK-MG and LGI1-AE patients (PBMC and antigen-specific B cells) both with or without IgG4-AID-related HLA haplotypes, will be co-cultured with T cells from patients carrying different HLA haplotypes. Lymphocyte proliferation will be assessed and, the measurement of cytokines associated with IgG4 production and class switching will be conducted. Selected cells and molecules from genomics and multiparametric data (from WP2) and lymphocyte culture studies will be targeted for suppression in MuSK-EAMG. Genes of interest (including IL4R) will be knocked down and tested for the effects on IgG4 class switch in human B-cell culture in vitro.

2) What are the pathogenic effects of LGI1/CASPR2 mAbs derived from patients?

Patient-derived mAbs (LGI1/CASPR2-Abs) will be performed in vitro for use in a passive transfer animal model. Pathogenicity of the patient mAbs in vivo will be determined through ICV administration of mAbs into mice and subsequent behavioral (Y maze, rotarod, open field etc.) and brain pathology studies.

The description of our research includes the following:

• Co-culture studies on PBMCs in a cohort with IgG4-related HLA genotype (healthy, IgG4-anti drug antibody, IgG4-autoimmune diseases).
• Characterizing the pathogenic roles of specific molecules, along with lymphocyte culture using MuSK-EAMG (experimental autoimmune myasthenia gravis) model (suppression) and human B cell (knock-down).
• Determination of IgG4 pathogenicity in an animal model by passive transfer of human LGI1/CASPR2-Abs.

Methods used in the project:

Cell sorting by MACS or FACS, antigenic stimulation in co-culture, CFSE for proliferation, ELISA/bead assay for cytokine level, an active and passive immunization mouse model of EAMG, shRNA application for gene silencing in an animal model, gene knocking down in B cells, isolation of antigen-specific specific B cells from PBMC. 

*The development of EAMG in C57BL6 mice immunized with MuSK antigen will primarily be assessed through clinical findings. Muscle strength will be measured using a dynamometer, and the weights of the mice will be recorded (at least twice a week). It is expected that these follow-ups and observations will be conducted by the PhD student.

Required project-specific qualifications and skills:

• M.Sc. degree in biology, biotechnology, molecular medicine or related fields
• English proficiency is required 
• Previous laboratory experience with cell culture, animal work, cell sorting or ELISA assays will be an advantage

Project duration: 36 months (40h/week)

Foreseen mobility: Secondments are planned in:

• Maastricht University (supervised by P. Martinez Martinez, the Netherlands): Production of patient-derived mAbs to be used in passive transfer studies for analyses of pathogenic effects of IgG4.
• UKSH (supervised by F. Leypoldt, Germany): Analysis and cloning of antigen-specific B-cells/B-cell receptors from CSF and antigen-specific B-cell enrichment from PBMCs in IgG4 LGI1 encephalitis.
• Sanquin (supervised by T. Rispens, the Netherlands): Analysis of effects of IL4R blocking in cultured human B-cells on de novo IgG4 class switch.

Hosting lab description:

The PhD candidate will work under the supervision of Associate Prof. Vuslat Yilmaz and the co-supervision of Prof. Erdem Tüzün in Aziz Sancar Institute of Experimental Medicine, Neuroscience Department of Istanbul University. Their research is focused on B cell-associated neuroimmunological diseases and antibody-mediated neurological disorders. They have extensive experience in experience in anti-neuronal antibody identification. Currently, their groups investigate animal models of Myasthenia Gravis for new therapeutic strategies. 

R6: Characterization of early IgG4 responses (WP2)

Research project description:

In this project, the central aim is to better characterize IgG4 responses both serologically and cellularly, starting at the onset of the immune response. We hope to identify key early events and unravel immunological processes that are instrumental in the development of IgG4 responses. As a model system for IgG4-AID, we will do most of these analyses in patients treated with different biologics (anti-TNF) that often develop a strongly IgG4-skewed anti-drug Ab response. This enables monitoring all early events leading towards an IgG4-dominated immune response (which develops slowly over time). Prospective sampling of patient material is already ongoing. The expected outcome of this project is molecular insight into pathways that control IgG4 switch (in particular precursor B cells and their phenotypes), and insight into the dynamics of IgG4-associated features such as enhanced Fab glycosylation (cause or consequence).

Methods used in the project:

Next-generation sequencing, Luminex assay, mass cytometry, scRNA-seq, Mass spectrometry, Glycan analysis, GWAS.

Required project-specific qualifications and skills:

• Interest in antibody structure, antibody function and general immunology

• Interest both in basic research as well as in its clinical application

• Enthusiastic, motivated and ambitious

• Master's degree in Biomedical or Biopharmaceutical Sciences, or equivalent education

• Strong communicational skills/team player

• Good command of written and spoken English (C1)

Project duration: 48 months* (36h/week)

*In The Netherlands a PhD project takes at least 4 years, therefore the project will continue one year beyond the duration of the consortium.  

Foreseen mobility: Secondments are planned in:

• UKSH (supervised by F. Leypoldt, Germany): GWAS genotyping of IgG4-ADA patients.

• Medical University of Vienna (supervised by I. Koneczny, Austria): Correlation of IgG1- and IgG4 response to the HLA genotype.

Hosting lab description:

At Sanquin, we have a long-standing interest in the immunobiology of IgG4 antibodies. Decades of research have contributed to uncovering the unique traits of IgG4 antibodies, including their ability of Fab arm exchange and the distinctive dynamics of IgG4 responses. We focus on elucidating the role of IgG4 in a clinical context, in particular in autoimmune diseases. We explore the molecular characteristics underlying IgG4's unique properties. Furthermore, we study the mechanistic details shaping the IgG4-switched B cell response. The research is embedded within the antibody structure & function research group. More on Theo Rispens research group can be found here.  

R7: Molecular characteristics of IgG4 contributing to disease mechanism (WP3)

Research project description:

In this project, you're going to investigate the role of IgG4 in IgG4-AID on a molecular level by isolating and characterizing monoclonal antibodies from patient-derived cells. The main disease studied here will be autoimmune encephalitis, specifically the IgG4 driven variants, such as CASPR2 and LGI1. The main objective is to successfully isolate monoclonal antibodies, and characterize them using a variety of biochemical and molecular techniques, as well as look at their effects in vivo using animal models.

Methods used in the project:

FACS, Single-cell sorting, PCR, Electrophoresis, Cloning, Gibson assembly, Cell culture, Protein purification, ELISA, immunofluorescence, microscopy, bioinformatics, Animal work (surgery, behavior, dissection).

Required project-specific qualifications and skills:

• Knowledge about molecular lab techniques, sterile work and animal modeling
• Hands-on experience with molecular lab techniques and cell culture (preferred)
  
• English language proficiency C1

Project duration: 36 months (40h/week)

Foreseen mobility: Secondments are planned in:

• Maastricht University (supervised by P. Martinez Martinez, the Netherlands): Inject CASPR2 mAbs and role of FAE in a passive transfer mouse model.

• TND (Tzartos NeuroDiagnostics, supervised by S. Tzartos, Greece): Test of in vitro apheresis with serum IgG4 isolated from the humanized IgG4 mouse model.

• Hellenic Pasteur Institute (supervised by K. Lazaridis, Greece): Passive transfer of mAbs to Lewis rats for IgG4 apheresis.

Hosting lab description:

This project will take place in the Autoimmune and Neurodegeneration group of the Department of Neurology and Experimental Neurology at the Charité-Universitätsmedizin in Berlin, Germany, under the guidance of Prof. Dr. med. Harald Prüβ. The lab of Prof. Prüβ lays its focus on the detailed analysis of autoantibody-mediated impairment of neuronal function through the use of patient-derived recombinant monoclonal autoantibodies, but also continues to develop novel diagnostics and innovative antibody-selective immunotherapies. For further information on this research, please visit the Prüβ lab website.  

R8: In vivo study of pathogenic mechanisms of IgG4-AID (WP3)

Research project description:

IgG4 autoimmune diseases (IgG4-AID) are a group of severe autoimmunopathies that are characterized by pathogenic IgG4 autoantibodies (Abs) against defined target antigens. While affecting different organs, they share important mechanistic and therapeutic commonalities. IgG4-AID are individually rare, but together affect up to 11 patients per 10,000 (cumulative prevalence) thus >490,000 patients in the EU. IgG4-AID are clinically severe, difficult to diagnose and challenging to treat. They affect different organs and were therefore previously not recognized to be related.

IgG4 Abs exert their effects by blocking protein-protein interaction thus disrupting signal transduction pathways and tissue architecture, instead of complement-mediated injury of target-organs. For example, MuSK Abs interrupt signal transduction pathways required for the maintenance of neuromuscular junctions resulting in impaired neuromuscular transmission and causing severe skeletal muscle weakness. Current treatment of IgG4-AID utilizes non-specific immunosuppression and plasma separation, but the response is often incomplete and patients tend to relapse. Chronic, therapeutic B-cell depletion only benefits a subgroup of patients and has relevant side effects. Importantly, there are no biomarkers that would predict treatment response and many IgG4-AID are not well characterized, and lack strategies and guidelines for successful clinical management. This may lead to reduced quality of life or even death for the patients.

IgG4 antibodies have unusual features distinguishing them from other immunoglobulins: They do not activate the classical complement pathway, do not recruit immune cells via Fcγ receptor binding, and undergo a process named "Fab-arm exchange" (FAE): IgG4 may split up into two half-molecules and recombine to form bi-specific Abs. We and others have started to unravel the mechanisms and consequences underlying IgG4 class switch and IgG4 predominance in IgG4-AID, yet many factors are still unknown. We lack an understanding of which cells and how the IgG4 subclass itself, including Ab structure, sequence and posttranslational modifications (PTM) such as glycosylation and FAE may contribute to its pathogenicity. FAE leads to bi-specific, functionally monovalent antibodies, changing Ab valency and avidity, and glycosylation may affect Ab interaction with Fcγ receptors and immune cells. The main objective of WP3 is to characterize the structural, molecular and functional characteristics of IgG4 Abs.

In collaboration with other members of the consortium, the PhD candidate in this project will:

• analyze the lipidomic profile and spectral imagining of IgG4+ B cells from IgG4-AID patients' PBMCs using (liquid and imagining) LC-MS. By comparing the IgG4+ B cells from IgG4-AID patients to IgG4+ B cells from healthy donors, IgG1-3+ B cells from AID patients and IgG1+ B cells from IgG4-AID patients.
• characterize the pathogenic mechanisms of different IgG4-AID patients' derived IgG4 autoantibodies against LGI1 and Caspr2 using different in vitro assays
• study the in vivo effect of the pathogenic of LGI1 and Caspr2 autoantibodies using a passive transfer model to describe the relevance of IgG4 valency and FAE on pathogenicity. Abs effects on neuronal firing and in vivo electrophysiology will help to characterize the model and develop more suitable animal models to test therapeutic strategies
• develop and characterize a chronic model for LGI1/Caspr2. Using a battery of biochemical and behavioural tests, we will study the functional effects of the valency in IgG4 Abs and characterize their effector mechanisms in an IgG4 humanized model.

Methods used in the project:

LC-MS, Animal work (surgery, behavior), Passive transfer mouse model,  Biochemical assays, In vitro assays, Electrophysiology, ELISA, MSI, Primary cell cultures, Immunohistochemistry, Flow cytometry

Required project-specific qualifications and skills:

We are looking for a young self-driven researcher with experience in the field of immunology, neuroimmunology and/or neuropsychiatry.

• M.Sc. in Biology, Biotechnology, Biochemistry, Immunology and/or Neuroscience or equivalent
• English language proficiency level corresponding to C1
• Background in neuroimmunology and antibody-mediated channelopathies of the central and peripheral nervous system
• Experience with animal models for neurological antibody-mediated autoimmune disorders, including model development, behavior and motor-function tests and electrophysiological measurements
• In possession of a Lab animal science certificate (Article 9, Felasa Accredited) or willing to obtain it
• Familiar with protein purification and quantification assays, phenotype characterization, primary cell cultures and disease effector mechanisms monitoring assays, drug efficacy assays
• In possession of TMS-VRS D certificate or willingness to obtain it
• Academic thinking and working level
• Excellent written and verbal communication and interpersonal skills
• Being able to set up and supervise independent research
• Capacity to work on several projects simultaneously and deliver results within established deadlines
• Being able to function well within teams and the ability to work together in a multidisciplinary manner
• Teaching skills or the willingness to learn
• International and national orientation and network

Project duration: 48 months* (36-40h/week)

*In The Netherlands a PhD project takes at least 4 years, therefore the project will continue one year beyond the duration of the consortium.   

Foreseen mobility: Secondments are planned in:

• Sanquin (supervised by T. Rispens, the Netherlands): Isolation of antigen-specific IgG4+ B-cells for lipidomics analysis.

• LUMC (supervised by M. Huijbers, the Netherlands): Development of new IgG4-AID models.

• TND (Tzartos NeuroDiagnostics, supervised by S. Tzartos, Greece): Role of FAE in passive transfer models.

Hosting lab description:

The Research group of Neuroinflammation and autoimmunity led by Prof. Martinez is working on understanding the pathophysiological mechanisms of neuroinflammatory and neurodegenerative disorders and nervous system autoimmunity as well as discovering biomarkers and developing new diagnostic assays and studying the potential of novel treatment strategies. The group has 2 main research lines; 1) Role of lipids and their transporters in Neuropsychiatric disorders and 2) Autoimmune factors in Neurological, Psychiatric and Neuropsychiatric disorders.

R9: Structural and functional characteristics of IgG4 autoantibodies (WP3)

Research project description:

IgG4 is the most fascinating IgG subclass, as it has unique structural and functional properties. While it retains >90% sequence homology to IgG1, single amino acid differences in the constant region have dramatic effects on the antibody effector mechanisms, including a loss of C1q binding, a strongly reduced interaction with activating Fcγ receptors and the ability of the two heavy chains to detach from each other, causing a random exchange of half-molecules (called Fab-arm exchange) and changing the valency of antibody binding, which in turn may affect pathogenicity of IgG4.

Further post-translational modifications, such as glycosylation, may also alter the characteristics of IgG4, such as antigen binding, activation of complement or interaction with Fcγ receptors and immune cells. However, we lack understanding if -and how- the IgG4 subclass itself, including the antibody structure, sequence and post-translational modifications may contribute to its pathogenicity. We want to gain a better understanding of the pathogenic mechanisms of IgG4 antibodies. Are post-translational modifications changing the properties of the antibodies- and if so, do they make the antibodies more or less pathogenic? Therefore, we aim to characterize post-translational modifications of IgG4 and study the functional characteristics of IgG4 antibodies in vitro.

Main objectives:

• To characterize post-translational modifications of the IgG4 constant region in patients with IgG4-AID, we will isolate IgG4 from patients and non-disease controls for analysis of IgG glycosylation (sialylation, galactosylation) by mass spectrometry-based proteomics. Further, we will analyse Fab glycosylation using lectin affinity chromatography and lipid modifications of patient IgG4 isolated from serum by affinity chromatography will be analyzed at UM using lipidomics.
• To study the role of Ab valency and glycosylation for the pathogenicity, we will use patient mAbs with different constant regions and valencies, and deglycosylated patient IgG4, and test them for pathogenicity in primary rat hippocampal neurons or similar cellular models. We expect antibody-specific effects on target interaction depending on the valency of the autoantibodies, which we will analyze using ELISA, co-immunoprecipitation and cell-based assays.

Methods used in the project:

Affinity chromatography, Mass spectrometry, ELISA, Fab-arm exchange in vitro, In vitro assays, Primary rat hippocampal neuron culture

Required project-specific qualifications and skills:

• Master's degree in Biochemistry/Molecular Biology/Immunology or related fields
• English language proficiency is required  
  
• Hands-on experience with cell culture, ELISA and in vitro assays
• Previous experience in bioinformatics or proteomics (preferred)

Project duration: 36 months (40h/week)

Foreseen mobility: Secondments are planned in:

• Maastricht University (supervised by P. Martinez Martinez, the Netherlands): Subcloning of mAbs in IgG1,4 or hinge-mutated IgG4, lipid modifications of patient antibodies.

• Sanquin (supervised by T. Rispens, the Netherlands): Analyze Fab glycosylation levels (using lectin affinity chromatography).

Hosting lab description:

All required methods, including affinity chromatography, primary hippocampal neurons, mass spectrometry and a range of functional assays are established in the laboratory. Inga Koneczny has expertise in the relevant methods, particularly in affinity chromatography, Fab-arm exchange, primary cells and functional studies. Further supervision at MUW will be provided by Romana Höftberger (primary hippocampal neurons), Klaus Kratochwill (mass spectrometry), and in the exchange labs by Pilar Martinez-Martinez (lipidomics) and Theo Rispens (lectin affinity chromatography).

R10: Development of a novel IgG4-specific immunoadsorption therapy for commercial exploitation (WP4)

Development of a novel IgG4 removal therapy for commercial exploitation

To develop a new approach for specific treatment of all IgG4-mediated autoimmune disease (AID) by removing the IgG4 Abs from the patients' circulation. IgG4-AID disease severity correlates with IgG4 titers, while removal of IgG through plasmapheresis in the majority of IgG4-AID patients results in fast improvement of the patient's symptoms. The PhD student R10 (with the supervision of the three PIs) will develop and optimize the IgG4 removal approach. The various parameters that may affect treatment efficiency will be examined (e.g. efficiency and speed of IgG4 removal etc.), and the therapeutic potency of the method will be assessed. The major advantages of the proposed therapy are that a. it is relatively specific and yet it aims to the whole group of IgG4-diseases, b. it should be feasible and not too expensive because the IgG4 is a small Ig fraction (~1/20 of the whole human Ig) c. selective IgG4 removal will not significantly deplete the patients from their protective immunoglobulins.

Main objectives:

We will determine the IgG4 removal efficiency, speed and capacity of the system for IgG4 Abs in vitro. Then (during a secondment to the Istanbul University), the approach's therapeutic potential will be assessed in vitro by testing the effect of whole serum or following IgG4 removal in cell cultures by disruption of AChR clustering in C2C12 cells in the case of MuSK Abs, or neuronal toxicity in the case of LGI1 Abs. Additionally, the calcium status of neurons will be assessed by the Ca++-imaging method. We will determine the therapeutic potency in vivo using passive transfer animal models induced by injection of patient sera in rats for MuSK-MG and autoimmune encephalitis (LGI1 and CASPR2 Abs) and by injection of cloned CASPR2 Abs. These will confirm the therapeutic potential of the approach in conditions similar to the clinical setting.

Methods used in the project:

Antibody production, Heterologous protein production, Affinity chromatography, Cell-based assays, Radioimmunoassay, Antibody function on cell cultures, Ca++-imaging method, SIMOA (Single molecule array) technology, Induction of experimental IgG4-disease in animal models, Rodent handling and surgical techniques

Required project-specific qualifications and skills

• The PhD candidate will be enrolled in the School of Medicine, National & Kapodistrian University of Athens (NKUA). Enrolment requires a Master's degree (students with MD degree, do not need a Master's).
• English language proficiency is required. This may be proved by appropriate degrees, studies in English programmes or during the interviews, or a combination of these alternatives.

Project duration: 36 months (40h/week)

*Should extra time be needed, the hosts will try to cover the student expenses for 1-2 years until completion of the study.

Foreseen mobility: Secondment is planned in:

• Istanbul University (supervised by V. Yilmaz and E. Tüzün, Turkey): Analyze pathogenicity of depleted sera in primary rat hippocampal neurons.
  

Hosting labs description:

The PhD candidate will work in 3 intimately collaborating places in Athens: the research and diagnosis laboratory Tzartos NeuroDiagnostics (PI and Co-supervisor: Socrates Tzartos), the Immunology Department of the Hellenic Pasteur Institute (PI and Co-supervisor: Konstantinos Lazaridis) and the 2nd Neurology Department of the School of Medicine, National & Kapodistrian University of Athens, NKUA (PI and Co-supervisor: John Tzartos). The collaborating groups have extensive and documented relevant expertise in the research topic and in PhD supervising.

Tzartos NeuroDiagnostics (TND) is a major private laboratory in Greece specializing in research and diagnosis of neuroimmune diseases, created in 2013. Some of its diagnostics are unique in Europe and it serves several European labs/clinics. It is also very active in research, discovering new antibodies and developing new diagnostics. The personnel of the lab includes 2 MDs, 2 post-docs (biologist and chemist), 2 Ph.D. students, 3 biologists with Masters' degrees and 4 technicians. TND is fully equipped with all needed instrumentation, including a complete cell culture facility, fluorescent microscope, the high-tech SIMOA HD-X instrument, Fluostar Omega, Lumipulse automatic analyzer, recombinant protein production unit with all needed instrumentation radioimmunoassay facilities with multichannel gamma counters, etc. In addition, it has easy access to instrumentation of nearby research establishments.

The Hellenic Pasteur Institute (HPI) is the oldest biomedical research institute in Greece. It has a well-established expertise in neuroimmune and autoimmune diseases, offering a vibrant research environment supporting young researchers. HPI has core bio-imaging and animal facilities equipped with behavioural and microsurgery units, enabling the execution of advanced studies in animal models. In addition, the host laboratory is fully equipped (e.g. tissue culture, radioactivity work-space, ELISA readers) for the proposed experiments. Konstantinos Lazaridis has expertise in the development of new animal models of autoimmune diseases, in vivo preclinical studies for testing therapeutic efficacy of novel treatments (including antibody aphaeresis) and in vitro characterization of antibody aphaeresis methods as treatment.

The University of Athens (NKUA) is the oldest and major Greek University. It teaches many disciplines. Its Medical School is also the oldest and major Greek Medical School. Its B' Neurology Department, part of the large University hospital "Attico", serves patients from almost half the Athens/Attica area (~2 million people). It has a strong clinical and research interest in neuroimmune diseases, the main expertise of the PI, including the IgG4-related ones. The contribution of John Tzartos will be mostly on clinical aspects on which the B' Neurology Dep. has extensive expertise and is fully equipped. In addition, laboratory infrastructure and instrumentation are available both at the clinic's laboratory and at the collaborating laboratories of the Atticon Hospital.