Cytotoxic T lymphocytes (CTLs) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse. The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAPs). We utilized 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 (SCGs) and multi core granules (MCGs), 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 released soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion released intact SMAPs. We envision 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.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 894 (E.K., U.B. and J.R; SFB1286 to H.U. and R.J.), IRTG 1830 (J.R.), European Commission (ERC-2014-AdG_670930 to S.B. and M.L.D. and ERC -2021-SyG_951329 to J.R., S. V., C.T.B. and M.L.D) and Kennedy Trust for Rheumatology Research (S.B. and M.L.D.) and Wellcome Trust for support of Diamond Light Source Ltd..