The phosphatidinositol-transfer protein Nir3 is a modulator of T cell development

Abstract

Abstract Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) functions as a critical source of second messengers implicated in T cell activation. In T cells, hydrolysis of PIP2 by the lipid phosphatase PLCγ1 generates the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), resulting in PKC and Ras/MAPK activation as well as calcium increases that contribute to T cell responses. A secondary consequence of PLCγ1 activation is a transient reduction in PIP2 levels. The PI transfer proteins Nir2 and Nir3 have been shown to replenish PIP2 levels at the PM through delivery of the precursor PI from the endoplasmic reticulum (ER) via non-vesicular transport. In particular, Nir3 has been shown to replenish PI levels during basal signaling and in response to weak signaling. Given the importance of weak signaling in thymocyte positive selection, as well as the elevated levels of Nir3 mRNA during the double positive (DP) stage of thymocytes development (Immgen), we hypothesize that Nir3 is a regulator of PI homeostasis during thymocyte development. Using CRISPR-Cas9 technology, we have generated a Nir3 knockout mouse. DP thymocytes from Nir3 knockout mice have reduced basal calcium levels, as well as reduced TCR-induced calcium flux. Furthermore, introduction of the OT1 transgenic TCR into Nir3 knockout mice resulted in elevated numbers of DP thymocytes and reduced numbers of CD8SP thymocytes, suggesting a block in positive selection. Analysis of TCR signaling in Nir3 knockout OT1 thymocytes revealed attenuated calcium flux as well as defective induction of pERK, pAkt, and pS6. These findings are the first to characterize the function of PI transfer proteins and their importance specifically in T cell development.