Differential Expression of Adhesion Molecules and Chemokine Receptors Confers Increased Migrative Capacity to ZAP-70-Positive Subclones in CLL Primary Cells,
Authors: Crespo Marta , Carpio Cecilia del Carmen , Calpe Eva , Abrisqueta Pau , Palacio Carlos , Purroy Noelia , Bosch Francesc 
Published: October 2019

Abstract
Abstract 3889
Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disease characterized by the accumulation and proliferation of mature B-lymphocytes. CLL clinical course is extremely heterogeneous; patients with worse prognosis can be identified by the presence of high ZAP-70 expression. Increasing evidence indicates that the microenvironment plays a critical role providing survival and proliferative signals to CLL cells. In this sense, ZAP-70 protein expression has been related to increased capability of the cells to respond to several survival and migration signals provided by the cellular microenvironment through chemokines and cell-to-cell direct contact.
We aimed to analyze the expression levels of several adhesion molecules and chemokine receptors potentially involved in CLL pathogenesis and progression in subclones of CLL cells with high or low ZAP-70 expression within the same patient. For this we obtained peripheral blood mononuclear cells from 40 patients diagnosed with CLL at our institution after informed consent. We then performed a flow cytometry analysis with 7 parameters that allowed for the measurement of the expression of different molecules in CD19+/CD5+ CLL cells with high or low ZAP-70 expression. The expression level of ZAP-70 protein in CD3+ T lymphocytes was used to set the threshold between CLL cells with low or high ZAP-70 expression (Figure 1). Using this approach we analyzed the differential expression of CCR7, CXCR4, CXCR5, CD44, CD49d and CD62L. Interestingly, we found that the expression levels of all the adhesion molecules and cytokine receptors analyzed were significantly higher in those CLL subclones with high ZAP-70 expression compared to CLL cells with low ZAP-70 expression within the same patient (Table 1), suggesting that the relationship with the microenvironment is not uniform across the CLL clone, but those CLL cells with higher expression of ZAP-70 have increased potential to receive signals from other cells. In order to analyze if this could translate into an increased capacity of the CLL cells with high ZAP-70 expression to migrate towards chemokines, we performed an in vitro transmigration assay across bare polycarbonate filters using primary CLL cells from 7 of the patients. We allowed the cells to migrate for 6 hours towards a media containing 1 μg/ml of CCL21 (the ligand of CCR7) and proceed to measure the percentage of CD19+/CD5+ CLL cells with ZAP-70 expression among those cells that had transmigrated and to compare it with the percentage of ZAP-70-positive cells within the ones that did not migrate. Of note, for all the cases analyzed, we observed that the percentage of ZAP-70-positive cells was significantly higher in the cells that had migrated compared to the cells present in the upper chamber (p=0.018) indicating that ZAP-70-positive CLL cells have an enhanced ability to respond to and to migrate towards CCL21. In conclusion, the differential expression of adhesion molecules and chemokine receptors in primary CLL cells with higher expression of ZAP-70 can influence their relationship with the microenvironment and confers them a higher migratory potential towards CCL21.Figure 1:Figure 1:. Table 1:MFI: mean fluorescence intensity SEM: standard error of the meanMean MFI ± SEM Low ZAP-70 CLL cellsMean MFI ± SEM High ZAP-70 CLL cellsPaired samples t-test p valueCCR7163.56 ± 11.3178.20 ± 13.40.007CXCR4263.01 ± 36308.95 ± 45.30.004CXCR5604.02 ± 62.6652.89 ± 67.50.001CD441081.11 ± 76.91210.8 ± 82.5>0.001CD49d58.43 ± 16.169.6 ± 17.6>0.001CD62L33.81 ± 8.947.63 ± 12.1>0.001

Disclosures:
No relevant conflicts of interest to declare.