Lenalidomide Promotes a Pro-Inflammatory Switch of Nurse-like Cells Derived from Chronic Lymphocytic Leukemia
Authors: Maffei Rossana , Fiorcari Stefania , Martinelli Silvia , Bulgarelli Jenny , Audrito Valentina , Zucchini Patrizia , Debbia Giulia , Colaci Elisabetta , Potenza Leonardo , Narni Franco , Deaglio Silvia , Luppi Mario , Marasca Roberto 
Published: October 2019

Abstract
Lenalidomide is an immunomodulatory agent clinically active in CLL patients. The specific mechanism of action is still undefined, but includes the modulation of microenvironment. In CLL patients, nurse-like cells (NLCs) differentiate from CD14+ mononuclear cells and nurture/protect CLL cells from apoptosis. NLCs resemble M2 macrophages with potent immunosuppressive functions.
We examined the ability of lenalidomide to mediate a pro-inflammatory switch of NLCs affecting the protective microenvironment generated by CLL into tissues.
NLCs were generated in presence or absence of lenalidomide: cell surface markers, phagocytosis and induction of T cell proliferation were analyzed after 10 days. NLCs activation was measured using a yellow tetrazolium MTT assay after 5 days of culture and NLCs proliferation was measured by CFSE staining. Microarray-based gene expression profiles of NLCs treated or not with lenalidomide were evaluated after 10 days and data were confirmed by real time PCR.
Lenalidomide modifies the immunophenotype and the biological characteristics of NLCs. First, treatment with lenalidomide 0.5µM and 1µM increased the number of NLCs to 268% and 309% compared to untreated control (100%) respectively (p<0.05). To explain the high number of NLCs generated by lenalidomide, we analyzed cell activation and proliferation. We observed a strong increase in NLCs activation after treatment with lenalidomide that correlated with stimulation of NLCs proliferation from 44% to 55% (% of dividing cells) (p<0.05).
In contrast with the high number of NLCs generated in presence of lenalidomide, we found that NLCs lost the ability to nurture and protect CLL cell from apoptosis reducing their viability from 54.2% to 44.5% (p<0.05), but they strongly attracted CLL cells reaching an increase of adhesion to 227% and 212% with the addition of 0.5µM and 1µM lenalidomide (p<0.05). Accordingly with these results, we investigated the ability of lenalidomide to interfere with leukemia-promoting activity of NLCs. Lenalidomide improved the ability of NLCs to engulf zymogen particles to 141% and 155% with dosage of 0.5µM and 1µM compared to control (100%) (p<0.05), further confirmed analyzing the uptake of FITC-dextran by NLCs that increased to 252% and 356% compared to untreated control (p<0.01). Moreover lenalidomide strongly improved the ability of NLCs to induce T cells proliferation from 19.5% to 35.0% (% of dividing cells) (p<0.05). Lastly, gene expression profiling showed a switch to a pro-inflammatory profile in NLCs induced by treatment with lenalidomide involving a modulation of pivotal genes for the immune response, activation/proliferation of T cells, complement activation as well as regulation of cellular movement, cytokine and chemokine activation. In particular, down-regulation of CCL2, CXCL12, IL-10, CD163 and up-regulation of IL-2 were apparent.
Collectively, our data provide new insights into the mechanism of action of lenalidomide that reverts NLCs polarization from M2 to M1-skewed phenotype affecting the supporting and protective microenvironment generated by CLL into tissues.

Disclosures
Maffei: Celgene: support for travel to congresses Other. Marasca:Celgene: Honoraria, Research Funding.