MICA: Investigating the pathogenic role of T follicular helper cells and their therapeutic targeting in primary Sjogren's syndrome

Lead Research Organisation: Queen Mary, University of London
Department Name: William Harvey Research Institute

Abstract

Sjögren's syndrome (SS) is the second most common rheumatic autoimmune disease with a prevalence affecting between 100,000-250,000 in UK and a predominance in women (female to male ratio is 9:1). SS is characterised by immune cell infiltration in the salivary and lacrimal glands leading to dry mouth and dry eyes. However, SS is a highly heterogeneous disease with 30/40% of SS patients developing moderate to severe extraglandular manifestations and 5% developing a form of cancer (lymphoma) which arise from B cells, a type of white blood cells. Currently, the treatment of SS is unsatisfactory with no effective therapies available.

Studies from our and other groups have identified a subset (30-40%%) of SS patients which present B cell clusters in the salivary glands called germinal centres. These patients have a more active disease and have a 16-fold higher risk of developing lymphoma than the remaining patients without germinal centres.

Previous work has demonstrated that another subset of very specialised white blood cells, called T follicular helper (Tfh) cell, play a critical role in supporting the survival and activation of B cell in germinal centres. While the role of Tfh is well established in germinal centres which arise in lymphoid organs (i.e. the spleen, tonsils or lymph glands) their role in aberrant germinal centres which form during chronic inflammatory disease is unknown.

Thus, in this application, which is a Partnership between 3 UK Universities and MedImmune, a pharmaceutical company, we aim 1) to investigate the role of Tfh in promoting the disease in a series of studies involving human samples and animal models; 2) to address whether blocking Tfh in preclinical studies with novel pharmaceutical compounds will impact on various features of the disease, such as the activation of B cell in the salivary glands and the amelioration of salivary flow and 3) to identify key factors which will allow us to understand which SS patients are more likely torespond to novel treatment targeting Tfh in SS.

This proposal have the objective to have immediate clinical impact as MedImmune and the Academic partners of this application are working closely together to bring novel treatments in the clinic for the benefit of patients with SS (pilot clinical studies planned for late 2015 and in 2016 in UK). It is envisaged that, if successful, this work will also impact on other medical conditions which might benefit from targeting Tfh cells. Specifically, we anticipate that this proposal will bring new knowledge and will identify key factors which will allow us to measure the level of activation of Tfh in different patients. This work will be essential because will inform on which SS patients are more likely to respond to the new treatments that we are developing and will be tested in upcoming clinical trials in SS. These trials will be based on the concept of "target validation", whereby the work done in this project will put us in the position to stratify patients based on the level of Tfh present in their salivary glands (or in their blood). We hope that this approach on one side will increase the response rate and on the other side will prevent SS patients who are unlikely to respond from being exposed to the potential side effects of the novel treatments.

Technical Summary

In this proposal we will test the hypothesis that T follicular helper (Tfh) cells fuel autoreactive B cell activation and autoimmunity in Sjogren's syndrome (SS), in particular in patients with germinal centres (GC) in the salivary glands (SG). Moreover, we aim to pursue with our industrial partner a translational program to develop Tfh blocking agents for clinical use in SS and to understand how Tfh-based patient stratification may inform randomised clinical trial (RCT) design. We will proceed following 3 progressive steps:

1) To investigate the presence of Tfh and their products in the circulation and SG of SS and characterise their functional role in promoting autoreactive B cell activation.
We will access the deeply phenotyped EMR SS Biobank with over 150 SG biopsies with matching histology, RNA and peripheral blood. To identify Tfh-related signatures and stratify samples according to the degree of lymphoid organization, we will use:
i) multicolour confocal microscopy
ii) Fluidigm microfluidic chips
iii) multicolour FACS analysis
iv) in vitro organ cultures

2) To test in vivo the efficacy of pharmacological blockade of Tfh in modulating sialoadenitis, autoimmunity and exocrine dysfunction in murine models of SS.
We will block key factors involved in Tfh function, such as the ICOS/ICOSL and the IL-21/IL21R pathways, in order to ameliorate disease in 2 animal models of SS:
i) spontaneous NOD.H2h4 mice
ii) novel human SS/SCID xeno-grafts

3) To define the contribution of Tfh to SG pathobiology and disease outcome through the analysis of biopsies pre- and post-treatment in order to inform Tfh-based patient stratification.
We will use matching SG biopsies and peripheral blood collected as part of the UK multicentre RCT of Rituximab in SS. Using the techniques summarised in Aim 1, we will investigate whether:
i) Tfh signatures are influenced by B cell depletion
ii) persistency of Tfh allows the escape of autoreactive B cells in the SG

Planned Impact

The project will have a significant impact both on basic research and applied translational science.

1. Impact on basic research and knowledge acquisition within and beyond the field of SS and rheumatology.
Tfh cells play a fundamental role in the regulation of adaptive immune responses, specifically on B cell activation and germinal centres in secondary lymphoid organs. Our proposal aims to investigate Tfh within ectopic germinal centres forming in the salivary glands of patients with SS. Thus, at the basic science level a better understanding of the role of key pathways involved in Tfh function, such as the ICOS/ICOSL and IL-21/IL-21R will broadly benefit the scientific community with considerable advance in knowledge not only in the specific field of SS but also other autoimmune diseases, characterized by ectopic germinal centres (i.e. rheumatoid syndrome, lupus nephritis, inflammatory bowel diseases, multiple sclerosis, autoimmune thyroiditis etc).

2. Impact on translational and clinical research
- Moving towards new therapeutics in SS: Demonstration that blocking Tfh cells ameliorate sialoadenitis, autoimmunity and exocrine dysfunction will provide supporting evidence that targeting Tfh-related pathways is a potential therapeutic strategy in SS. The opportunity to dissect the molecular functions in vitro and in vivo (SCID/SS chimeras) using human tissues will provide invaluable data for the translational relevance of this pathway to the disease. Our aim is to impact on clinical trial design by identifying biomarkers that could be used to stratify patients prior to inclusion in clinical studies. MedImmune, the Industrial partner of this application, has invested significant resources to bring preclinical compounds towards early phase clinical trials. The PI and Academic co-applicants of this application are working closely with MedImmune within the NIHR NOCRI translational research partnership to develop RCTs in SS. This project will impact in defining inclusion criteria and clinical and biological endpoints in view of starting patient recruitment in late 2015 (anti-ICOS) and from 2016 (anti-IL-21).

- Broader impact on translational and clinical science through the development of Tfh blocking agents for clinical use: MedImmune is a world player in antibody therapeutics with significant expertise in the development of novel therapeutics in autoimmune diseases. SS as a first indication is highly ranked in the translational pipeline. The opportunity to develop unique reagents in partnership with MedImmune will further accelerate the developmental programme to bring Tfh targeted therapies for clinical use not only in patients with SS, but also in other autoimmune conditions characterized by autoreactive B cell activation.

3- Commercial impact on the public and private sector. A defined developmental programme described in this application coupled with a strong Academic Industry partnership will maximise the opportunity to translate novel scientific and clinical observation into commercially valuable assets such as diagnostic or therapeutic agents. The equitable Head of Terms agreed between QMUL and MedImmune will also ensure that economic benefits will profit both private and public partners.

4- Impact on patient health. It is hoped that the ultimate beneficiaries of this research will be patients with SS, a disease with prevalence in UK between 0.2-0.5% (100,000-250,000 patients) which currently lacks disease modifying drugs. This application will impact directly on patient health as the pathways that we intend to study in this proposal will be targeted in early phase clinical trials during the proposed tenure of this application. The definition of Tfh-related tissue and/or peripheral biomarkers of disease activity as well as response (or lack thereof) to B cell depletion will make immediate impact on SS through patients stratification guiding "intelligent" clinical trial design.

Publications

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Astorri E (2016) Ultrasound of the salivary glands is a strong predictor of labial gland biopsy histopathology in patients with sicca symptoms. in Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology

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Barone F (2015) IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs. in Proceedings of the National Academy of Sciences of the United States of America

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Bombardieri M (2017) Ectopic lymphoid neogenesis in rheumatic autoimmune diseases. in Nature reviews. Rheumatology

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Corsiero E (2016) NETosis as Source of Autoantigens in Rheumatoid Arthritis. in Frontiers in immunology

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Corsiero E (2016) Ectopic Lymphoid Structures: Powerhouse of Autoimmunity. in Frontiers in immunology

 
Description BSR and BHPR Guideline for the Management of Adults with Primary Sjögren's Syndrome
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
URL http://www.rheumatology.org.uk/includes/documents/cm_docs/2016/f/full_guidline_open_cons_sjogrens.do...
 
Description Horizon 2020 funding for the HarmonicSS project on the harmonization of Sjogren's patients cohorts to change policy at european level
Amount € 283,000 (EUR)
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 01/2017 
End 07/2020
 
Description Recombinant monoclonal antibodies from single lesional B cells isolated from the salivary glands of patients with Sjogren's syndrome 
Organisation Stanford University
Country United States of America 
Sector Academic/University 
PI Contribution This is a collaborative effort with Prof WH Robinson at Stanford University aimed at dissecting the autoantigen specificity of B cells infiltrating and forming ectopic germinal centres in the salivary glands of patients with Sjogren's syndrome. In this collaboration, single B cell subsets (i.e. naive, vs memory vs plasmablasts) will be single cell sorted at our lab. Monoclonal antibodies will be then generated using either 1) traditional single cell PCR for the H and L Ig variable region as already established in my lab or 2) see below
Collaborator Contribution 2) in collaboration with Stanford following NGS and barcoding whereby individual IgH V region will be first sequenced and dominant clones established, including analysis of intra-lesional clonal diversification as indication of an antigen-driven response. Selected sequences will be then cloned into expression vectors for antibody expression and characterization of their antigenicity.
Impact This collaboration formed the basis for the recent award of an ARUK PhD Scholarship.
Start Year 2016
 
Description Role of IL-27 in regulating TFh 
Organisation Cardiff University
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We entered a formal collaboration to conduct experiments in Cardiff University with a novel model of inducible ectopic lymphoid neogenesis that we previously developed via adenoviral-delivery in murine submandibular glands through retrograde cannulation of the excretory duct.
Collaborator Contribution Our collaborator is making available IL-27Ra KO mice to run the cannulation model
Impact The collaboration has just been formally set up, further details will be included in the next report period
Start Year 2016
 
Description T cell profiling in Sjogren's syndrome 
Organisation University of Cambridge
Department Department of Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have provided matched PBMC collected from patients with Sjogren's syndrome before and after treatment with Rituximab as part of the TRACTISS trial. We will be analysing T cell phenotype using a CyTOF panel that have finalised which will be compared with molecular signatures identified by the Cambridge group.
Collaborator Contribution Dr Paul Lyons in Cambridge will generate molecular signatures via RNA NGS on CD4 and CD8 populations sorted from PBMC.
Impact Analysis is under way
Start Year 2015
 
Title Clinical Trial in Sjogren's syndrome 
Description This is a phase 2a clinical trial in primary Sjogren's syndrome using a anti-ICOSL antibody thus targeting interaction between Tfh cells and B cells. It is a co-development between Amgen and MedImmune. I am the CI for the UK and I will be intimately involved in the analysis of salivary gland biopsies taken before and after treatment in order to 1) identify biomarkers able to predict response to treatment and 2) elucidate mechanisms of action of the IP. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Early clinical assessment
Year Development Stage Completed 2017
Development Status Under active development/distribution
Clinical Trial? Yes
Impact Clinical trial is completing recruitment. Impact will depend on the clinical outcomes. 
 
Title Phase II clinical trial in primary Sjogren's syndrome 
Description This is a phase 2a clinical trial in primary Sjogren's syndrome blocking the CD40/CD40L interaction thus influencing the crosstalk bwtween Tfh cells and B cells. It is sponsored by Novartis and adopted by the NOCRI musculoskeletal translational research partnership (TRP). 
Type Therapeutic Intervention - Drug
Year Development Stage Completed 2017
Development Status Under active development/distribution
Clinical Trial? Yes
Impact Recruitment in progress, impact depending on clinical outcomes.