investigating-murine-cd4-t-cell-differentiation-using-crispr-cas9

Investigating Murine CD4 T Cell Differentiation Using CRISPR-Cas9 Ribonucleoprotein Complex-mediated Gene Ablation

The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells ...

PI3K signalling is required for activation, differentiation, and trafficking of T cells. PI3Kδ, the dominant PI3K isoform in T cells, has been extensively characterised using PI3Kδ mutant mouse models and PI3K inhibitors. Furthermore, characterisation of patients with Activated PI3K Delta Syndrome (APDS) and mouse models with hyperactive PI3Kδ have shed light on how increased PI3Kδ activity affects T cell functions. An important function of PI3Kδ is that it acts downstream of TCR stimulation to activate the major T cell integrin, LFA-1, which controls transendothelial migration of T cells as well as their interaction with antigen-presenting cells. PI3Kδ also suppresses the cell surface expression of CD62L and CCR7 which controls the migration of T cells across high endothelial venules in the lymph nodes and S1PR1 which controls lymph node egress. Therefore, PI3Kδ can control both entry and exit of T cells from lymph nodes as well as the recruitment to and retention of T cells within inflamed tissues. This review will focus on the regulation of adhesion receptors by PI3Kδ and how this contributes to T cell trafficking and localisation. These findings are relevant for our understanding of how PI3Kδ inhibitors may affect T cell redistribution and function.

PI3K in T Cell Adhesion and Trafficking.

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). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP-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.

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

The immunologic mechanisms underlying pulmonary type 2 inflammation, including the dynamics of eosinophil recruitment to the lungs, still need to be elucidated.

Eosinophil trafficking in allergen-mediated pulmonary inflammation relies on IL-13-driven CCL-11 and CCL-24 production by tissue fibroblasts and myeloid cells.

Following stimulation, the T cell receptor (TCR) and its coreceptors integrate multiple intracellular signals to initiate T cell proliferation, migration, gene expression, and metabolism. Among these signaling molecules are the small GTPases RAS and RAP1, which induce MAPK pathways and cellular adhesion to activate downstream effector functions. Although many studies have helped to elucidate the signaling intermediates that mediate T cell activation, the molecules and pathways that keep naive T cells in check are less understood. Several recent studies provide evidence that RASA2 and RASA3, which are GAP1-family GTPase-activating proteins (GAPs) that inactivate RAS and RAP1, respectively, are crucial molecules that limit T cell activation and adhesion. In this review we describe recent data on the roles of RASA2 and RASA3 as gatekeepers of T cell activation and migration.

Mind the GAP: RASA2 and RASA3 GTPase-activating proteins as gatekeepers of T cell activation and adhesion.

While inputs regulating CD4 T helper cell (Th) differentiation are well-defined, the integration of downstream signaling with transcriptional and epigenetic programs that define Th-lineage identity remain unresolved. PI3K signaling is a critical regulator of T cell function; activating mutations affecting PI3Kδ result in an immunodeficiency with multiple T cell defects. Using mice expressing activated-PI3Kδ, we found aberrant expression of proinflammatory Th1-signature genes under Th2-inducing conditions, both  and . This dysregulation was driven by a robust PI3Kδ-IL-2-Foxo1 signaling loop, fueling Foxo1-inactivation, loss of Th2-lineage restriction, altered chromatin accessibility and global impairment of CTCF-DNA interactions. Surprisingly, ablation of , a Foxo1-repressed gene, restored normal Th2 differentiation, TCR signaling and CTCF expression. BioID revealed Fas interactions with TCR-signaling components, which were supported by Fas-mediated potentiation of TCR signaling. Our results highlight Fas-FasL signaling as a critical intermediate in phenotypes driven by activated-PI3Kδ, thereby linking two key pathways of immune dysregulation.

Article	Year
PI3K in T Cell Adhesion and Trafficking. Frontiers in immunology\| PubMed ID: 34421914	2021
A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1-mediated adhesion in T cells. Science signaling\| PubMed ID: 35857633	2022
Eosinophil trafficking in allergen-mediated pulmonary inflammation relies on IL-13-driven CCL-11 and CCL-24 production by tissue fibroblasts and myeloid cells. The journal of allergy and clinical immunology. Global\| PubMed ID: 37781651	2023
Mind the GAP: RASA2 and RASA3 GTPase-activating proteins as gatekeepers of T cell activation and adhesion. Trends in immunology\| PubMed ID: 37858490	2023
A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 T helper cell signaling, differentiation and epigenetic remodeling. bioRxiv : the preprint server for biology\| PubMed ID: 39803425	2024

Dominic P. Golec

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