Regulation of LFA-1 on T cells by phosphoinositide signalling

Abstract

Lymphocyte Function-Associated Antigen 1 (LFA-1) is the major integrin in T cells and binds Intercellular Adhesion Molecule 1 and 2 (ICAM-1 and ICAM-2) expressed on endothelial cells and antigen presenting cells. LFA-1 affinity for ICAM is increased following chemokine and T cell receptor (TCR) engagement by inside-out signalling. This process coordinates T cell migration, adhesion, and activation of T cells. LFA-1 activation is mediated by phosphoinositide 3-kinase (PI3K) and the downstream phosphoinositide PtdIns(3,4,5)P3 (PIP3). To investigate how PI3K regulates LFA-1, I optimised CRISPR/Cas9-mediated mutagenesis in T cells, and designed a retroviral library of CRISPR/single guide RNAs targeting all known and potential PIP3-binding proteins. Using this library, I screened the targeted genes for effects on ICAM-1-binding by a flow cytometry-based ICAM-1-binding assay, using Cas9-expressing primary mouse T cells. I identified multiple proteins regulating LFA-1-mediated adhesion to ICAM-1, including the RAP1/RAS GTPase-activating protein RASA3. I found that RASA3 is a critical negative regulator of LFA-1 activation and that RASA3 is inhibited by PI3K signalling. T cells without RASA3 have greatly increased ICAM-1-binding. Mice with conditional deletion of Rasa3 in T cells have altered T cell homeostasis including increased numbers of mature thymocytes in the thymus, but decreased T cells in lymph nodes, spleen, and especially in the blood. Further, Rasa3 deletion in T cells resulted in reduced germinal centre and antigen-specific antibody responses to immunisation, likely as a result of decreased T follicular helper (TFH) cell numbers. The presented results thus describe a novel genetic screen in T cells which has uncovered a critical role for RASA3 as a PI3K-regulated inhibitor of T cell adhesion and migration that is required for T cell homeostasis and function.