Defective LFA-1 Mediated T Cell Motility In Chronic Lymphocytic Leukemia Is Mediated by Defects In the Rho GTPase Signaling Pathway
Authors: Ramsay Alan G. , Evans Rachel , Svensson Lena , Kiaii Shahryar , Hogg Nancy , Gribben John G. 
Published: October 2019

Abstract
Abstract 914
T lymphocytes have an essential role in adaptive immunity and rely on tightly regulated signaling through integrin lymphocyte function-associated antigen (LFA)-1 to migrate into lymph nodes and interact with antigen-presenting cells. Malignant cells modify their immune microenvironment to prevent effective host anti-tumor responses, promote tumor progression, and suppress the therapeutic benefit of immunotherapy treatments. Here we assessed LFA-1-mediated cell migration of highly purified T cells from treatment naïve chronic lymphocytic leukemia (CLL) patients compared to age-matched healthy donor T cells using CXCL12 stimulation and immobilized ICAM-1, the principal integrin ligand. Video microscopy with motility tracking analysis identified that both CD4 and CD8 T cells from CLL patients (n=14) exhibited significantly reduced migration rates (P &lt; .01) compared to healthy donor T cells (5.5 ± 0.3 (SEM) μm/min and 4.4 ± 0.2 μm/min compared to 8.2 ± 0.3 μm/min and 7.5 ± 0.3 μm/min respectively). We further identified that direct CLL cell contact, and not soluble factors alone, induced similar T cell motility dysfunction in previously healthy CD3 T cells. Primary co-culture of healthy donor T cells with CLL cells caused a significant decrease in the speed of migration on ICAM-1 compared to coculture with control healthy B cells (6.2 ± 0.3 μm/min versus 9.5 ± 0.6 μm/min) (n=9) (P &lt; .05). Next we sought to repair this T cell defect in CLL using a clinically relevant agent. We identify that treatment of CLL patient T cells (n=9) with lenalidomide restores rapid LFA-1 mediated migration on ICAM-1. Ex vivo treatment of CLL T cells with lenalidomide (1μM for 24 hours) significantly increased the speed of T cell migration compared to untreated patient T cells (7 ± 0.4 μm/min versus 2.5 ± 0.7 μm/min) (P &lt; .05) and the rescued T cell migratory function of lenalidomide exposed patient T cells was comparable to healthy donor T cells treated with or without drug. Interference reflection microscopy (IRM) examining the contact zone between migrating T cells and ICAM-1 identified a significant CLL patient T cell adhesion defect (P &lt; .05) with reduced spreading area and strength of adhesive contacts (pixel density) compared to healthy donor T cells. IRM was further utilized with pharmacological inhibitors to demonstrate that exposure to lenalidomide rescued CLL T cell adhesion by acting on the Rho family GTPases that are dysregulated in cancer patient T cells. Lenalidomide significantly increased (P &lt; .05) levels of active RhoA in CLL patient T cells compared to untreated cells. In addition, untreated CLL patient T cells adhering to ICAM-1 exhibited significantly reduced expression levels of phosphorylated myosin light chain (MLC) compared to healthy donor T cells (P &lt; .05) and this defect was repaired following lenalidomide treatment. MLC is normally phosphorylated by MLC kinase at the T cell leading edge and by the RhoA target, ROCK at the trailing edge, and is an important downstream signaling molecule during LFA-1-mediated T cell motility. Further expression analysis identified that lenalidomide significantly increased (P &lt; .01) ICAM-1-engaged high-affinity LFA-1 in CLL patient T cells to levels comparable to healthy donor T cells. Overall, our results show that T cells in CLL patients have dysfunctional tumor-induced cytoskeletal signaling via the Rho GTPase signaling pathway, and this is reversed by lenalidomide, rescuing dynamic LFA-1 mediated outside-in signalling and migration. Lenalidomide's immunomodulatory activity was highly cancer T cell specific: rescuing defective LFA-1 migration and signaling in CLL T cells, but with no detectable effects on healthy donor T cells. These findings provide important mechanistic insight into the action of lenalidomide, and highlight the potential clinical utility of immunomodulatory drugs to rescue normal immune function in cancer.

Disclosures:
Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.