4 Discussion
The effectiveness of rituximab depends on several factors, including its
activity and concentration in the environment of the tumor. To maximize
therapeutic efficacy and to optimize dosing, a reliable assay for
accurate measurement of rituximab levels in the bloodstream of patients
is needed.18 Here, we show that serum rituximab levels
vary highly among patients, who received standard doses of rituximab. In
different studies, higher serum rituximab levels correlated with more
favorable clinical response.19 In addition, initially
measured tumor burden was reported to inversely correlate with serum
rituximab levels.20 We show here that patients with
limited disease (risk groups R1 and R2) have higher serum rituximab
levels compared to patients of advanced disease (risk groups R3 and R4)
at day five after rituximab administration. This correlates with
previous findings of significantly better rituximab response in patients
of risk group R3/R4.21 The advanced disease stage in
R3/R4 patients translates into more available CD20 epitopes, where
rituximab can attach. Consequently, less unbound rituximab molecules can
be measured in the R3/R4 patient’s serum. This rituximab ELISA is a
reliable method to measure serum rituximab levels on a routinely basis
in the future and can easily be implemented in daily routine.
ADCC, mediated by FcγRs of NK cells, is thought to be the main rituximab
mechanism of action.22 FcγRI expression on classical
monocytes was shown to be increased at day five after rituximab
treatment, while FcγRII expression significantly decreased. FcyRII can
consist of two isoforms, FcyRIIa and FcyRIIb.23 The
antibody binding to FcγRII used for these experiments binds against both
isoforms, FcγRIIa and FcγRIIb. However, both isoforms have different
functions in B cell depletion, FcγRIIa being activatory on target
effector cells and FcγRIIb being inhibitory.23 It is
tempting to speculate that monocytes expressing the activatory FcγRIIa
might be exhausted at day five after rituximab treatment. In DLBCL
patients, low FcγRIIa expression levels at diagnosis were described to
lead to impaired rituximab-mediated cytotoxicity of NK
cells.24 Also, expression of inhibitory FcγRIIb might
be reduced on the circulating effector cells after treatment since this
Fc receptor type was described to negatively influence
rituximab-mediated B cell depletion.23
FcγRI is the main receptor that mediates trogocytosis, describing the
process where effector cells remove rituximab-CD20 complexes, which can
lead to rituximab resistance but also to target cell
death.25 The mechanism was described to be less
effective in rituximab mediated B cell depletion but is initiated when
other mechanisms are exhausted.26 This supports the
upregulated expression of FcγRI at day five after treatment with
downregulated FcγRII expression, due to exhaustion.
Although being the main receptor mediating ADCC, expression of FcγRIII
remained unchanged in monocytes and NK cells. This might indicate that
FcγRIII mediated B cell depletion via ADCC or ADP is not exhausted at
day five after rituximab treatment and plays a role in an earlier time
of the treatment. FcγR expression of neutrophils did not change,
probably because neutrophils are not the predominant effector cells at
day 5 after treatment. In neutrophils, only FcγRIIIb is expressed on the
surface, rather mediating phagocytosis than ADCC.27
S100A9 and S100A12 belong to the S100 family and act via Toll-like
receptors (TLRs) and receptors for advanced glycation end products
(RAGEs).28-30 Expression of S100A9 is highest in
monocytes and neutrophils, while S100A12 is mainly expressed by
neutrophils.31,32 High serum levels of S100A8, S100A9
and S100A12 are associated with enhanced proinflammatory activity,
predominantly found in patients with inflammatory or autoimmune
diseases.33,34 Therefore, S100A9 and S100A12 can be
used as valuable serum inflammatory markers.33,34Furthermore, S100A9 could be used as a prognostic factor of survival in
advanced ENKL patients.35 Additionally, a combination
of serum S100A8/A9 and S100A12 was shown to predict rituximab treatment
response in lupus nephritis.36 The expression of the
proinflammatory proteins S100A9 and S100A12 was found to be
significantly increased in monocytes and neutrophils at day five after
rituximab treatment. Conversely, S100A8/A9 and S100A12 serum levels
significantly decreased and therefore secretion by effector cells is
lower at day five after treatment. Serum S100A8/A9 and S100A12 levels
are similar to healthy individuals after rituximab treatment, concluding
they are slightly increased before treatment indicating signs of
inflammation.37,38 Increase in intracellular
expression is accompanied with decrease in secretion of S100A9 and
S100A12 by effector cells and vice versa, probably depicting the
location of the proteins at the measured timepoint.
Among others, S100 proteins may, directly or indirectly, lead to
recruitment and activation of NK cells that are the main effector cells
that deplete B cells. It will be interesting to examine these mechanisms
in future studies to point to new possibilities of activation and
inhibition.
CD57+ cells express more perforin (PRF1) and granzyme B (GrzB), which
are crucial for lytic activity of NK cells and are primed effectors for
ADCC.39 Interestingly, expression of CD57, PRF1 and
GrzB was significantly decreased at day five after rituximab
administration. In other studies, high expression of CD57, PRF1 and GrzB
correlated with highly active NK cells.39,40 Since
GrzB and PRF1 both predominantly contribute to ADCC and we identified a
decrease in expression, it is tempting to speculate that ADCC by NK
cells might be the main mechanism of action in patients suffering from
B-NHL.40 This could indicate that the NK cell
population with high B cell depletion activity might be exhausted at day
five after rapid NK cell recruitment in the first days after rituximab
administration. By measuring the lytic activity of NK cells from patient
samples, this effect could be studied further. However, the predominant
NK cell population after rituximab treatment might be less mature since
expression of GrzB and PRF1 directly correlates with cellular
maturity.41 Additionally, different mechanisms of
action may play a role at various timepoints. In future studies, it
would be feasible to monitor the expression after administration of
rituximab over time to improve rituximab therapy and to identify new
diagnostic markers.