Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes

Hsin-Fang Chang,Maria Harkiolaki,Henning Urlaub,Cosima T Baldari,Claudia Schirra,Štefan Bálint,Keerthana Ravichandran,Reinhard Jahn,Salvatore Valitutti,Ulrike Hahn,Jens Rettig,Michael L Dustin,Ute Becherer,Momchil Ninov,Elmar Krause

doi:10.1038/s41467-022-28596-y

Abstract

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets.

Highlights

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS)
Analyses by super-resolution and total internal reflection fluorescence microscopy demonstrates that both classes of cytotoxic granules (CG) fuse at the IS and that multi core granules (MCG) are the source of supramolecular attack particles (SMAP)
We found that about 60% of granules were MCGs co-labeled with wheat germ agglutinin (WGA) and granzyme B (GzmB), 20% were single core granules (SCG) only containing GzmB, while the rest were small single WGA-positive organelles distributed throughout the cell cytoplasm and at the IS (Fig. 5d)

Summary

Introduction

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). We employ a synaptobrevin2-mRFP knock-in mouse[8] in an unbiased approach to purify mature, fusion-competent CGs from mouse CTLs. Using density gradient centrifugation followed by immuno-isolation and mass spectrometry we find two classes of fusion-competent CGs. Transmission and scanning electron microscopy analysis discerned different morphologies, with one CG class containing a single dense core and uniform diameter (single core granule, SCG) and the other class containing multiple cores and varying diameters (multi core granule, MCG). Analyses by super-resolution and total internal reflection fluorescence microscopy demonstrates that both classes of CGs fuse at the IS and that MCGs are the source of SMAPs. Our results show an additional type of cytotoxic granule nearly 40 years after the discovery of SCG

Methods

Results

Conclusion