Understanding B cell biology in auto-immune diseases.

Background
This protocol is part of a longstanding research line of our department that
investigates the pathophysiology of rheumatic diseases. Our studies on
auto-reactive B cells have so far focused on one prototypic rheumatic
autoimmune disease: rheumatoid arthritis (RA). The majority of RA patients
harbour antibodies against citrullinated protein antigens (ACPA). ACPA are
highly disease specific biomarkers that associate with destruction of joints,
the pathological hallmark of RA. ACPA presence pre-disease prognosticates RA
development, implicating crucial involvement of ACPA in relevant
disease-initiating processes. Experimental data support this notion as infusion
of ACPA in mice exacerbates arthritis. Moreover, ACPA trigger a variety of
inflammatory processes in vitro. Likewise, genetic variants in genes that
encode peptidyl arginine deiminases, the enzymes that generate citrullinated
antigens, are risk factors for RA. Together, these observations suggest that
ACPA and/or the citrullinated antigen-specific B cell response have a central
role in RA pathogenesis.

Aim of the study
We intend to study in detail the different aspects of B cell biology in the
auto-immune diseases outlined above. With the sample material obtained from
patients (see below), we will be able to analyze (auto-reactive) B cells and
their functional requirements, the interaction with other cells of the immune
system or with stromal cells, the characteristics of the autoantibodies in
serum and those produced in culture, as well as functional and molecular
aspects of relevant autoantibodies. The studies will include phenotypic
analysis of B cells and their interaction partners by flow cytometry and mass
cytometry, transcriptomic analyses of isolated B cells, B cell receptor
sequencing analyses, single cell cultures and co-culture systems, migration
assays, and functional studies including the analysis of secreted
autoantibodies by proteomics/glycomics techniques such as mass spectrometry,
UPLC and comparable methods. The data obtained need to be analyzed in the light
of clinical characteristics such as disease activity, disease duration,
treatment and age and sex of the patient. Of note, it is crucial to perform
these studies on material from patients, as suitable murine models are not
available for the types of auto-reactive B cell responses that we intend to
study.

To achieve our goals, we intend to collect peripheral blood from consecutive
patients with clinically suspect arthralgia, rheumatoid arthritis, systemic
lupus erythematodes, ANCA-associated vasculitis, and systemic sclerosis. In
case patients display arthritis as one of the clinical symptoms and
arthrocentesis is performed as part of the clinical diagnostic or therapeutic
work-up, also synovial fluid will be studied. Both the cellular and humoral
components will be isolated from the material collected. The cellular component
will be used to isolate immune cells such as total PBMCs, B cells, T cells and
others. Isolated cells will be characterized phenotypically using cell-surface
markers (FACS) and functionally in cell type-specific assays (e.g.
proliferation, cytokine secretion, in-vitro culture system for
autoantibody-producing B cells). The humoral component (serum/plasma/synovial
fluid) will be used for measurement of various soluble factors related to the
function of B cells, such as (auto-) antibodies, complement factors,
coagulation factors, cytokines etc.

Potential risks and benefits
Blood sampling will occur at the central blood draw facility *prikpost*
of the LUMC (currently located at C2). Therefore, the risks of this study are
limited to the collection of peripheral venous blood and includes pain at the
place of puncture, hematoma formation and, rarely, short moments of
hypotension. These symptoms are usually mild and recover fully. The central
blood draw facility is trained and equipped to handle these events
appropriately. The aspiration of the synovial fluid will be performed by a
rheumatologist but only when he/she decides that this is required for optimal
medical care. The participants do not benefit from this study but their
participation could lead to improved future therapeutic care.