Alternatively Spliced Variants of Murine CD247 Influence T Cell Development and Activation, Revealing the Importance of the CD3ζ C-Terminal Region

Abstract CD247, also known as CD3ζ, is a crucial signaling molecule that transduces signals delivered by TCR through its three ITAMs. CD3ζ is required for successful thymocyte development. Three additional alternatively spliced variants of murine CD247 have been described, that is, CD3ι, CD3θ, and CD3η, that differ from CD3ζ in the C terminus such that the third ITAM is lost. Previous studies demonstrated defects in T cell development in mice expressing CD3η, but the TCR signaling pathways affected by CD3η and the impacts of the CD3ι and CD3θ on T cell development were not explored. In this study, we used a retrovirus-mediated gene transfer technique to express these three isoforms individually and examined the roles of them on T cell development and activation. Rag1−/− mice reconstituted with CD3θ-expressing bone marrow failed to develop mature T cells. CD3ι-expressing T cells exhibited similar development and activation as cells expressing CD3ζ. In contrast, thymic development was severely impaired in CD3η-reconstituted mice. Single-positive but not double-positive CD3η-expressing thymocytes had reduced TCR expression, and CD5 expression was decreased at the double-positive stage, suggesting a defect in positive selection. Peripheral CD3η-expressing T cells had expanded CD44hi populations and upregulation of exhaustion markers seen by flow cytometry and RNA sequencing analysis. Analysis of early signaling events demonstrated significantly reduced activation of both the PLCγ1 and Akt/mTOR signaling pathways. There was also a reduction in the frequency of activation of CD3η-expressing T cells. These studies reveal the importance of the CD3ζ C-terminal region in T cell development and activation.