Various Stages of Immune Synapse Formation Are Differently Dependent on the Strength of the TCR Stimulus.

Hsin-Fang Chang,Michael Estl,Pascal Blatt,Varsha Pattu,Xuemei Li,Ute Becherer,Jens Rettig

doi:10.3390/ijms21072475

Hsin-Fang Chang, Michael Estl + Show 5 more

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https://doi.org/10.3390/ijms21072475

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Abstract

Cytotoxic T lymphocytes (CTL) are key players of the adaptive immune system that target tumors and infected cells. A central step to that is the formation of a cell–cell contact zone between the CTL and its target called an immune synapse (IS). Here, we investigate the influence of the initial T cell receptor (TCR) trigger of a cytolytic IS on the distinct steps leading to cytotoxic granule (CG) exocytosis. We stimulated primary CTLs from mouse using lipid bilayers with varying anti-CD3 but constant ICAM concentrations. We fluorescently labeled molecular markers of distinct IS zones such as actin, CD3, granzyme B, and Synaptobrevin2 in CTLs and imaged cytolytic IS formation by total internal reflection fluorescence microscopy (TIRFM). We found that an intermediate anti-CD3 concentration of 10 µg/mL induces the fastest adhesion of CTLs to the bilayers and results in maximal CG fusion efficiency. The latency of actin ring formation, dwell time, and maximum surface area at the IS exhibit different dependencies on the stimulatory anti-CD3 concentrations. The number and surface area of CD3 clusters at the IS seem to show a different dependency to the TCR trigger when compared to their dwell time. Finally, the mode of full CG exocytosis appears to be independent of the TCR trigger.

Highlights

Cytotoxic T lymphocytes (CTLs) are part of the adaptive immune system and function to kill infected and tumorigenic cells
Since exocytosis of cytotoxic granule (CG) is one of the most important consequences of immune synapse (IS) formation and effector function of CTLs, we investigated the dependency of this process on the strength of the T cell receptor (TCR) stimulus
We isolated CTLs from a recently generated Granzyme B-mTFP (GzmB-mTFP) knock-in mouse to endogenously label CGs and allowed them to adhere to such bilayers to form an IS

Summary

Introduction

Cytotoxic T lymphocytes (CTLs) are part of the adaptive immune system and function to kill infected and tumorigenic cells. Similar to helper T cells, they first need to be activated via the T cell receptor which forms a complex with antigenic major histocompatibility complexes (MHC) on the target cells. This triggers a signaling cascade inside the T cell leading to the formation of an immune synapse (IS) [1]. IP3 binding to IP3 receptors on the membrane of the endoplasmic reticulum initiates Ca2+ depletion from the ER lumen which triggers Ca2+ influx from the extracellular space This store operated calcium entry (SOCE) is essential for CTL effector function [3]

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