Diamond Annual Review 2020/21

34 35 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Biological Cryo-Imaging Group Beamline B24 A ‘protein bomb’used by the immune systemto kill infected and cancerous cells Related publication: Bálint, Müller S., Fischer R., Kessler B. M., Harkiolaki M., Valitutti S. & Dustin M. L. Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells. Science. 368 , 897–901 (2020). DOI: 10.1126/science.aay9207 Publication keywords: Immunology; Immunotherapy; Apoptosis; Secretion; Synapse; 3D imaging; Cryo Soft X-ray Tomography (CSXT); Correlative Light and X-ray Tomography (CLXT) C ytotoxic T cells (CTLs) kill infected and cancerous cells by secreting cytotoxic proteins. An international team of researchers investigated howthese cytotoxic proteins avoid dilution in the vast extracellular space and enter target cells that need to be killed. The question they were seeking to answer was whether these proteins organise into particles. Diamond Light Source's 3D X-ray tomography and super resolution fluoresence microscopy for the life sciences beamline (B24) uses soft (low-energy) X-rays to generate 3D maps of organic material in cells and small particles released from cells. For this research, B24 provided correlative information about ‘protein bombs’ released by cytotoxic T cells and within the cells. Soft X-rays were better than electrons in this case as they allow imaging of entire T cells, although the resolution is lower. The results allowed the team to confirm that the ‘protein bombs’ had a ‘core-shell’structure and identify potential storage sites for the ‘protein bombs’in the T cells. Whereas most biological drugs are single protein molecules, ‘protein bombs’ are ‘supramolecular’, a loose assembly of hundreds or thousands of proteins that deliver the active components to targets in the body. These ‘protein bombs’ have the potential to be used in immunotherapy applications, which help the body’s immune system to fight cancer and other diseases. One of the next steps for the research team is to try to make synthetic ‘protein bombs’. B24will be helpful in characterising these ‘supramolecular’drugs. CytotoxicT lymphocytes (CTL) and Natural Killer (NK) cells are components of the immune system that help cure viral infections and prevent the progression of cancers. CTL and NK cells migrate through the body to the location of target cells, directly embrace these targets and secrete cytotoxic proteins onto their surface. Cytotoxic proteins were identified 30 years ago and it was determined that one of them, perforin (Prf1), forms holes in the surface of the target cells and the others, granzymes (for example, Gzmb), enter the cytoplasmand trigger a chain reaction leading to target cell death. The prevailing hypothesis over the past 15 years is that when CTL and NK cells embrace targets, they create an immunological synapsewithan isolatedextracellularcompartment intowhichcytotoxicproteins can be released while maintaining a high, active concentration 1 . However, single proteins disperse rapidly and the functional impact of Prf1 and Gzmb may be better achieved if they can be transferred to targets in functional quanta without dilution as they traverse the gap between cells. A challenge of studying such entities is they may be consumed rapidly by target cells or destroyed on interactionwith incompatible surfaces, whichmakes their recovery impossible. A breakthrough in this regard was achieved by using supported lipid bilayers (SLB) assurrogatetargetstocaptureputativesupramolecularattackparticles(SMAPs) 2 . SLBareformedby liposomefusionontoaglasssubstrateandenablecoupling ofproteinstotheirsurfacethroughthelipidheadgroups.Tcellscanbeactivatedby SLBs presenting the adhesionmolecule I CAM1 and a ligand for theT cell antigen receptor (TCR), which can be a histocompatibility antigen-peptide complex or anti-TCR antibodies.These SLB induce liveT cells to form immunological synapses characterised by a ring of adhesion molecules surrounding a central cluster of antigen receptors, which is also the location of the secretory domain. The CTL then attempt to kill these mock targets, releasing Prf1 and Gzmb into the centre of the immunological synapse on the SLB. We have previously shown that T cells can then be removed to expose any released particles that are adherent to the SLB surface. This approach has enabled analysis of proteins and lipids from the synaptic output of helper T cells 3 and similar experiments were performed with CTL 2 . Total Internal Reflection Fluorescence Microscopy on CTL attached to SLB showed that T cells release particles containing Gzmb and Prf1 and that these particles accumulated ICAM1 at the point of particle contact with the SLB. Proteomics analysis revealed the presence of thrombospondin-1 (TSP1) in the particles,whichwasverifiedbystainingoftheparticleswithanti-TSP1antibodies and by expression of green fluorescent proteins (GFP) conjugated TSP1, which co-localised with Gzmb and Prf1. We also verified that TSP1 had a role in CTL mediated killing using Crispr/Cas9 targeting. Super-resolution localisation microscopy revealed that the particles had a shell of TSP1 surrounding a core of Gzmb and Prf1 with overall particle size of 120 nm. The properties of these particles were unexpected in that antibodies to Gzmb and Prf1 access the core of the particles without permeabilisation. These data collectively suggested that the shell had static pores or that the shell was sufficiently dynamic to allow entry of antibodies into the core without allowing the Prf1 and Gzmb to escape. The particles were physically characterised by Cryo-Soft X-ray Tomography (CSXT) at beamline B24. We developed methods to activate CTL on electron microscopy grids of the type used for plunge freezing and CSXT. Analysis of released particles on these grids confirmed a core-shell structure with a dense shell having a diameter of 110 nm and a relatively translucent core. The discrepancy in size in comparison to previous localisation microscopy imaging could be explained by the increased diameter contributed by the ~10 nm antibodies used for staining in the localisation microscopy studies, which were not present in the unstained samples analysed by CSXT. Another feature identified by CSXT was that the CTL contain multicore cytoplasmic granules with many translucent voids consistent with densely packed core-shell particles (Fig. 1). Cryo-Structured Illumination Microscopy (CSIM), performed using the newly developed cryoSIM microscope at beamline B24, correlated with CSXT data revealed that the core-shell particles hadsignalsforbothPrf1andGzmb(Fig.2),althoughthisdata isnotyetpublished in a peer reviewed journal and is thus labelled as preliminary. Biochemical analysis of the cytotoxic particles detected a ~60 kDa fragment of TSP1asthemajorformof TSP1inSMAPsalongwithPrf1andGzmb. Thesedata suggest that the cytotoxic particles are built around a network of non-covalent interactions of TSP1 C-terminus with glycoproteins that are readily detected by wheat germagglutinin staining.Thus, we refer to the particles as supramolecular attack particles or SMAPs.The structures can be likened to‘protein bombs’as they are large structures with many copies of Prf1 and Gzmb that may be left by CTL to disrupt entiremicroenvironments when conditions do not favour classical one- to-one synaptic killing 4 . SMAPs are also released fromNK cells 2,5 . CSXT at beamline B24 has great potential for the study of cellular structures relevant to T cell mediated killing. Future work will focus on correlative imaging inside T cells transfected with fluorescently tagged TSP1 and Gzmb. This will determine if the observed multicore granules are the cellular source of SMAPs andmay open up experiments to understand SMAP biogenesis. References: 1. Stinchcombe J. C. et al. The immunological synapse of CTL contains a secretory domain andmembrane bridges. Immunity 15 , 751–761 (2001). DOI: 10.1016/S1074-7613(01)00234-5 2. Bálint et al. Supramolecular attack particles are autonomous killing entities released from cytotoxicT cells. Science (80-. ). 368 , 897–901 (2020). DOI: 10.1126/science.aay9207 3. Saliba D. G. et al. Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand fromhelperT cells. Elife 8 , e47528 (2019). DOI: 10.7554/eLife.47528 4. Immune cells blast infections and cancer with protein‘bombs’. Science Magazine (2020). DOI:10.1126/science.abc6849 5. Ambrose A. R. et al. Synaptic secretion fromhuman natural killer cells is diverse and includes supramolecular attack particles. Proc. Natl. Acad. Sci. U. S. A. 117 , 23717–23720 (2020). DOI: 10.1073/pnas.2010274117 Funding acknowledgement: This work was funded by the European Commission Advanced Grant ‘Immunological synapse derived ectosomes inT cell effector function (SYNECT)’ ERC-2014-AdG_670930 andWellcome Principal Research Fellowship ‘Translation of the Immunological synapse’100262Z/12/Z.This work was carried out with the support of the Diamond Light Source, beamline B24 (proposals BI26084, BI23581 and BI23000). Corresponding author: Prof. Michael Dustin, Kennedy Institute of Rheumatology, University of Oxford, [email protected] Figure 1: Image from a tomogram of a CTL interacting with an EM grid and highlighting a multicore granule (arrow). Captured during a rapid access visit to B24. Figure 2: Preliminary demonstration of Correlative Light and X-ray Tomography (CLXT) for cytotoxic particles released onto an EM grid by a CTL. A focus of Prf1 staining in the cryo-SIM image (A) and a cluster of SMAPs in the CSXT image (B) are well correlated (C). Captured during a standard access session to beamline B24. S. Balint, University of Oxford.