Generation of a non-cleavable allele of Pik3ip1/TrIP

Phosphoinositide 3-Kinases (PI3Ks) are key regulators of cellular activation, metabolism, and survival, including in lymphocytes like T cells. In recent years, we and others have studied a novel negative regulator of PI3K, which is particularly highly expressed in immune cells, and which we refer to as Transmembrane Inhibitor of PI3K or “TrIP” (gene: PIK3IP1- phosphoinositide-3-kinase interacting protein 1). TrIP contains an extracellular kringle domain and an intracellular p85-like domain. The p85-like domain of TrIP can associate with p110 family catalytic subunits of PI3K, resulting in modulation of the allosteric activation of PI3K by p85α/β adaptor proteins. Conditional KO of TrIP from mouse T cells resulted in enhanced T cell activation and clearance of Listeria infection, as well as enhanced anti-tumor responses. Although there is substantial evidence documenting the negative regulatory role of TrIP on T cell activation, there remain challenges to dissecting the downstream effects of TrIP on the molecular pathways that regulate T cell function. Chief among these is the proteolytically driven down-regulation of TrIP from the surface of activated T cells, i.e. within several hours of stimulation through the TCR. As a result, studies examining the effects of TrIP on TCR signaling, metabolism and gene regulation have thus far been limited by the “moving target” of TrIP expression that changes during the course of activation in WT cells. This makes comparisons to TrIP KO cells more complicated to interpret. We and others recently demonstrated that TrIP downregulation requires TCR signaling-induced cleavage by ADAM10/17 proteases. We have also determined that this cleavage occurs in the “stalk” region of TrIP, which lies between the N-terminal kringle and transmembrane domains, since deletion of part of this domain ablates activation-induced TrIP cleavage. Importantly, this protease-resistant TrIP construct still retains its inhibitory activity, indicating that its function is still intact. With this project, we propose to generate novel genetically modified mice that can inducibly (i.e. in the presence of Cre recombinase) express a mutated form of TrIP that cannot be proteolytically cleaved. We will also perform preliminary analysis of these animals to validate proper expression of the modified TrIP allele and effects on T cell development and activation in vitro. Project Number: 1R03AI196379-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Lawrence Kane | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $135,586 | Activity Code: R03 | Study Section: Adaptive Immunity Study Section[AI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R03AI19637901