Abstract 18229: HIV-NEF Modulates T-Cell Interaction With Endothelial Cells in a Rac-Dependent Pathway to Enhance Transfer of Nef Protein to Endothelium
Introduction: Anti-Retroviral therapy (ART) has reduced the incidence of AIDS but HIV+ patients face an increased risk of developing vascular pathologies including pulmonary hypertension and cardiovascular diseases. We found Nef protein in PBMCs of HIV patients on ART and that this protein is transferred to vascular endothelium where it subsequently causes reactive oxygen species production and apoptosis. The mechanism through which Nef protein) transfers to endothelial cells (EC) is poorly understood. In this study, we will study how Nef affects T Cell-EC interaction in a Rac-dependent fashion and confirm these findings in PBMCs from HIV+ patients on ART.
Methods: SupT1 cells expressing a Nef-ER fusion protein (AIDS reagent repository) serves as a model of inducible Nef activity. For adhesion assays, Nef-ER cells (stained green with calcein AM) were allowed to adhere to endothelial cell monolayer for 1 hr. Non-adhered cells were washed 1x with PBS and adhered cells quantified using flow cytometry & microscopy. For transmigration assays, Nef-ER SupT1 cells were allowed to transmigrate through an EC monolayer. The number of transmigrated cells were quantified using CyQuant Cell quantification kit. To study Nef-Rac interactions, Nef-ER cell lysate was immuneprecipitated using Rabbit anti-Nef serum (AIDS reagent repository) and then analyzed using western blotting.
Results: Nef expression significantly increased T-Cell adhesion (1.5 fold, p = 0.01) to EC and T-cell migration (1.5 fold, p = 0.0005) through EC monolayer in response to 10% FBS gradient, CXCL12 and CCL21. These effects were neutralized when Nef+ T cells were treated with Rac inhibitor, NSC23766 and Atorvastatin (which pleotropically inhibits Rac activity). Immunoprecipitation of Nef from the cell lysate also pulled down Rac as shown by western blot.
Conclusion: By upregulating adhesion and transmigration of T cells, Nef increases the time Nef+ T cells spend in contact with ECs allowing Nef to transfer into the vascular endothelium. We are currently confirming these assays in PBMCs of HIV patients on ART to characterize T-Cell - EC interaction in an ex vivo model of HIV.
Author Disclosures: S. Chelvanambi: None. B. Meier: None. P. Zorlutuna: None. S. Gupta: None. M. Clauss: None.
- © 2016 by American Heart Association, Inc.