Does anti-CD20 treatment affect vaccination
responses
in RA?
B cells are an important part of the adaptive as
well as the innate immune system since they are
able to produce immunoglobulins, present antigens,
and secrete cytokines. In humans, the B cells are
produced in the bone marrow by pluripotent stem
cells. Their further development can be divided
into two phases: the initial phase that occurs in
the primary lymphoid organs (foetal liver or adult
bone marrow), where the pro-B cells are formed [1].
After gene rearrangement the IgH and IgL chains are
expressed and form the first functional surface
bound immunoglobulin M receptor, and the B cells
progress to immature B cells. Thereafter follows a
clonal selection process [2] where the B-cell
receptor is ascertained not to react with self
antigens. Later, the B cells migrate out to the
peripheral lymphoid organs for further maturation
[1]. Once in the peripheral lymphoid organs, the B
cells encounter specific antigens reacting with
their surface Ig receptor and this interaction
triggers clonal B-cell expansion. After this step
the B cells further mature to finally become memory
B cells or plasma cells.
It is believed that B cells may play an important
role in the development as well as maintenance of
rheumatoid arthritis (RA) by their production of
autoreactive antibodies, e.g. rheumatoid factors
and antibodies to citrullinated peptides. These
autoantidodies can be detected in almost 80% of all
patients [3]. In addition, B cells migrate and
accumulate in the zone of synovial membrane
junction with the cortical bone participating in
pannus
formation
and
cartilage
destruction
processes in RA.
It has recently been shown that B cells may
contribute
to
the
RA
pathogenesis
in
some
additional ways. In fact, Silverman and Carson [4]
showed
that B cells present immune-complexed
antigens to autoreactive T cells, express adhesion
and other co-stimulatory molecules that promote Tcell activation, synthesize chemokines that induce
leukocyte infiltration, and produce factors that
initiate and sustain angiogenesis and granulation
tissue formation.
B cells can be found in large quantities in the
rheumatoid synovial tissue in all RA patients. This
synovial tissue have been found to look and behave
as germinal centers and is therefore called ectopic
germinal centers [5]. These ectopic germinal
centers contain T cells and B cells arranged around
a network of dendritic cells. However, in certain
other patients these lesions contain aggregates of
B cells and T cells, and finally, the third type of
lesions displays diffuse arrangement of B cells and
T cells [6].
Furthermore, Takemura et. al. [7]
reported
that
B
cells
produce
and
express
lymphotoxin (LT)- and LT-. Analysis of LT- mRNA
in synovial tissues indicated a strong correlation
with IgG production, another marker closely related
to synovial architecture. In fact, RA-patients with
ectopic
germinal
center
formations
displayed
concomitant high levels of LT- mRNA as well as
high IgG production. In addition, a recent study by
Brown et. al. [8] suggests that LT-12 may be
responsible for several more effects than just
arranging B cells. LT-12 has been shown to cause
profound changes in the function of synovial
fibroblasts, resulting in induction of a number of
factors that contributes to inflammation and T-cell
recruitment.
By use of anti-CD20 monoclonal antibodies Takemura
et. al. [9] treated mice engrafted with synovial
tissue
from
RA-patients
resulting
in
total
dissociation of the B-cell follicular structures.
In addition, the levels of IL-1 and IFN-
dramatically decreased, suggesting that B cells had
also profound effect on T-cell differentiation in
synovial tissue.
We have previously shown that immunization trigged
response to vaccines (e.g. influenza vaccine) is
decreased in patients with RA [10]. This finding
may be ascribed both the innate immunity caused by
the disease itself as well as treatment remedies
including
use
of
cytostatic
drugs.
The
new
immunomodulatory
treatment
has
successfully
diminished both inflammation and tissue destruction
in diseases such as RA and SLE. However, the price
of this success is increased risk of infections. In
case of CD20 antibody treatment the risks of
decreasing immune responsiveness to viral and
bacterial infections is evident.
Questions and hypotheses
The long-term effects on the vaccination status
regarding the major viral (e.g. influenza) and
bacterial (e.g. Streptococcus pneumoniae, HiB,
meningococcal polysaccharides) antigens following
anti-CD20 therapy are unknown. We would like to
assess in RA-patients, treated with Rituximab, the
antibody response to the above immunogens before,
during, and after treatment with Rituximab. This
has to our knowledge not been performed in the
setting of anti-CD20 treatment in RA.
1) This study is a long-term study where the RApatients will be immunised with viral and bacterial
vaccines and the specific immune responses using
the ELISPOT-technique and regular ELISA will be
analysed. The vaccination will be performed in (a)
a group of RA patients (n=10-20) prior to start
with Rituximab, (b) just after finished Rituximab
treatment (n=10-20) and (c) 6-12 months later
(n=10-20).
2) In the second study we will analyse, using flow
cytometry, the intracellular cytokine expression
(in B cells, T cells, and circulating monocytes)
during the same intervals as in point 1. This is to
clarify
the
immunomodulatory
properties
of
Rituximab on circulating T cells and monocytes. In
addition, we will analyse the CD25 expression on B
cells before and after Rituximab treatment since we
have found that human CD25-positive B cells are
extremely potent antigen presenting cells (M.
Brisslert, manuscript).
3) Moreover, we would like to investigate the in
vivo and in vitro effect of anti-CD20 treatment
regarding antigen presentation properties of B
cells since this property might be of importance
for disease progression. This will be performed by
collecting bone marrow, or peripheral B cells,
treating
them
in
vitro
with
Rituximab
and
thereafter performing a mixed lymphocyte reaction
where
we
will
evaluate
CD4-positive
T-cell
proliferation as a readout system. The ability to
proliferate following stimulation with B cell
mitogens will also be investigated.
4) In addition, the ability of B cells, from
peripheral blood and bone marrow, to survive and
differentiate after Rituximab treatment will be
assed by analysing surface marker expression (i.e.
IL-7Ra, CD43, CD19, and Ig light chain, as well as
expression of Bcl-6, Blimp-1, apoptosis proteins
such as Bcl-2, Smac, survivin and cytochrome C).
5) Finally, we aim to investigate the in vivo
effect of anti-CD20 infusion regarding the numbers,
and maturation level of B cells in the bone marrow
of RA patients by analysing bone marrow aspirate
before
Rituximab
infusion,
followed
by
reaspiration after 1 month, and 6 months after the
treatment in the same manner as described above.
Conclusion
The outcome of the proposed investigations will,
apart from better understanding of the physiology
of B-cell compartment in RA patients, provide
insights as to the potential need of vaccinations
and when to apply them with respect to the course
of anti-CD20 therapy.
Indeed, recently published data [11] show that in a
mouse model of RA, treatment with B cell depleting
therapy does not affect immunisation responses.
REFERENCES
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Lam, K.-P. and K. Rajewsky, B-cell
development, in Inflammation: Basic principles and
clinical correlates, J.I. Gallin, I.M. Goldstein,
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New York. p. 151-166.
2.
Bernet, F., The clonal selection theory
of aquired immunity. 1959, London: Cambridge
University Press.
3.
Parham, P., Disruption of Healthy
Tissue by the Immune Response, in The Immune
System. 2004, Garland Pub.
4.
Silverman, G.J. and D.A. Carson, Roles
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5.
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Immunol,
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adversely affect humoral responses in a respiratory
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