Title: Transcriptomic deconvolution to dissect the immunological mechanism of B cell depletion therapy in ANCA associated vasculitis.
Funder: Medical Research Council

Abstract: Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is an autoimmune condition characterised by inflammation of blood vessels, which can affect any organ in the body. The condition used to be fatal but, with the introduction of immune-suppressing drugs, can now be treated as a life-long condition with periods of remission (control) and relapse (flare-up). _x000D_ Rituximab is a newer drug, which targets a particular subset of immune cells, called B cells. B cells have many functions, including the production of ANCA antibodies which can lead to flare-ups in people with ANCA-associated vasculitis. Repeated dosing of Rituximab can effectively stop these flare-ups and control the disease for many patients, but it can also limit the ability to produce other antibodies. This can weaken the immune system and lead to increased risk of infection; it can also affect the body’s ability to respond to vaccinations. For example, many patients treated with Rituximab who have received the Covid-19 vaccination remain vulnerable to infection._x000D_ It is not clear why Rituximab is a successful long-term treatment for some patients while other patients relapse when it is stopped. It is likely due to the interactions of B cells with other cells in the immune system. This study will analyse blood samples collected from participants with relapsing ANCA-associated vasculitis who took part in a previous clinical trial (RITAZAREM). In this trial, repeated doses of Rituximab were compared with another drug (Azathioprine), to see which was better at controlling the disease. We will use new, sophisticated computer methods to analyse how the B cells interacted with other immune cells when treated with either Rituximab or Azathioprine, and how this changed over time. Researchers will compare this information with the clinical information collected as part of the trial to see if there is overlap between changes in the B cell interactions and disease flare-ups. _x000D_ This study will help us understand the effect of Rituximab on the immune system and potentially explain why some patients react differently to treatment. Researchers hope this knowledge will help clinicians prescribe more effective personalised treatment for people with ANCA-associated vasculitis.

Lay abstract: Dissecting immunological mechanisms of B cell depletion to explain disease course following rituximab in ANCA associated vasculitis(AAV) using transcriptomic deconvolution methods_x000D_ Background: Rituximab, a B cell depleting monoclonal antibody is an established treatment for AAV. But the effect is variable and often not sustained with relapses occurring on discontinuation of therapy. B cell return and rising ANCA levels are associated with relapse, but neither have sufficient sensitivity or specificity to be used as biomarkers to inform treatment decisions. T cell dysregulation persists during clinical remission and is associated with relapse. The longer-term response to rituximab is likely a combination of these B cell factors, as well as the complex relationship between B and T cells that underpins the pathophysiology of AAV. Yet, the effect of rituximab on T cell populations is not known._x000D_ Aims/Objectives: To understand processes leading to establishment of long-term remission or disease recurrence on stopping rituximab in AAV. Specifically, to_x000D_ 1. Identify and quantify longitudinal changes in deconvoluted immune cell proportions following B cell depletion therapy and relate to clinical outcomes._x000D_ 2. Directly query the longitudinal trajectory of a previously identified T cell exhaustion signature associated with clinical outcome at baseline in AAV._x000D_ Methods: Using a transcriptomic approach, the effect of rituximab on cell populations, using samples collected from 150 patients at 4 key time points as part of the RITAZAREM clinical trial will be undertaken. RNA will be extracted in collaboration with the Cambridge Stratified Medicine Core Laboratory using established protocols and sequencing performed using an Illumina HiSeq4000 platform to generate a target of 20M 75bp paired-end reads per sample for bulk mRNAseq. Using cutting-edge machine learning based deconvolution methods of whole blood transcriptomes, cell intrinsic profiles will be analysed at multiple time points in each individual. Existing postulated predictors of sustained remission, including delayed reconstitution of memory B cells and a CD8 T cell exhaustion signature will be evaluated, in the context of high-quality clinical data collected as part of this clinical trial._x000D_ Timelines: The RITAZAREM trial completed in November 2019. All samples have been collected and are stored in a single repository. RNA extraction and sequencing could commence immediately, allowing time for transcriptomic analyses to be conducted and clinical data integrated, and the project to be completed within 12 months._x000D_ Impact/dissemination: Further characterisation of the immune impact of rituximab with potential identification of new therapeutic targets of relevance to AAV and other autoimmune diseases. Optimisation of relapse prevention strategies with rituximab leading to more personalised therapy, efficient use of rituximab and better patient outcomes. The Cambridge vasculitis group have a long record of generating data that is incorporated into management guidelines. They also lead on national and international guideline statements and communicate advances directly to vasculitis specialists through the UKIVAS ands EUVAS networks. There is considerable interest around optimising rituximab dosing and translatable advances will be rapidly adopted. This proposal is supported by Vasculitis UK, the leading patient charity for AAV and results will be disseminated via them to reach patients.

Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CARDIOVASCULAR;INFLAMMATORY AND IMMUNE SYSTEM