TMEM16F regulates bystander TCR-CD3 membrane binding at the immunological synapse

Abstract

Abstract The signaling cascade induced by T cell receptor (TCR) triggering has been extensively studied, but the steps separating peptide recognition from TCR phosphorylation remain elusive. In resting T cells, the TCR CD3ɛ and ζ chains dynamically associate with the plasma membrane (PM) through electrostatic interactions with anionic lipids such as phosphatidylserine (PS). Upon ligand recognition, the CD3 chains dissociate from the PM, enabling robust signaling initiation. However, the mechanisms regulating PM dissociation remain obscure. Recent studies have highlighted the role of TMEM16F, a PS-specific scramblase, in regulating TCR signaling, but the molecular steps involved in this activity have not been identified. To study the role of TMEM16F in regulating TCR activation, we knocked-down its expression using shRNA targeting. Our results show that TMEM16F knockdown inhibited PS redistribution, which significantly reduced TCR activation. To showcase TMEM16F activity in relation to TCR signaling, we transduced T cells with a mutant form of the scramblase that constitutively redistributes PS. TMEM16F-mutant T cells showed a significant increase in proximal TCR signaling. This was directly linked with an increase in PS redistribution upon TCR engagement, which enabled the dissociation of bystander CD3ɛ chains and their participation in signaling. Our results show that PS redistribution by way of TMEM16F activity is critical for TCR signaling. Our results also establish our ability to modulate PS redistribution by targeting TMEM16F activity in order to regulate T cell activation. These findings may ultimately lead to novel approaches enabling the modulation of TCR and other immune receptor activity in T cell-based immunotherapies.