Modulation of PI3K Signaling to Improve CAR T Cell Function

During CD8+ T cell activation, engagement of the T cell receptor (TCR) along with costimulatory receptors triggers signaling pathways that lead to T cell expansion and differentiation. Among these, activation of phosphoinositide 3-kinase (PI3K) has a critical effect on T cell proliferation, survival, migration, and effector/ memory subset formation. Class I PI3Ks are composed of one of three isoforms of the p110 catalytic subunit (p110α, p110β, or p110δ), that constitutively associate with a p85 regulatory subunit. Class I PI3Ks catalyze the phosphorylation of phosphatidylinositol 4,5-bisphosphate, generating phosphatidylinositol (3,4,5)-trisphosphate, which recruits proteins containing pleckstrin homology (PH) domains to the plasma membrane. PH domaincontaining targets, including AKT, initiate signaling and activate downstream effectors of cellular differentiation and metabolism [1]. During T cell activation, signaling through the T cell receptor, costimulatory molecules, and IL-2 receptor can all activate PI3Kδ. TCR ligation induces zeta-chain associated ZAP70-mediated phosphorylation of LAT, which is required for the recruitment of PI3K to the membrane [2]. The costimulatory molecules CD28 and ICOS contain the consensus YxxM PI3K binding motif in their cytoplasmic tails. The mechanism of PI3Kδ activation through IL-2 signaling may involve LCK/FYN activity and control of the accumulation of phosphatidylinositol (3,4,5)-trisphosphate [3]. PI3Kδ signaling after T cell activation leads to AKT-dependent inactivation and nuclear exclusion of FOXO1, which has been implicated in the downregulation of memory T cell markers such as IL-7Rα and CD62L. PI3Kδ also promotes mTOR signaling, leading to increased T cell metabolic activity which facilitates effector T cell differentiation and function [2]. While loss of PI3K activity is detrimental to immune function, constitutive activation of PI3K also impairs immunity because it preferentially promotes formation of short-lived terminally differentiated effector T cells at the expense of long lived memory T cells. Control of T cell activation and differentiation by PI3K is particularly relevant to Chimeric Antigen Receptor (CAR) T cell immunotherapy. CARs retarget genetically modified T lymphocytes through hybrid receptors that incorporate a tumor antigen-specific scFv, one or more costimulatory domains (most commonly 41BB or CD28), and the CD3-zeta domain. CAR-T cells have experienced a surge in interest due to the now proven effectiveness of CD19-specific CAR-T cells in the treatment of precursor B cell malignancies. CAR-modified T cells are not merely retargeted conventional T lymphocytes. The presence of a CAR on a T cell’s surface alters its activation and differentiation, even in the absence of a complementary ligand. Constitutive self-signaling through CAR, related to both the scFv framework and the signaling domains, can lead to aberrant T cell behavior, including altered differentiation and decreased survival. This is significant as the effectiveness of CAR-T cells in patients is directly associated with their in vivo longevity. Long et. al. demonstrated that the presence of the CD28 costimulatory domain increased CAR-T cell exhaustion induced by persistent CAR self-signaling; the 4-1BB costimulatory domain had a lesser effect [4]. Using a panel of mutant CAR, our group identified a dominant role of the CAR CD3-zeta ITAMs in self-signaling. CD3-zeta significantly enhanced the constitutive activation of the PI3K, AKT, mTOR, and glycolysis pathways, and fostered formation of short-lived effector cells over central/stem memory cells [5]. Manipulation of PI3K signaling can be used to prevent altered CAR-T cell differentiation due to constitutive CAR self-signaling and foster longlived memory T cell development. We demonstrated that pharmacologic blockade of PI3K during CAR-T Editorial