The Fourth Intracellular Src Homology 3 Binding Motif of CD2 is Required for Durable T Cell Activity

Abstract

Recent research from both the bench and clinic have demonstrated the importance of the CD2/CD58 axis in the effectiveness of T cells in diseases where chronic antigen exposure is common. Despite this understanding, the specific mechanisms by which CD2 exerts its effects is uncertain. This study was undertaken to determine the role of specific intracellular polyproline rich motifs of CD2 in T cell signaling and activation after primary challenge as well as activity after multiple rounds of antigen stimulation. Like other costimulatory receptors, CD2 exists in two distinct locations in the Immune Synapse, a structure that is formed in the area of contact between a T cell and an APC. CD2 can be found in the central portion, the cSMAC, where it is believed to be most heavily involved in signal transduction, as well as a peripheral zone referred to as the corolla, where it binds to the cytoskeleton. CD2 contains 4 intracellular polyproline motifs that act as ligands for SH3 domain containing proteins. To study the function of the polyproline motifs, CD2 constructs were designed to disrupt three of these motifs, the first, second and fourth, which have known binding partners involved in signal transduction and interaction with the cytoskeleton. Mutations to motifs 1 and 2 resulted in significant decreases in the ability to bind the Src family kinase Lck, reduced signal transduction downstream of the TCR as well as reducing the activation of the transcription factor NFAT. Mutations to motif 4 had little to no effect in any of these assays. However, the main goal of this study was to determine mechanisms of resistance to exhaustion, so a repeat challenge assay was used to evaluate the effects of the mutants in a model of chronic antigen exposure. In this assay, motif 4 was critical for maintaining the ability to kill target cells, motifs 1 and 2 were largely dispensable. This suggests that changes to the T cell during a model of chronic antigen exposure increase reliance on motif 4, not motif 1 or 2, which are more heavily involved in signal transduction after initial exposure to antigen. The exact mechanism of this dependency still needs to be determined.