The aim of this
study was to investigate the role of microenvironmental
interactions in the regulation of CD20 expression, since
down-modulation of CD20 in the context of immune niches would be a
straightforward explanation for the stroma-mediated rituximab
resistance described by us and others (Mraz
et al., BJH,
2011; Buchner
et al., BJH, 2010; Marquez
et al.,
BJH, 2015). Firstly, we co-cultured primary CLL cells with the bone
marrow stromal cell line HS-5 for 24/48/72 hrs and analyzed CD20
surface expression by flow cytometry. Suprisingly, CD20 levels were
significantly up-regulated (P=0.03) on CLL cells co-cultured with
HS-5 in comparison to CLL cells cultured on plastic. The changes in
CD20 expression levels are likely to play a direct role in
microenvironmental interactions and especially BCR signaling in
immune niches (Uchida
et al., Int Immunol, 2004). To test
this hypothesis we assessed the CD20 expression on CLL cell
populations defined according to CXCR4 and CD5 levels. The
low-level surface expression of chemokine receptor CXCR4 and
high-level expression of activation molecule CD5 can characterize B
cells that have recently exited the lymph nodes (Calissano
et
al., Mol Med, 2011). CXCR4
dimCD5
bright
cells had ~2-fold higher surface CD20 expression when compared to
CXCR4brightCD5dim CLL cells
circulating in the blood of the same patient for a longer time
(P<0.0001, N=21) and CD20 expression clearly decreased with the
transition of CLL cells from CXCR4
dim to
CXCR4
bright together with the decrease of CD5
expression. Further, sorted CXCR4
dimCD5
bright
CLL cells had also ~2 times higher CD20 mRNA expression (P=0.002,
N=8) suggesting that changes in CD20 expression were due to changes
in its mRNA levels rather than a surface modulation. This led us to
hypothesize that CXCR4/SDF-1 is directly implicated in CD20
regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a
ligand for CXCR4 which is produced by HS-5, up-regulated surface
CD20 (P=0.03). We also observed that CLL cells with higher
expression of CD20 had higher levels of surface Ig (P=0.0015, N=12)
and this was coupled with higher responsiveness to BCR crosslinking
with anti-IgM as measured by calcium flux (P=0.005). Therefore, we
investigated the effect of ibrutinib on CD20 levels as ibrutinib is
a BCR-signaling pathway inhibitor and prevents CLL cells from
responding to microenvironmental stimuli, including chemokine
signaling. We observed that treatment of CLL cells with ibrutinib
in vitro resulted in CD20 down-modulation (P<0.001).
The levels of CD20 in CLL cells were also strongly down-modulated
in samples obtained before vs. during ibrutinib treatment of CLL
patients
in vivo (pre- ibrutinib vs. day 15 and week 5/12,
N=8; P=0.02, P=0.01, P=0.07, respectively). These data suggest
that a reduction of the target antigen (CD20) is not the cause for
rituximab resistance in the context of immune niches, and the
sustained activity of ibrutinib and rituximab combinations in
clinical trials (Burger
et al., Lancet Oncol, 2014)
suggests that ibrutinib has other mechanisms that allow for
rituximab efficacy. We focused on the regulation of anti-apoptotic
molecules, namely Mcl1, since that was shown to protect from
rituximab-induced apoptosis and to be rapidly down-modulated by
rituximab infusion
in vivo (Byrd
et al., Blood,
2002; Hussain
et al., Clin Cancer Res, 2007). Indeed, we
observed higher levels of Mcl1 mRNA in the
CXCR4
dimCD5
bright CLL subpopulation (N=8,
P=0.04), and in CLL cells co-cultured with stromal cells.
Importantly, Mcl1 was down-regulated after ibrutinib treatment both
in vitro and in ibrutinib treated patients.
Conclusion: Microenvironmental interactions
up-regulate CD20 expression in CLL B cells through the CXCR4/SDF-1
axis, and the ibrutinib-mediated impairment of microenvironmental
interactions down-regulates CD20 expression. This study reveals a
novel regulation of CD20 levels in the context of immune niches,
which has important implications for CD20-targeting antibodies and
the use of BCR-inhibitors in combination.
Supported by: SoMoPro II-no.4SGA8684 (cofinanced by the European
Union and the South-Moravian Region); NGS-PTL(306242); EHA
Fellowship award; IGA MZ CR NT11218-6/2010; MUNI/A/1180/2014;
CZ.1.05/1.1.00/02.0068; G.P. is a city of Ostrava scholarship
holder. # G.P. and V.S. contributed equally to this
study. contact:marek.mraz@email.cz
