Abstract

Trafficking and homing of lymphocytes between the blood, bone marrow, and lymphoid tissues is a multistep process that involves selectins, integrins, and chemokine receptors. We previously demonstrated the importance of CXCR4 chemokine receptors for CLL migration to stromal cells, such as mesenchymal stromal cells and monocyte-derived nurselike cells (NLC). However, the importance of other molecules for CLL cell trafficking is less defined. Expression of CD38 and ZAP-70 by the leukemia cells recently has been described to enhance CLL cell migration in response to the CXCR4 ligand, CXCL12 which may explain the poor prognosis of CLL patients expressing these molecules. In stroma co-cultures, a fraction of input CLL cells spontaneously migrates beneath and underneath the stromal cells, where they are protected form spontaneous and drug-induced apoptosis. This migration phenomenon is termed “pseudoemperipolesis” (PEP) and could explain the evasion of a subpopulation of CLL cells from cytotoxic therapy, leading to minimal residual diseases commonly seen after conventional CLL therapy. To determine which molecules are involved in CLL cell migration beneath stromal cells, we evaluated the expression of migration-related markers on CLL cells in pseudoemperipolesis assays. CLL PBMC were isolated by Ficoll and plated onto marrow stromal cell monolayers in 12-well plates. After 5 hours incubation, supernatant cells were washed off. The stromal layer containing the migrated cells was documented photographically, and then detached with trypsin. Supernatant and migrated CLL cells were stained with saturating concentrations of the following antibodies: anti-CD5, anti-CD38, anti-CD40, anti-CXCR5, anti-CD44, anti-CXCR4, anti-CD62L, anti-CD49d, anti-CD80, anti-CD54, anti-CD86, anti-CXCR3, anti-CCR7, and anti-CD19. Aliquots of supernatant and migrated cells also were subject to cell counting by FACS to determine the relative proportion of migrated CLL cells. Comparing mean fluorescence intensities (MFI) of migrated versus non-migrated cells, we noticed a significant down-regulation of CD62L expression on the migrated cells (10 ± 2 vs. 34 ± 19, MFI mean ± standard deviation, n = 15, P < 0.05), down-regulation of CXCR4 (570 ± 188 vs. 819 ± 400, n = 15, P < 0.05), higher expression of CD49d (116 ± 81 vs. 62 ± 42, n = 15, P < 0.05), and higher expression of CD38 (19 ± 11, vs. 12 ± 7, n = 15, P < 0.05). No significant differences where observed for the expression of the other markers. In conclusions, this study demonstrates that spontaneous migration of CLL cells beneath marrow stromal cells involves selectins, CXCR4, and VLA4 integrins. CD62L is an adhesion molecule that recognizes carbohydrate groups on endothelial cells as well as high endothelial venules of lymphoid tissues. Down-regulation of CD62L after migration indicates that L-selectin is important for this process which also involves down-modulation of CXCR4 in response to CXCL12 secreted by stromal cells. The higher expression of CD49d on migrated CLL cells confirms the role of this integrin in the interaction with VCAM-1 or fibronectin on marrow stromal cells. On the other hand the higher expression of CD38 on migrated CLL cells suggests that CD38 pathway may be involved in this migratory response to stromal cell. In conclusion, our findings suggest that CD62L, CXCR4, CD49d, and CD38 are involved in cognate interactions between CLL cells and stromal cells in the multistep process of CLL migration beneath marrow stromal cells.

Author notes

Disclosure: No relevant conflicts of interest to declare.