Upregulation of CD20 on Human Acute Lymphoblastic Leukemia Cells by IL-4 and CpG Motif Containing Oligonucleotides Increases Susceptibility to Rituximab.
Authors: Nijmeijer Bart , Van Schie Marianke L.J. , Willemze Roelof , Falkenburg J.H. Frederik 
Published: October 2019

Abstract
Monoclonal antibodies are emerging modalities in the treatment of hematologic malignancies. Rituximab (RTX), a chimeric antibody that recognizes CD20, shows therapeutic efficacy in non-Hodgkin lymphoma (NHL). Precursor-B ALL (pB-ALL) may also express CD20. However, little is known on the activity of RTX in this disease. We determined CD20 surface expression levels on primary pB-ALL cells by flow cytometry following a standardized protocol in which cells are stained with RTX and secondary antibody. CD20 on primary ALL cells was generally absent or expressed at lower levels than on primary NHL cells (median mean fluorescence intensity (MFI): 35.1, range 5–423 in 17 pB-ALL samples, and median MFI 1082, range 440–1818 in 6 NHL samples). The ALL cells that expressed the highest levels of CD20 were similarly susceptible to RTX mediated complement dependent cytotoxicity (CDC) as NHL cells (median 17.8% lysis, range 9.5–98% and 41% lysis, range 13–72%, respectively). However, in pB-ALL cells, RTX activity was strongly limited by CD20 expression levels as lysis strictly correlated with CD20 surface expression (P=0.89), and CD20 negative pB-ALL cells were not killed. Despite apparent lack of surface expression, quantitative RT-PCR (qPCR) demonstrated that all samples expressed CD20 mRNA. Transcriptional expression levels correlated with surface CD20 expression (P=0.72). Therefore we explored upregulation of CD20 as a strategy to augment activity of RTX in ALL. We previously established an in vitro culturing system that supports long-term proliferation of ALL cells. Using the LeidenALL cell lines that were generated in this system, we evaluated the effect of various agents on expression of CD20. Ten LeidenALL pB-ALL cell lines were cultured in the presence or absence of IL-2, IL-3, IL-5, IL-7, IL-15, a CpG motif containing oligonucleotide (CpG), TNFa or IFNg. Culture of LeidenALL cells in the presence of IL-4 resulted in upregulation of CD20 in 4 out of 10 samples, 24 hours after incubation. Culture of LeidenALL cells in the presence of CpG resulted in upregulation of CD20 in another 4 out of 10 samples, 48 hours after incubation. None of the other cytokines affected expression of CD20. IL-4 and CpG displayed synergistic action as co-incubation of LeidenALL cells with IL-4 and CpG resulted in further increased expression of CD20 in 9 out of the 10 samples after 48 hours of co-incubation. Mean CD20 expression increased from MFI 406 (range 14–2668) to MFI 1572 (range 70–4394). qPCR revealed that CD20 was upregulated on a transcriptional level in all of the 10 cell lines. Upregulation of CD20 augmented RTX-mediated CDC (from 41% lysis, range 8–95% to 78% lysis, range 3–93%). Three CD20-negative LeidenALL cell lines that initially were not susceptible to RTX-mediated CDC were lysed after upregulation. In an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay, using peripheral blood mononuclear cells from healthy donors, rituximab-mediated ADCC was increased in 7 out of the 10 samples (from 18% lysis, range 4–48% to 25% lysis, range 6–51%). In conclusion, these results demonstrate that RTX may possess activity in pB-ALL. Activity of RTX in ALL can be potentiated by modulation of the leukemic cells. These insights may allow successful application of rituximab in ALL.