The different aspects of B cell biology in arthritis.

Arthritis, i.e. inflammation of joints, is a common symptom of several
autoimmune or auto-inflammatory disorders. The most common forms of arthritis
are osteoarthritis (OA) and rheumatoid arthritis (RA). While OA is regarded as
primarily degenerative disease, RA is a systemic, chronically-progressive
inflammatory disease that specifically targets the synovial tissue of
diarthrodial joints. RA is characterized by joint pain, swelling and
destruction of bone and cartilage, leading to functional failure of the joint
and impairment of quality of life. This disease afflicts approximately 1 % of
the population worldwide.1 The etiology of RA remains unresolved. However, two
main observations point to a crucial role for B cells in the pathogenesis of
RA. First, the majority of patients harbor autoantibodies that, in part, are
highly associated with disease risk, disease severity and progression. 2Second,
B cell depleting therapy has proven to be an effective treatment for RA.3 Both
aspects, however, are not necessarily related, as the depletion of B cells
ameliorates disease while the levels of autoantibodies remain high. Therefore,
multiple functional aspects of B cells as mentioned above (i.e.: antibody
production, cytokine production, antigen presentation) need to be considered to
understand the role of B cells in the pathogenesis of RA. In addition, it is
important to study the characteristics of the autoantibodies themselves, as
these allow to conclude on characteristics of the B cells that produced the
antibodies.

This project intends to study the biology of B cells in arthritis. B cells are
important components of the human immune system and have several functions.
Next to the production of antibodies, B cells can function as efficient antigen
presenting cells and as producers of cytokines and of other mediators relevant
to the immune system. In order to make use of human material as efficiently as
possible, it is intended that the material obtained for this project (see
below) will be used to study several aspects of B cell biology in the context
of rheumatoid arthritis (to analyze autoreactive B cells and their functional
requirements, the interaction with autoreactive T cells, the characteristics of
the autoantibodies in serum and those produced in culture, as well as
functional aspects of relevant autoantibodies) These data need to be analyzed
in the light of clinical characteristics such as disease activity, disease
duration, treatment and age and sex of the patient.

Autoantibody-specific B cells:
Several autoantibodies have been described in RA. Among those,
anti-citrullinated protein antibodies (ACPA) exhibit the highest specificity
for the disease, predict disease onset and severity, and identify a subgroup of
patients with distinct genetic background and requirement for aggressive
treatment.4 Because of these associations, ACPA-specific B cells and ACPA
themselves are the focus of intense research efforts. Recently, we have shown
that particular subsets of B cells, called plasmablasts and plasmacells, are
capable of producing ACPA and circulate in peripheral blood of patients with
RA. 5Preliminary data also indicate that these cells are present in high
frequency at the site of inflammation, i.e. the inflamed joint. Therefore, the
first part of the project described will focus on further characterization of
the phenotype of these cells and on compounds that might be able to inhibit
their function.

Autoantibody characteristics:
As described above, multiple autoantibody reactivity*s have been described in
sera of patients with RA. ACPA, the most specific and potentially clinically
most relevant reactivity, comprise a group of polyclonal antibodies targeting
citrullinated proteins. Recent studies by our group and others have
demonstrated that ACPA have distinct properties that indicate that ACPA
producing B cells are different from *conventional* B cells. Specifically, we
found that ACPA carry distinct glycans which are absent from the majority of
other antibodies.6-8 As glycans play a fundamental role in modulating pro- or
anti-inflammatory effector functions of antibodies, studying ACPA-specific
glycosylation is crucial for understanding ACPA pathogenicity and the biology
of the underlying B cell response. Therefore, another, second part of the
project described will focus on the characteristics of autoantibodies in RA
with a specific focus on ACPA. This also includes studies on the functional
consequences of these autoantibody characteristics, e.g. for the interaction
with other immune cells, with relation to the pathogenesis of RA.

Development/Generation of autoreactive B cells:
Another research focus is to understand the pathogenesis of RA and more
specifically the mechanisms by which individuals develop ACPA. In the context
of RA, it is still unclear why, how and where autoreactive B cells develop. B
cells require help from T cells to develop into antibody producing plasmacells.
Therefore, also the interaction between helper T cells and B cells from RA
patients will be studied. The most important genetic risk factor for the
development of ACPA positive RA is the HLA class II locus. HLA class II
molecules present peptides to CD4+ T-cells and it is proposed that the HLA
class II association with ACPA positive RA is explained by (autoreactive) CD4+
T-cells providing help to citrulline-specific B-cells. We are currently
studying different implicated (autoreactive) CD4+ T-cells populations and there
role in ACPA positive RA.

Our in-vitro culture system offers the possibility to assess the influence of
therapeutic and/or immunomodulatory agents on the generation of
autoantibody-producing B cells. The amount of autoantibody positive culture
wells as well as the mean titer of autoantibodies produced serve as readouts.
Changes in the ratio between autoantibodies and total IgG can serve as
indicator for effects specifically affecting autoantibody production. We
propose to add different inhibitory compounds relevant to B cell function in
grading concentrations to our cultures, e.g. an anti-CD40 monoclonal antibody
or a JAK/STAT-inhibitor. In addition, different B cell subsets will be isolated
to high purity by FACS sorting and stimulated separately with or without such
compounds in order to assess the individual effects of these compounds on
autoantibody producing B cells. In all cultures, we will measure the production
of total IgG as control.

Furthermore, relevant phenotypic markers will be examined on various B cell
subsets using flow cytometry to determine which subset of B cells is
potentially affected by individual compounds and to determine if there is a
correlation between the phenotype of B cells and autoantibody titers. In
addition, platelet poor plasma will be collected to specifically determine
soluble CD40 ligand (sCD40L) levels in correlation to autoantibody titers. As a
beneficial side effect of the preparation of platelet poor plasma, a pure
population of platelets will be obtained. As activated platelets (CD62P+) are
the main circulating resource of sCD40L, platelet phenotype will be determined
using flow cytometry (expression of CD62P, soluble and membrane bound CD40L,
platelet-platelets aggregates). Furthermore, platelets will be activated
in-vitro using platelet agonists (TRAP, ADP) to prompt sCD40L production and
the interaction between platelets and B cells will be studied.
With regard to autoantibody characteristics, we will study the glycosylation
autoantibodies in detail, with a specific focus on the Fab region of ACPA. The
predominant function of the Fab region of an antibody is the binding of its
cognate antigen. Therefore, glycans located in the variable region of IgG Fab
are likely involved in increasing or decreasing affinity for antigen binding
and could serve as a potential new biomarker in rheumatoid arthritis. To this
end, we will determine the structure and precise localization of ACPA
Fab-linked glycans and we will investigate the biological relevance of ACPA Fab
glycosylation for RA pathogenesis. These studies will also involve in-vitro
culture of B cells isolated from these compartments for the stimulation of ACPA
production as described before and subsequent glycosylation analysis.

In relation to the presence of autoreactive CD4+ T cells we are now able to
stably produce HLA class II tetramers that can be used to quantify and
characterize specific populations of antigen-specific T-cells. This enables us
to track vinculin-DERAA specific T-cells and other autoreactive T-cells in the
peripheral blood and the synovial fluid of patients. We will follow these
T-cells during disease progression and determine if there is a correlation
between these specific T cells and ACPA production. Furthermore, these specific
tetramers enable us to isolate autoreactive T cells from patients and to study
their T-cell repertoire and capacity to activate B-cells. A biased TCR
repertoire could provide us with a new therapeutic strategy by specifically
targeting only those immune cells contributing to the pathogenesis of ACPA
positive RA.

Blood sampling will occur at the *Prikpost* B4 at the LUMC. Therefore, the
risks of this study is limited to a minimal bruise due to the blood collection.
The aspiration of the synovial fluid will be performed by a rheumatologist only
when he/she decides that this is required for optimal medical care. The
participants do not benefit from this study but could lead to improved future
therapeutic care.