Abstract
Purpose :
A key factor in the pathophysiology of dry eye disease (DED) is the interaction between intercellular adhesion molecule 1 (ICAM1) on antigen-presenting cells (APCs), such as dendritic cells (DCs), and lymphocyte function-associated antigen 1 (LFA-1) on T cells, resulting in immunological synapse (IS) formation and T cell activation. Previous in vitro studies have shown that lifitegrast may inhibit LFA-1 to ICAM1 interaction, thus preventing IS formation or disrupting established IS’s between T cells (CD3+ or CD4+) and human DCs, thereby inhibiting T cell activation. The exact mechanism of action of lifitegrast in DED is unclear. The present analysis aimed to visually demonstrate the kinetics of lifitegrast’s effects on APCs to T cell binding using a live cell imaging assay.
Methods :
Two cell lines (Raji [as APC] and Jurkat [as T cells]) were used. Raji cells were infected with a lentivirus encoding human ICAM1 fused to enhanced green fluorescent protein, and Jurkat cells were labeled with Hoechst 33258 (blue).To detect prevention of IS formation, live cells were pretreated with either lifitegrast (10µM) or vehicle control followed by staphylococcal enterotoxin B (SEB). Data were captured as time-lapse movies to show kinetic changes and indicate IS formation and the effect of lifitegrast in real-time. Presence of IS’s in cells was evaluated using fluorescence microscopy. To detect disruption of established IS, live cells were stimulated with SEB and subsequently treated with lifitegrast and IS was assessed in real-time.
Results :
In activated cells, lifitegrast prevented IS formation and disrupted the established IS’s (Fig. 1A-C). In control cells, IS formation was evidenced by the green signal unevenly distributed between cells (white arrow; Fig. 1A). Pretreatment with lifitegrast prevented IS formation as shown by the green signal distributed uniformly around cells (Fig. 1B). Post-treatment with lifitegrast disrupted the established IS as evidenced by the green signal (arrow) that became more uniform with the increasing time of lifitegrast action (Fig. 1C).
Conclusions :
Our findings show kinetic changes, indicating that lifitegrast can prevent IS formation and disrupt established IS between APCs and T cells. A study with primary cells or LFA-1-labeled T cells is ongoing. These findings may further validate the mechanism of action of lifitegrast in DED.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.