Development and characterization of a physiologically relevant model of lymphocyte migration in chronic lymphocytic leukemia

Abstract

Key Points• We have developed a novel in vitro system to model how shear force and transient interaction with endothelial cells alter chronic lymphocytic leukemia cell phenotype and behavior.• We have used our model to investigate chronic lymphocytic leukemia cell migration and have determined the critical role for integrin a4b1 in this process.There is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL), but to date, only static in vitro cultures have been used to study these phenomena. To address this lack of data, we have developed a dynamic in vitro model in which CLL cells experience shear forces equivalent to those in capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells. CLL cells treated in this way increased their expression of CD62L and CXCR4 (both P < .0001) and of CD49d and CD5 (both P 5 .003) directly as a result of the shear force. Furthermore, CLL cells migrated through the endothelium into the "extravascular" space (mean migration, 1.37% 6 2.14%; n 5 21). Migrated CLL cells had significantly higher expression of CD49d (P 5 .02), matrix metallopeptidase-9 (P 5 .004), CD38 (P 5 .009), CD80 (P 5 .04), and CD69 (P 5 .04) compared with CLL cells that remained in the circulation. The degree of migration observed strongly correlated with CD49d expression (r 2 , 0.47; P 5 .01), and treatment with the CD49d-blocking antibody natalizumab resulted in significantly decreased migration (P 5 .01). Taken together, our data provide evidence for a novel, dynamic, and tractable in vitro model of lymphocyte migration and confirm that CD49d is a critical regulator of this process in CLL. (Blood. 2014;123(23):3607-3617)

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration

“…In keeping with our previous report, 23 a small percentage of CLL cells migrated into the EVS after 48 hours (1.37% 6 2.32%). Compared with CLL cells remaining in the circulating compartment, migrated cells showed lower expression of CXCR4 (P 5 .0058), as well as increased expression of CD49d (P , .0002), CD38 (P , .0001), HLA-DR (P 5 .0002), CD5 (P 5 .0044), CD80 (P , .0001), CD86 (P 5 .0006), and CD69 (P 5 .0007) ( Figure 3B).…”

“…It has been previously shown that CD49d expression identifies CLL cells that have an increased capacity to undergo TEM 12,23 and interestingly, it is also a co-stimulatory molecule. 26 In order to investigate whether there is a link between migration and costimulation in CLL, we correlated the expression of CD49d with a variety of activation and co-stimulatory molecules in the LN and PB of patients with CLL, and in the circulating and extravascular compartment of the in vitro circulation system.…”

“…We used a physiologically relevant in vitro circulation system of CLL ( Figure 3A) 23 BLOOD, 28 JULY 2016 x VOLUME 128, NUMBER 4…”

“…We have previously demonstrated that blocking CD49d using natalizumab prevented CLL migration in our circulating system. 23 Here, we investigated whether natalizumab could also inhibit the ability of CLL cells to activate alloreactive T cells in an MLR. CLL cells from 6 CD49d hi CLL patients were pre-incubated with natalizumab prior to irradiation and coculture with purified CD3 1 T cells from a healthy donor.…”

“…6,11 Here, we used fine-needle aspiration (FNA) to perform functional assays and determine whether matched CLL cells from LN-CLL and PB-CLL had a distinct compartment-specific phenotype and T-cell activation function. In addition, we used our novel circulation system to study migration 23 in order to compare LN-derived CLL cells with CLL cells that migrated in vitro.…”