Rituximab Preferentially Eliminates BCR Signaling Proficient Chronic Lymphocytic Leukemia B Cells In Vivo
Authors: Pavlasova Gabriela , Borsky Marek , Musilova Katerina , Seda Vaclav , Cerna Katerina , Osickova Jitka , Vojackova Eva , Brychtova Yvona , Doubek Michael , Pospisilova Sarka , Mayer Jiri , Mraz Marek 
Published: October 2019

Abstract
The use of anti-CD20 antibody rituximab has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab has been shown to act through several mechanisms including antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct induction of apoptosis, and sensitization to chemotherapy. However, the exact contribution of each of these mechanisms to the clinical efficacy of rituximab in vivo and the exact mechanism of its action remain unclear. Importantly, the levels of cell surface CD20 expression were shown to associate with the efficacy of rituximab.
We and others have described that CLL cells that have recently exited the lymph node microenvironment to the peripheral blood express lower cell surface levels of the chemokine receptor CXCR4 and higher levels of the activation marker CD5 (Callisano et al., 2011). We observed that these CLL cells (CXCR4dimCD5bright) have also higher cell surface CD20 expression (~2-fold; P<0.0001) compared to CLL cells from the same patient circulating in the blood stream for a longer time (CXCR4brightCD5dim), and we showed that CD20 is up-regulated by stromal cell interactions through the binding of SDF-1α to CXCR4 (P<0.001). We further hypothesized that higher levels of CD20 on CXCR4dimCD5bright cells will make them the primary target for rituximab in vivo. To investigate this, we obtained peripheral blood samples from CLL patients (N=17) before rituximab administration (day 0) and 24 hours (day 1) after rituximab administration (375 mg/m2, single agent). This showed that rituximab primarily and nearly completely eliminates the CXCR4dimCD5bright subpopulation (8.3 % pre-rituximab vs. 2.1 % post-rituximab, P<0.0001), which harbors the highest levels of CD20 (P<0.005).
It has been previously suggested that the CD20 plays a direct role in microenvironmental interactions and especially in B cell receptor signaling (BCR; Uchida et al., 2004). Therefore, we investigated the BCR signaling propensity of CXCR4dimCD5bright subpopulation. We have observed that CXCR4dimCD5bright CLL cells have higher surface immunoglobulin (Ig) expression than CXCR4brightCD5dim cells from the same patient (~2-fold, P<0.005). This was also coupled with higher responsiveness of CXCR4dimCD5bright cells to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Moreover, CXCR4dimCD5bright cells also had higher levels of CD19 (1.8-fold, P<0.0001), which is an important docking molecule in PI3K signaling, and its higher levels lower the threshold for BCR signaling activation.
Altogether, this study shows that rituximab primarily and effectively eliminates, at least in the short term, the BCR signaling proficient CLL B cells. It is likely that one of the mechanisms of rituximab action is the indirect inhibition of BCR signaling by eliminating CLL cells with strongly activated BCR pathway. These observations might have important implications for combinatorial therapies with BCR signaling inhibitors such as ibrutinib or idelalisib.
This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Republic, grant No. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. This work was supported by the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16), MUNI/A/1028/2015, and G.P. is a city of Ostrava scholarship holder. contact: marek.mraz@email.cz

Disclosures
No relevant conflicts of interest to declare.