Abstract
Purpose :
Interaction between the adhesion molecules ICAM-1 and LFA-1 on dendritic cells and T cells, respectively, is believed to mediate immunological synapse (IS) formation. ICAM-1 is overexpressed in dry eye disease (DED) and is a key factor in the development of ocular surface inflammation. Lifitegrast ophthalmic solution 5% is an LFA-1 antagonist indicated for the treatment of the signs and symptoms of DED. A high content imaging assay was developed to verify that lifitegrast prevents the ligand interaction between ICAM-1 and LFA-1 within the IS.
Methods :
Cultures of human CD4+ T-cells in the presence of either activated (with LPS or IFNgamma) or non-activated normal primary human dendritic cells were plated in 384-well plates and exposed to either lifitegrast, efalizumab (an anti-LFA-1 antibody, control), or compound 4 (lifitegrast analog, positive control) at increasing concentrations. Antibodies against ICAM-1 and LFA-1 were used to detect IS by wide-field fluorescence imaging. Positive IS formation (synapse count) was defined by regions of ICAM-1 and LFA-1 accumulation and proximity to nearest ICAM-1 or LFA-1 positive neighbour (cells not proximal to other positive cells were omitted). Half maximal inhibitory concentration (IC50) values were separately calculated for LFA-1 and ICAM-1 positive cells.
Results :
In activated cells, inhibition of IS formation with lifitegrast (IC50 of LFA-1, 1.781 µM and ICAM-1, 3.842 µM) or the lifitegrast analog (IC50 of LFA-1, 0.043 µM and ICAM-1, 0.136 µM) was more potent than with efalizumab (Figure).
Conclusions :
Lifitegrast displayed potent concentration-dependent prevention of IS formation between ICAM-1 and LFA-1 in a robust high-content imaging assay. This interaction is important for downstream T-cell activation and is a key factor in the pathophysiology of DED. The rapid clinical effect of lifitegrast observed in efficacy trials may be attributable to this dose-dependent inhibition of IS formation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.