A lentiviral screen for novel regulators of IL-7R[alpha] in a pre-B cell line

Abstract

IL-7R[alpha] is one component of the heterodimeric IL-7R[alpha] receptor, and signaling through this receptor is essential for murine T and B cell development as well as human T cell development. IL-7R[alpha] signaling is also responsible for homeostatic proliferation of T cells in lymphopenic hosts, as well as maintenance of naïve and memory CD8+ T cells in the periphery. A number of regulators of IL-7R[alpha][alpha] have been identified, but the complex processes underlying fine control of IL-7R[alpha][alpha] expression are poorly understood. The RNAi Consortium's lentivirus-based shRNA library targeting murine kinases and phosphatases has allowed large scale screening for modulators of IL-7R[alpha][alpha] surface expression. This library provides shRNAs in the pLKO. 1 lentiviral vector targeting 1278 known kinases and phosphatases. Analysis of the FACS-based assay identified 38 potential regulators of IL-7R[alpha][alpha] in a pre-B cell line. Subsequent validation of five of the hits confirmed known pathways of IL-7R regulation and also pointed to new potential points of regulatory control. Phosphoinositide 3-kinase (PI3K) regulates a number of cell signaling pathways that promote survival, proliferation and increased metabolism. In mammals the Class I family of PI3Ks consists of four different catalytic subunits, which can pair with any of six different regulatory subunits. PI3K was recently shown to regulate the expression of the interleukin-7 receptor alpha chain (IL-7R[alpha][alpha]) via the Foxol transcription factor.

(cont.) Because IL-7R signaling is vital for murine B and T cell development, cells use a variety of mechanisms to maintain tight control of the expression of IL-7R[alpha][alpha]. Here we utilize sequential knockdown of the four Class I PI3K catalytic subunits to demonstrate that each plays a distinct role in separate pathways leading to the activation of Akt and the expression of total Foxol protein.