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Chronic lymphocytic leukemia

Rituximab primarily targets an intra-clonal BCR signaling proficient CLL subpopulation characterized by high CD20 levels

Leukemia volume 32, pages 2028–2031 (2018)Cite this article

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The use of the anti-CD20 antibody rituximab has improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab was shown to act via various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct apoptosis, or sensitization to chemotherapy (reviewed in ref. [1]). However, the biological function of CD20 and the reasons for the impressive activity of rituximab and other anti-CD20 antibodies remain elusive. It has been suggested, but remains controversial, whether CD20 functions as a calcium channel, couples with CD40 and MHCII, or B-cell receptor (BCR), and whether it has a role in T cell-dependent and -independent immunity [1,2,3,4]. This is also of great clinical interest for the design of combinatorial treatment of rituximab with BCR inhibitors, DNA damaging or immunomodulatory drugs, or CAR T cells. We have previously shown that microenvironmental interactions upregulate the CD20 levels on CLL cells through the CXCR4/SDF-1 axis [5]. Here we describe that higher CD20 expression has a direct role in the BCR signaling in CLL cells, and a BCR-proficient intra-clonal CLL cell subpopulation is more efficiently eliminated by rituximab in vivo due to higher CD20 levels.

Based on the findings above and the fact that CD20 is upregulated in the microenvironment, we hypothesized that higher levels of CD20 might be of direct importance for BCR signaling. Notably, we have observed that CXCR4dimCD5bright CLL cells not only have a 2-fold higher cell-surface CD20 levels, but also have a 2.2-fold higher cell-surface immunoglobulin (sIg) expression than CXCR4brightCD5dim cells from the same patient (N = 48, P < 0.0001; Fig. 2b). This was also coupled with higher responsiveness of CXCR4dimCD5bright(CD20bright) cells to BCR crosslinking with anti-IgM as measured by calcium flux (P = 0.0015, Suppl. Fig. 14). Further, we asked whether CD20 is of direct functional importance for BCR signaling, since CLL cells with high CD20 have also higher sIg levels (see Suppl. Fig. 15). Silencing CD20 in MEC1 cells revealed that CD20 affects the phosphorylation of multiple BCR-associated kinases and proteins (LYN, SYK, GAB1, and ERK) after Ig-ligation (Fig. 2c, Suppl. Fig. 16). Silencing CD20 also impaired LYN kinase phosphorylation, which is the first kinase phosphorylated in response to BCR ligation. This suggests that upregulation of CD20 in the microenvironment is required for proper activity of BCR, and that CD20 is very “intimately” connected with BCR. This is also supported by the impaired BCR-induced calcium flux in MEC1 cells with silenced CD20 (Suppl. Fig. 19), and by a similar phenotype in mice deficient for CD20 in B cells [12]. Our data show that the subpopulation of CLL cells that interact in the immune niches (CXCR4dimCD5brightCD20bright cells) represents the “aggressive” cells with the capacity for BCR signaling [13]. Interestingly, we observed that the effect of CD20 on phosphorylation of BCR-associated kinases is not entirely dependent on its calcium-channel function [2, 12], since the effect of CD20 silencing was present also in calcium-free media (Suppl. Fig. 20).

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Fig. 1
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Acknowledgements

This work was supported by Ministry of Health of the Czech Republic, grant nr. 16–29622A. All rights reserved. We would like to thank Thomas J. Kipps and Laura Z. Rassenti (UCSD) for inspiring discussion and comments to the study.

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  1. These authors contributed equally: Gabriela Pavlasova, Marek Borsky, Veronika Svobodova.

Authors and Affiliations

  1. Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic

    Gabriela Pavlasova, Veronika Svobodova, Jan Oppelt, Katerina Cerna, Vaclav Seda & Marek Mraz

  2. Department of Internal Medicine, Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic

    Gabriela Pavlasova, Marek Borsky, Veronika Svobodova, Katerina Cerna, Jitka Novotna, Vaclav Seda, Yvona Brychtova, Michael Doubek, Sarka Pospisilova, Jiri Mayer & Marek Mraz

  3. Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology and Faculty of Science, Masaryk University, Brno, Czech Republic

    Miloslava Fojtova & Jiri Fajkus

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  1. Gabriela Pavlasova
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Correspondence to Marek Mraz.

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Pavlasova, G., Borsky, M., Svobodova, V. et al. Rituximab primarily targets an intra-clonal BCR signaling proficient CLL subpopulation characterized by high CD20 levels. Leukemia 32, 2028–2031 (2018). https://doi.org/10.1038/s41375-018-0211-0

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