A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1–mediated adhesion in T cells

Abstract

The integrin lymphocyte function–associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP 3 -binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.

The critical role of Rap1-GAPs Rasa3 and Sipa1 in T cells for pulmonary transit and egress from the lymph nodes

“…We examined the impact of the loss of Rasa3 and Sipa1 in T cells in vivo . Compared to WT mice, Rasa3 -KO mice exhibited decreases in both the percentages and numbers of CD3 + T cells in superficial LNs (SLNs) and blood, while T-cell numbers were normal in Sipa1 -KO mice ( Figures S1A, B ), as previously reported ( 26 , 33 ). The absence of Rasa3 and Sipa1 decreased the numbers of CD3 + T cells in SLNs and to a greater extent in the spleen and blood ( Figures 2A, B ; S1A, B ).…”

“…T-cell–specific deletion of Rasa3 did not appear to affect T-cell homeostasis in the thymus or periphery but did impair Th17 production in experimental autoimmune encephalomyelitis model mice ( 32 ). A recent study reported that the lack of Rasa3 in T cells increased T-cell receptor (TCR)–triggered binding to soluble ICAM1 and impaired T-cell entry into and egress from peripheral LNs ( 33 ). However, the precise mechanisms by which Rap-GAPs regulate T-cell trafficking have not been fully elucidated.…”

Butyrate Protects against SARS-CoV-2-Induced Tissue Damage in Golden Hamsters

“…Alveolar capillary endothelial cells not only functioned as gas exchangers, but were also capable of recruiting immune cells through adhesion molecules and activating CD4 + T cells [ 31 ]. By binding to T cell integrin, ICAM1 increased T cell receptor (TCR) signaling to mediate the activation, adhesion, and migration of T cells [ 32 , 33 ]. Patients with COVID-19 showed pulmonary vascular injury associated with intracellular presence of SARS-CoV-2 and endothelial cell destruction [ 34 ].…”

High-throughput CRISPR technology: a novel horizon for solid organ transplantation

“…Schwartzberg et al. conducted a CRISPR knockout screen in CD8 + T cells using a PIP3-binding protein CRISPR library to identify genes influencing the binding ability of primary mouse T cells to ICAM1 ( 122 ). RASA3 was identified as the potential target gene by comparing sgRNA frequencies between scICAM1 neg (not binding ICAM1) and scICAM1 pos (ICAM1-binding) cells.…”