CD20 is dispensable for B-cell receptor signaling but is required for proper actin polymerization, adhesion and migration of malignant B cells.

Veronika Kozlova,Pavlina Janovska,Vitezslav Bryja,Adriana Ladungova,Aneta Ledererova,Sarka Pospisilova,Jiri Mayer,Michal Smida,Michael Doubek,Aleksander Slusarczyk,Viera Vakulova,Jan Oppelt,Joanna Domagala,Pavel Kopcil,Helena Peschelova,Yulia Komarova

doi:10.1371/journal.pone.0229170

Abstract

Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20. On the contrary, we found a strong defect in actin cytoskeleton polymerization and, consequently, defective cell adhesion and migration in response to homeostatic chemokines SDF1α, CCL19 and CCL21. Mechanistically, we could identify a reduction in chemokine-triggered PYK2 activation, a calcium-activated signaling protein involved in activation of MAP kinases and cytoskeleton regulation. These cellular defects in consequence result in a severely disturbed homing of B cells in vivo.

Highlights

Protein CD20 on the surface of B cells is used in the clinic as a key target of therapy with monoclonal antibodies to treat B-cell malignancies [1]
Burkitt lymphoma cell lines Ramos and Raji were obtained from ATCC, diffuse large B-cell lymphoma cell line Oci-Ly1 and chronic lymphocytic leukemia cell line MEC1 were obtained from DSMZ
To functionally evaluate the loss of CD20, we assessed the response of CD20-knockout cells to the therapeutic CD20 antibodies Rituximab (RTX) or Obinutuzumab (OBI)

Summary

Introduction

Protein CD20 on the surface of B cells is used in the clinic as a key target of therapy with monoclonal antibodies to treat B-cell malignancies [1]. We investigated the effect of CD20 loss upon the activation of individual BCR signaling proteins in both Ramos and MEC1 cell lines in response to the triggering of IgM-BCR as the major type of B-cell receptor (Fig 1D and S3 and S4 Figs).

Results

Conclusion