Project 1: Serological, histological, cellular and genetic characteristics of the IgG4 immune response

Host laboratory: Inga Koneczny,

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.

Figure 1: Immunohistochemical characterization of inflammation in brain autopsies of LGI1 and CASPR2 encephalitis patients. (A-C) LGI1 encephalitis is characterized by only moderate B- and T-cell infiltration at disease onset, with a focus on the limbic system (A, CD8; asterisks mark distribution of inflammatory infiltrates, rectangle enlarged in B, CD20 and C, CD8; 72-year-old man, 3 weeks disease duration). (D and E) In a later disease stage inflammation is more pronounced, with numerous CD8 positive T cells invading the parenchyma (D) and microglia activation (E, HLADR; 70-year-old man with anti-CASPR2 encephalitis, 11 weeks disease duration). F. Dura with resident B-/Plasma cells (CD79a). A: 0.98x, B-E: 400x. (Höftberger et al., unpublished data).

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 (Figure 1), and characterization of these cells may identify dysregulated or dysfunctional IgG4 regulators, and constitute novel biomarkers.

• 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 tissues bases assay, histopathological characterization of patient biopsy and autopsy material and correlation to the clinical characteristics.

• Characterization of tissue-resident immune 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.

Expected Results:

We expect to comparatively characterize IgG4-AID patients and identify clinicopathological commonalities. Further, we expect to characterize disease-associated tissue resident immune cells in biopsy and autopsy material from affected organs.

Methods:

Cell -/tissue based assays, Immuno-histochemistry, Imaging mass cytometry, Flow cytometry, ELISA, Biobanking, Western blot, PBMC isolation,HLA genotyping

Host lab:

The PhD candidate will work in the laboratory at MUW, and will receive supervision by Inga Koneczny and Romana Höftberger. The lab is located in the Division of Neuropathology and Neurochemistry, Department of Neurology, and is the Austrian reference centre 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, Western blot, immunohistochemistry and PBMC isolation,  and imaging mass cytometry are established at MUW. Bonus: Vienna has the best coffee.

1. INSERM (R. Le Panse): Validation of dysregulated tissue immune cells by flow or spectral cytometry

2. TND (S.Tzartos): Validation of diagnostic findings by complementary methodology, rodent model

Project 2: Identification of specific serum biomarkers and immune cell dysregulation commonly associated with IgG4 autoimmune diseases

Host laboratory: Rozen Le Panse

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 around 1,536 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 IU (supervised by V. Yilmaz and E. Tüzün, Turkey).

Project 3: Identification of genetic predisposition and transcriptional factors involved in isotype-switching of IgG4-AID B-cell clones

General description/introduction:

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 other 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 by 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 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 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 blood of affected patients using labelled, recombinant LGI1 domains, cultivate B-cells in vitro and characterize their functional effects.

Objectives:

(1) To identify genetic predisposition for IgG4-mediated AID within and between

subgroups.

(2) 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 sequzencing 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 cell and B-cell receptor sequencing (CSF), direct labelling 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 class-switch from IgG1 to IgG4. This will shed light on genetic and transcriptional mechanisms relevant for initiation and propagation of IgG4-mediated autoimmunity and help in designing targeted therapies.

Planned secondment(s):

1. INSERM (R. Le Panse): M31-M41 (10 months): Analyze LGI1-AE samples by flow cytometry to confirm scRNAseq and CytOF observations in switched B-cells from patients with LGI1-AE.

2. Sanquin (T. Rispens): M18-20 (2 months): Pilot experiments analyzing feasibility of blocking pathogenic IgG4 binding to LGI1 by using a hexamerized decoy LGI1-Fc fusion protein in vitro.

The Ph. D. candidate will work in the neuroimmunology group at the UKSH Kiel 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. Alle procedures are established and instruments available in the group. Our group has extensive national and international collaborations in the field of autoimmune encephalitis. The PhD will be awarded by Kiel University.

Project 4: New animal models for studying IgG4-AID

Host laboratory: Maartje Huijbers

Project description

In vivo studies toward IgG4 function and its role in disease are limited because of a 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 developing 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.

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

Host lab

The project will be executed in the Translational Neuroimmunology lab lead 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 our website!

Exchange labs

In this project you will visit three labs for secondments:

The lab of prof. dr. Pilar Martinez (Maastricht University, The Netherlands)

The lab of prof. dr. Socrates Tzartos (Tzartos Neurodiagnostics, Greece)

The lab of dr. Kostas Lazaridis (Hellenic Pasteur institute, Greece)

Project 5: Investigation of potential IgG4 regulators in vivo and in vitro

Host laboratory: Vuslat Yilmaz

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

Research goal is to identify new therapeutical target of immunological molecules associated with IgG4.

The questions of our 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 from 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;

1. Co-culture studies on PBMCs in a cohort with IgG4-related HLA genotype (healthy, IgG4-anti drug antibody, IgG4-autoimmune diseases).
2. 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).
3. Determination of IgG4 pathogenicity in an animal model by passive transfer of human LGI1/CASPR2-Abs.

The methods to be used are; cell sorting by MACS or FACS, antigenic stimulation in co-culture, CFSE for proliferation, ELISA/bead assay for cytokine level, active and passive immunisation mouse model of EAMG, shRNA application for gene silencing in animal model, gene knocking down in B cells, isolation of antigen specific B cell from PBMC

Project 6: Characterization of early IgG4 responses

Host Lab: Theo Rispens

Project Description

In this project, the central aim is to better characterize IgG4 responses both serologically and cellularly, starting at onset of immune response. We hope to identify key early events and unravel immunological processes that are instrumental in the development of IgG4 responses. As 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 Bcells and their phenotypes), and insight into the dynamics of IgG4-associated features such as enhanced Fab glycosylation (cause or consequence).

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

Host Lab

At Sanquin, we have a long-standing interest in the immunobiology of IgG4 antibodies. Decades of research has 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 (https://www.amsterdamumc.org/en/research/researchers/theo-rispens.htm)

Planned secondment(s):

1.UKSH (F.Leypoldt): genotyping of IgG4-ADA patients

2.MUW (I. Koneczny): correlation of IgG1- and IgG4 response to the HLA genotype

Project 7: Isolation and characterization of monoclonal antibodies

Host lab: Harald Prüss

Project Description

Autoantibodies targeting a variety of neuronal surface proteins have recently been discovered in a multitude of neurological diseases, and continuously define novel autoimmune disorders, such as CASPR2 autoimmune encephalitis. However, characterization of these autoantibodies and their effects have thus far been limited to polyclonal samples, meaning that while the disease specific antibodies were present, other non-pathogenic antibodies were present as well and could have interfered. To overcome this limitation, we have developed a multi-step approach to isolate single (monoclonal) antibodies from patient derived B cells, which can be found both in the blood as well as the cerebrospinal fluid within this group of disorders. This methodology uses molecular biological techniques, such as polymerase chain reaction (PCR) DNA amplification, together with custom made bioinformatic analysis to analyze the antibody repertoire. From this information, we are able to recombinantly express these monoclonal antibodies and investigate their potential pathogenicity in several assays, both in vitro and in vivo. Thus far, within our lab, we have been able to successfully isolate a range of monoclonal antibodies within several diseases, such as autoantibodies targeting NMDA receptor and GABAA receptor. However, the focus has always laid upon IgG1, and other subtypes were often overlooked. In this project, the goal is to isolate IgG4 monoclonals in the context of autoimmune neurological diseases, such as CASPR2 autoimmune encephalitis, and then further characterize these antibodies in vitro and in vivo. For this, you will use a wide variety of techniques, such as single cell sorting, PCR, cloning, cell culture and animal modeling.

Host Lab

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: https://expneuro.charite.de/en/department_of_experimental_neurology/team_pruess/.

Secondments

As part of this project, you will get the opportunity to visit the following labs:

• The lab of Prof. Dr. Pilar Martinez (Maastricht University, The Netherlands)
• The lab of Prof. Dr. Socrates Tzartos (Tzartos Neurodiagnostics, Greece)
• The lab of Dr. Kostas Lazaridis (Hellenic Pasteur institute, Greece)

Project 8: In vivo study of pathogenic mechanisms of IgG4-AID

Host laboratory: Pilar Martinez-Martinez

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 of protein-protein interaction thus disrupting signal transduction pathways and tissue architecture, instead of complement-mediated injury of target-organs5. For example, MuSK Abs interrupt signal transduction pathways required for the maintenance of neuromuscular junctions7 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: It does 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.

Methods used in the project:

The main objective of this study is to characterize the structural, molecular and functional characteristics of IgG4 Abs.

In collaboration with other members of the consortium, the PhD candidate 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.

Host lab:

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 discover biomarkers and develop new diagnostic assays and study the potential of novel treatment strategies. The group has 3 main research lines; 1) Role of lipids and their transporters in Neuropsychiatric disorders and 2) Autoimmune factors in Neurological, Psychiatric and Neuropsychiatric disorders.

Exchange labs:

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

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

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

Project 9: Structural and functional characteristics of IgG4 autoantibodies

Host laboratory: Inga Koneczny

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.

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.

Expected results:

We will characterize post-translational modifications (glycosylation, lipid modifications) in IgG4 autoantibodies and identify the relevance of Fab-arm exchange and IgG subclass for blocking of protein-protein / cell-cell interaction and/or cellular toxicity. This will help us understand the relevance of the structural characteristics of IgG4 and their relevance for pathogenicity.

Methods:

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

The PhD candidate will work in the laboratory at MUW, and will receive supervision by Inga Koneczny as main supervisor. 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 Kratochwil (Mass spectrometry), and in the exchange labs by Pilar Martinez (lipidomics) and Theo Rispens (lectin affinity chromatography). Bonus: Vienna has the best coffee.

Exchange labs:

1. UM (P.Martinez): Subcloning of mAbs in IgG1,4 or hinge-mutated IgG4, lipid modifications of patient antibodies

2. Sanquin (T.Rispens): Analyze Fab glycosylation levels (using lectin affinity chromatography)

Project 10: Development of a novel IgG4 removal therapy for commercial exploitation

Host laboratory: Socrates Tzartos, Konstantinos Lazaridis, John Tzartos

Objectives: 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 patient's symptoms. To develop a new and fast treatment for all IgG4-AID, the Ph.D student R10 (with the supervision of 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.

Expected Results: We will determine the IgG4 removal efficiency, speed and capacity of the system for IgG4 Abs in vitro. Then, 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. The latter will be done by annexin V-PI staining (flow cytometry) and expression level measurements of apoptosis-related molecules (WB and real time PCR). Additionally, calcium status of neurons will be assessed by 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 (to be developed in WP3). These will confirm the therapeutic potential of the approach in conditions similar to the clinical setting.

The Ph.D candidate will be enrolled in the School of Medicine, National & Kapodistrian University of Athens, and will work in 3 intimately collaborating places in Athens: the research and diagnosis laboratory Tzartos NeuroDiagnostics (Socrates Tzartos) the immunology Department of the Hellenic Pasteur Institute (Kostas Lazaridis) and the 2nd Neurology Department of the University of Athens (John Tzartos). The collaborating groups have extensive και documented relevant expertise in the research topic and in Ph.D supervising. Part of the work will be also performed in collaboration with V. Yilmaz and E. Tuzun of the Department of Neurosciences, Istanbul University.