Ultrarapid lytic granule release from CTLs activates Ca ²⁺ -dependent synaptic resistance pathways in melanoma cells

Abstract

Human cytotoxic T lymphocytes (CTLs) exhibit ultrarapid lytic granule secretion, but whether melanoma cells mobilize defense mechanisms with commensurate rapidity remains unknown. We used single-cell time-lapse microscopy to offer high spatiotemporal resolution analyses of subcellular events in melanoma cells upon CTL attack. Target cell perforation initiated an intracellular Ca 2+ wave that propagated outward from the synapse within milliseconds and triggered lysosomal mobilization to the synapse, facilitating membrane repair and conferring resistance to CTL induced cytotoxicity. Inhibition of Ca 2+ flux and silencing of synaptotagmin VII limited synaptic lysosomal exposure and enhanced cytotoxicity. Multiplexed immunohistochemistry of patient melanoma nodules combined with automated image analysis showed that melanoma cells facing CD8 + CTLs in the tumor periphery or peritumoral area exhibited significant lysosomal enrichment. Our results identified synaptic Ca 2+ entry as the definitive trigger for lysosomal deployment to the synapse upon CTL attack and highlighted an unpredicted defensive topology of lysosome distribution in melanoma nodules.

Greek Fire, Poison Arrows, and Scorpion Bombs: How Tumor Cells Defend Against the Siege Weapons of Cytotoxic T Lymphocytes

“…While the process of MTOC repolarization and granule convergence towards synapse has been shown to be very rapid, monitoring Ca 2+ entry into target cells at high time resolution as a marker of plasma membrane perforation provided the surprising result that lytic granule secretion can start even earlier than MTOC re-positioning at the LS. Pore formation-dependent Ca 2+ entry into target cells was indeed detected as early as 30-40 seconds after human CTL contact with target cells in many conjugates ( 57 , 58 ), while other studies on human NK cells have shown perforation of the target cell membrane (as detected by propidium iodide penetration) within a similar time frame ( 59 ). These results are intriguing as they imply that the entire process of lethal hit delivery comprising TCR-coupled signal transduction, Ca 2+ -dependent lytic granule secretion, and perforin-mediated pore formation can occur within seconds, making CTL-mediated cytotoxicity an extraordinarily rapid biological phenomenon.…”

“…It is conceivable that microtubules (MT)-initiation sites ( 62 ) might be formed at the IS during the first seconds following productive TCR engagement, leading to microtubule nucleation at the synaptic area and docking of few nearby lytic granules. As will be discussed in the following sections, while ultra-rapid lytic granule secretion confers flexibility and efficacy to the CTL killing behavior, this rapid exocytosis might also be detrimental for killing efficacy under some circumstances ( 57 ).…”

“…In this way, tumor cells mimic some of the strategies adopted by CTLs to protect themselves from their own cytotoxic molecules ( 142 ), though it is worth noting that the role of cathepsins in protecting CTLs from bystander toxicity is not universally accepted ( 143 ). One mechanism for removing perforin pores once they have formed is the ultra-rapid Ca 2+ -dependent synaptic lysosomal/late endosomal (LLE) membrane repair pathway, which is engaged extremely rapidly (within seconds) upon CTL attack ( 44 , 57 , 141 ). Upon perforation, melanoma cell lysosomes are relocated towards the IS, and this exposure of lysosomes on the melanoma cell surface serves to remove the damaged membrane and reduce CTL-mediated cytotoxicity in a SNAP-23-depenedent manner ( 141 ).…”

“…Deacidification of the lysosomal compartment effectively disables this defense mechanism ( 141 ). Importantly, this process of synaptic membrane repair is ultra-rapid and calcium-dependent; high spatio-temporal resolution single-cell imaging has demonstrated that a calcium signal propagates outwards from hotspots in the IS within milliseconds and that calcium chelation drastically increases CTL-mediated cytotoxicity by inhibiting synaptic repair mechanisms ( 57 ). In a similar scenario, actin remodeling has also been shown to mediate breast cancer cell resistance to NK cell-derived cytotoxic molecules ( 144 , 145 ); in these studies, a live F-actin probe was utilized to demonstrate the massive accumulation of actin at the IS in resistant but not susceptible target cells and this synaptic actin accumulation occurred very rapidly (<2min) ( 144 ).…”

T cell-mediated additive cytotoxicity – death by multiple bullets

“…While likely not being directly toxic, perforin pore-dependent Ca 2+ elevation occurs in 50% of all CTL-melanoma cell interactions and in 90% of eventually lethal events [13]. In human melanoma cells, the Ca 2+ influx triggers lysosome accumulation and, likely, membrane insertion at the immune synapse and resealing of the perforin-mediated membrane defects, which increases melanoma resistance to CTL-induced killing [25,26]. Plasma membrane defects are repaired, within minutes, by internalization or shedding of the damaged site, by the endosomal sorting complex required for transport [ESCRT(III)] machinery, Rab 27 and annexin A7 [27].…”