Summary
Cytotoxic T lymphocytes (CTL) eliminate tumor target cells in an antigen and cell-contact dependent manner. Lethal hit delivery occurs as a rapid and binary, “yes/no” process when immunogenicity is very high1–3, however in vivo CTL often fail to kill solid tumor cells during 1:1 conjugations4–6. Using long-term time-lapse microscopy in three distinct tumor cytotoxicity models and statistical modeling, we here show that migrating CTL transit between target cells and initiate apoptosis by a series of sublethal interactions (‘additive cytotoxicity’), while individual conjugations rarely induced apoptosis. Sublethal damage included perforin-dependent membrane pore formation, nuclear lamina rupture and DNA damage, and these events resolved within minutes to hours. In immunogenic B16F10 melanoma tumors in vivo, frequent serial engagements and sublethal hit delivery of CTL was largely confined to interstitial niches in the invasion front, resulting in eradication of invading tumor cells. Thus, additive cytotoxicity is a probabilistic process achieved by a series of CTL-target cell engagements and sublethal events. The need for additive “hits” has implications for the topographic mechanisms of elimination or immune evasion of tumor cells and microenvironmental regulation of CTL accumulation and cooperation by targeted therapy.
Cytotoxic T lymphocytes and NK cells can bind to and attack more than one target cell, termed “serial killing”7–9. Estimations from bulk killing assays and mathematical modeling suggest, that single CTL are capable of eliminating up to 20 target cells of the hematopoietic lineage per day both in vitro and in vivo10,11. This high efficacy of CTL-mediated serial killing, however, has not translated to the killing of solid tumors in mice4,5,12 and, likewise, is rarely observed in patients receiving adoptive transfer of tumor-specific TCR-engineered or CAR T cells13. To understand the discrepancy between CTL effector function against model target cell lines and solid tumors, we quantified the killing kinetics of single CTL in solid tumor models in 3D collagen-matrix based organotypic culture and the mouse dermis in vivo14. In these 3D environments, CTL mediated target cell killing depends on CTL motility and transient tumor interactions, not unlike kinetic priming models for CTL activation15.
Serial conjugation and effector function
In vitro activated OVA-specific OT1 CTLs were confronted with transformed mouse embryonic fibroblasts expressing the OVA peptide (MEC-1/OVA) and the co-stimulatory molecule B7.113. In contrast to tumor cells that evolved in vivo, this engineered model lacks natural immune escape modifications (e.g. down regulation of MHC-I or apoptosis resistance) and, thus, represents an idealized model for maximized CTL efficacy at a single cell level. After 30 h of co-culture, killing efficacy was near 100% at high effector-target (ET) ratios and reached background level below ET ratios of 1:128 (Extended Data Fig. 1a). To assess the serial killing efficacy of individual CTL, we analyzed antigen-specific CTL-target cell interactions and outcome in long-term time-lapse microscopy recordings by tracing individual CTL during interaction with target cells. The duration of individual CTL-target cell interactions was variable (lasting minutes to hours), with lag times between initial CTL binding and target cell death lasting 1.8 ± 1.5 h, and was followed by a subsequent period of ongoing CTL engagement with the dead cell body (‘necrophilic phase’) (Extended Data Fig. 1 b-d). This extended lag phase until apoptosis differs from lag phases obtained for CTL-mediated killing of target cells from leukocyte lineages, lasting <5 to 25 min1–3. In regions of low local CTL density, repetitive contacts resulted in the serial killing of multiple neighboring target cells by a single CTL (Fig. 1a; Movie 1). On the population level, 50% of the CTL acted as serial killers (maximum of 11 killed target cells/24 h), whereas a small CTL subset (15%) repeatedly contacted target cells without inducing apoptosis (Extended Data Fig. 1e). The percentage of CTL with killing capacity correlated with the surface expression of Lamp-1 by 85% of CTL, indicating recognition of the target cells and lytic vesicle exocytosis by the majority of CTL (Fig. 1b). The lag phase to apoptosis was neither compromised nor accelerated over consecutive killing events (Fig. 1c), which resulted in a consistent eradication frequency of 1 kill every 2 hours (Extended Data Fig. 1f). This excludes gain of cytotoxicity by kinetic priming through repetitive antigenic interactions. Thus, OT1 CTL serially eliminate highly immunogenic target cells over 24 h and in a non-exhaustive manner.
CTL induce sublethal damage
To compare effector function against solid tumor cells, which typically retain resistance to CTL mediated killing, OT1 CTL were confronted with mouse melanoma B16F10 cells expressing the OVA peptide (B16F10/OVA) (Extended Data Fig. 2 a-d; Movie 2). As a second model, IL-2 activated human SMCY.A2 CTL14, which recognize an HLA-A2 restricted antigen encoded on the y chromosome, were confronted with male human melanoma cell lines BLM or MV3 (Extended Data Fig. 2 e-i; Movie 3). Compared to the MEC-1/OVA cells, these three melanoma models show delayed, but ultimately effective target cell elimination at the end-point after 24 hours, whereas OVA-negative B16F10 or female MCF-7 cells survived (Fig. 1e). Thus, the endpoints of both murine and human models for probing CTL effector function show comparable target cell elimination in 3D culture. Notably, across all cell types tested, only a minority of individual CTL-target cell contacts induced apoptosis at first encounter, while 60-70% (MEC-1/OVA, BLM) or >90% (B16F10/OVA, MV3) of individual conjugations were followed by target cell survival (Fig. 1e).
CTL degranulation induces transient perforin-mediated pores in the target cell membrane, which facilitates diffusion of extracellular factors into the target cell, including CTL-derived granzyme B16 and extracellular calcium17. OT1 CTL deficient in perforin expression failed to kill B16F10/OVA cells in organotypic culture (Extended Data Fig. 2j), while CTL effector function remained intact after interference with Fas-FasL interaction (Extended Data Fig. 2k). Further, adding perforin-deficient OT1 CTL to a fixed number of wt OT1 CTL did not increase killing efficiency, indicating that perforin-independent mechanisms delivered by excess CTL, including soluble mediators, do not induce or enhance cytotoxicity against B16F10/OVA cells (Extended Data Fig. 2l). Thus, elimination of B16F10/OVA cells by OT1 CTL in 3D culture critically depends on perforin.
To visualize perforin-pore formation and to discriminate sublethal cytotoxic hits from functionally inert interactions, MEC-1/OVA and B16F10/OVA cells were engineered to express the calcium sensor GCaMP6s18, and monitored for transient Ca2+ influx through perforin pores (Fig. 2a, panel 1; Extended Data Fig. 3a; Movie 4). A substantial fraction of antigen-specific but non-lethal CTL-target cell contacts showed CTL associated intracellular Ca2+ events in B16F10/OVA cells (40%; Fig. 2b, panel 2), which were transient (median: 30 s; Fig. 2c) and in 90% followed by target cell survival (Fig. 2d). Ca2+ events originated at CTL - tumor cell contact regions, and differed from unspecific intracellular Ca2+ fluctuations by signal intensity and duration (Extended Data Fig. 3 b,c).
To test whether the variability of perforin release and consecutive Ca2+ events in B16F10/OVA cells were a consequence of heterogeneous TCR engagement, we quantified Ca2+ signals in OT1 CTL upon target cell contact. OT1 CTL showed comparably high rates of Ca2+ signaling when contacting MEC-1/OVA and B16F10/OVA cells (80% to 85%), typically within seconds after contact initiation (Extended Data Fig. 3 d). When co-registered with Ca2+ events in target cells, 40% of Ca2+ positive-CTL contacts with B16F10/OVA coincided with, or were immediately followed by a Ca2+ event in the target cell (Extended Data Fig. 3 e,f). In conclusion, while TCR triggering in OT1 CTL occurs reliably, the induction of perforin-events in the target cell varied.
Structural damage in target cells
To address whether transient transmembrane pores were associated with structural intracellular damage, B16F10/OVA cells were engineered to express NLS-GFP19 or 53BP1trunc-Apple20. NLS-GFP leakage into the cytoplasm was detected in 25% of CTL-target cell contacts and absent when CTL lacked perforin expression (Fig. 2a, panel 2; b, Extended Data Fig. 3 g-j; Movie 5). Recovery, as indicated by diminishing NLS-GFP signal in the cytosol and recovery in the nucleus, occurred in 75% of events within minutes to hours (median: 49 min; Fig. 2 c,d). 53BP1 initiates DNA damage repair complexes, which can be visualized as repair foci by 53BP1trunc-Apple20 (Extended Fig. 3 k; Movie 6). 53BP1 foci were induced in 35% of CTL contacts, in dependence of perforin expression in OT1 CTL (Fig. 2 a,b) and CTL density (Extended Fig. 3 l,m). CTL-induced 53BP1 foci persisted for several hours (median: 4 h) and were resolved in 73% of events (Fig. 2 c,d). These data indicate that CTL contacts induce reversible sublethal damage to the nuclear lamina and DNA.
Death induction by multiple CTL
Across all tested tumor models, sequential or simultaneous contacts by multiple CTL with the same target cell occurred before target cell death (Fig. 3a). In the B16F10 model, >90% of successful kills were preceded by 2-9 CTL encounters by distinct CTL (Extended Data Fig. 4a). When Ca2+ events in target cells were recorded, death induction was preceded by serial Ca2+ events with variable onset and frequency between hits (Fig. 3b; Movie 4, 7). The lag time between contact initiation and first Ca2+ event was 6 min in MEC-1/OVA cells and 16 min in B16F10/OVA cells (Extended Data Fig. 4b). Killing of the MEC-1/OVA cells was preceded by Ca2+ events with 5 min median interval, while Ca2+ events in B16F10/OVA cells occurred with longer intervals of 20 min (Extended Data Fig. 4c). While >50% of contacts induced only one Ca2+ event in B16F10/OVA cells, in 40% of CTL contacts yielded at least 2 repetitive Ca2+ events (Extended Data Fig. 4d). Thus, compared to MEC-1/OVA cells, delayed killing of B16F10/OVA cells correlated with delayed delivery of Ca2+ events (Extended Data Fig. 4e).
Additive cytotoxicity
We then addressed whether apoptosis could have been induced by rare deadly CTL hits (‘stochastic killing’) instead of sublethal contacts that add up over time (‘additive cytotoxicity’). Therefore, we analyzed whether the lethality of the final hit was enhanced by, or independent of, previous Ca2+ events, by plotting the lag time to apoptosis and target cell survival probability in relation to the number of pre-final Ca2+ events. Target cells which received two or more hits prior to the lethal one showed accelerated apoptosis induction, together with a sharp decrease in survival probability (Fig. 3c; Extended Data Fig. 5a,b). The dependence of the lag time to apoptosis on prior CTL hits is expected when additive effects exists, but it is inconsistent with the stochastic killing hypothesis. To explore stochastic killing directly, we performed the same analysis on simulated data, using randomly swapped times between hits and between the last hit and apoptosis. Here, cell death induction was gradual, and neither the lag time to apoptosis nor the survival probability was dependent on the number of prior hits (Fig. 3d).
To address how long sublethal events remain relevant, we estimated the time required to repair the damage caused by a single Ca2+ hit, using a Cox regression model based on additive killing. This resulted in an estimated damage half-life of 56.7 minutes (95% CI: 33.1-112.2) of Ca2+ events to contribute to lethal outcome. This interval was consistent with the recovery kinetics of nuclear lamina defects (Fig. 2c). Using the Bayesian Information Criterion (BIC) to compare model fits, showed that the model which integrates serial damage and decay explained the data better (BIC difference >10) than a model based on the number of Ca2+ events alone.
Multi-hit delivery as a function of CTL density
To test whether apoptosis induction under conditions of high CTL density facilitated additive, multi-hit interactions or, instead, a higher probability of lethal single-hit interactions of few CTL21, we titrated CTL density and monitored individual CTL-target cell interactions by time-lapse microscopy. At high CTL density (ET 1:2) target cell death was frequent and preceded by multiple CTL interactions (Fig. 3e), whereas at low CTL density (ET 1:16) infrequent apoptotic events were near-exclusively preceded by single-contact engagements (Fig. 3e). The CTL density effect was not enhanced by the addition of perforin-deficient CTL (Extended Data Fig. 2l). This indicates that high CTL density (“swarming”) enables efficient apoptosis induction by favoring serial perforin-dependent hits, whereas the poor killing at low CTL density largely relies upon single encounters.
Sublethal hit delivery in vivo
To address whether multiple encounters by CTL mediate anti-tumor cytotoxicity in vivo, activated OT1 CTL were adoptively transferred into C57BL/6 J mice bearing intradermal B16F10 melanoma and monitored longitudinally by intravital microscopy through a skin window (Extended Data Fig. 6 a, b). A single application of OT1 CTL caused a transient, dose-dependent growth delay of the OVA antigen-expressing but not of control tumors (Extended Data Fig. 6c). B16F10 tumors invade the dermis as multi-cellular strands22. Concomitantly, OT1 CTL first accumulated in the tumor periphery and subsequently redistributed towards the invasive tumor front (Fig. 4a). This resulted in local ET ratios of 1:1 along the tumor-stroma interface, whereas ET ratios in the tumor core remained below 1:250 (Extended Data Fig. 6 d,e). To identify where and by which contact mechanism eradication of tumor cells was achieved, CTL contacts and outcome were mapped using histone-2B/mCherry (H2B/mCherry) to detect nuclear fragmentation in B16F10/OVA cells23. Despite comparable ET ratios, high fragmentation rates occurred in the invasion niche but not the tumor rim (Fig. 4b). In either zone, >95% of CTL contacts were transient, short-lived (median duration 15 min) and non-lethal (Fig. 4c). When aggregated, >86% of apoptotic events were preceded by multiple CTL contacts, and a minority (<14%) by individual CTL conjugation (Fig. 4d). In the tumor invasion niche, high local CTL density coincided with confined migration along tissue structures with enhanced speed compared to the main tumor mass (Extended Data Fig. 7a, b; Movie 8). This supported frequent contacts to B16F10/OVA cells with cumulative contact duration reaching >1h and nuclear fragmentation in tumor cells in a time-dependent manner (Fig. 4e, Movie 9).
To discriminate functional from non-functional, irrelevant interactions, we analyzed the occurrence of Ca2+ events using GCaMP6s expressing tumors. Most Ca2+ events (80%) in B16F10/OVA cells were associated with CTL contacts, but rare without interacting CTL (Extended Data Fig. 7 c,d). In invasive tumor subregions with high apoptosis rates, the frequency of CTL contacts inducing Ca2+ events was 5-fold increased, compared to the non-invading tumor rim (Fig. 4 g,h; Movie 10, 11). Thus, in vivo eradication of tumor regions by CTL is a function of local CTL density and frequent sublethal interactions.
Conclusions
Compared with the deterministic and reliable elimination of B cells1–3, CTL effector function against solid tumor cells is inefficient, with a high failure rate, and rarely completed by a single CTL but dependent on a sequence of sublethal hits. Consequently, apoptosis induction is not a binary event but instead implies the probabilistic accumulation of a death signal within the target cell over time, not unlike the accumulation of activation signal in naïve T cells by successive encounters with APCs24–26. The reversible structural damage in the plasma and nuclear membrane and DNA integrity induced by CTL resembles damages induced by chemical and mechanical assault19,27,28 which are also incremental with challenge and reversible by repair. Thus, irrelevant interactions may alternate with variably damaging events and become integrated over time in the target cell until apoptosis is induced or recovery achieved (Extended Data Fig. 9). Additive cytotoxicity may provide a mechanism by which dense CTL infiltrates induce apoptosis in a viral infection model29 or, here, the tumor invasion zone. Alternatively, inefficient sublethal hit delivery may explain failed eradication despite high CTL numbers in solid tumors in mice5, during alloimmune response against transplants30 or, here, subregions of the tumor margin.
CTL density control may, thus, provide a filter limiting accidental cell damage by single, miss-targeted CTL. Additive cytotoxicity may enable cooperation of CTL with differing TCR-specificity or different cytotoxic recognition strategies, such as CTL and NK cells. Lethal hit delivery by serial CTL engagements, as gradual, tunable process, may further respond to microenvironmental and therapeutic immunomodulation, including stabilization of CTL-target cell contacts31,32, modulating local concentration of soluble factors33, activation of immunostimulatory pathways34 and/or blockade of immune checkpoints35. In conclusion, serial conjugation and delivery of sublethal hits define the efficacy of CTL effector function which can be exploited by targeted therapy to increase both single contact efficacy and CTL cooperativity.
Methods summary
Organotypic three-dimensional cytotoxicity assay and time lapse microscopy
Sub-confluent target cell monolayers were overlaid with 3D fibrillar collagen (PureCol, concentration: 1.7 mg/ml) containing in vitro activated OT1 CTL (cell models and activation protocols described in the Methods section). CTL migration, interactions with target cells and apoptosis induction was monitored by time-lapse bright-field microscopy frame intervals of 30 sec for 24 h followed by manual cell tracking and quantitative population analysis.
Monitoring sublethal damage
Target cells were lentivirally transduced to stably express the calcium sensor GCaMP617, NLS-GFP19 or 53BP1trunc-Apple36. OT1 CTL-target cell conjugation and consecutive reporter activity were coregistered by confocal 3D time-lapse microscopy (Leica SP8 SMD) at 488 nm and 561 nm excitation (both 0.05 mW) at frame intervals ranging from seconds to minutes and total observation periods of up to 30 h (GCaMP6s: 10 sec/12 h; NLS-GFP: 2 min/30 h; 53BP1: 5 min/40 h). Intravital microscopy of GCaMP6s activity was performed by simultaneous scanning with 910 nm (eGFP and Alexa750, 15 mW) and 1140 nm (mCherry, dsRed and SHG, 30 mW) with a sampling rate of 1 frame / 10-15 sec over periods of 1-2 h. CTL position and reporter activity were obtained by manual or semi-automated image segmentation and quantification using ImageJ/FIJI.
Intravital multiphoton microscopy
Histone-2B/mCherry expressing B16F10/OVA melanoma cells (1×105) were injected into the deep dermis of C57/B16 J mice (Charles River) carrying a dorsal skin-fold chamber and were repeatedly monitored for up to 15 days28. With the onset of tumor invasion and angiogenesis at day 3 after implantation, in vitro activated eGFP or dsRed OT1 CTLs (0.5-1 ×106) were injected intravenously. Multi-parameter intravital multiphoton microscopy was performed on anesthesized mice (1-3% isoflurane in oxygen) on a temperature-controlled stage (37°C) recording up to 5 channels simultaneously to visualize blood vasculature (70kD dextran/ AlexaFluor750) and peri-tumor stroma (SHG).
Statistical modeling
Survival curves of target cells receiving serial Ca2+ events were computed in GNU R using the ‘survival’ and ‘rms’ packages.
Statistical analysis
Unpaired student’s t-tests or Mann-Whitney U-tests, as appropriate, were applied using GraphPad Prism 8.
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
Author contributions
B.W., P.F. designed the experiments, interpreted the data and wrote the paper. A.d.B. designed and performed experiments, B.W., E.W., quantitatively analyzed the data, K.B. isolated, characterized and cultured the human SMCY.A2 CTL, J.T. and R.d.B performed mathematical analyses, H.D. and C.F. contributed to data interpretation. All authors read and corrected the manuscript.
Author Information
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to peter.friedl{at}radboudumc.nl.
Supplementary videos
Supplementary movie 1. Serial killing of seven MEC-1/OVA target cells by one OT1 CTL (3D collagen assay). Time, hours:min. Field size 80 × 80 μm.
Supplementary movie 2. B16F10/OVA mouse melanoma target cells cocultured with OT1 CTL. Time, hours:min. Field size 340 × 280 μm.
Supplementary movie 3. BLM human melanoma target cells cocultured with SMCY.A2 CTL. Time, hours:min. Field size 280 × 270 μm.
Supplementary movie 4. B16F10/OVA target cell expressing the Ca2+ sensor GCaMP6s (Fire LUT) contacted by an OT1 CTL (green). A single short-lived Ca2+ event followed by target cell survival (part 1). Repetitive Ca2+ events preceding target cell apoptosis (part 2). Time, hours:min:sec. Field size 60 × 55 μm (part 1), 60 × 45 μm (part 2).
Supplementary movie 5. Confocal time sequence of B16F10/OVA target cell expressing the NLS-GFP reporter (green) and H2B-mCherry (red) during contact by OT1 CTL (unlabeled, brightfield channel), causing sequential NLS-GFP leakage events with or without apoptosis induction. Circles indicate leakage events. Time, hours:min. Field size 180 × 180 μm.
Supplementary movie 6. Confocal time sequence of B16F10/OVA target cell expressing the 53BP1trunc-Apple reporter (Fire LUT) attacked by OT1 CTL (unlabeled), causing 53BP1trunc-Apple focalization followed by resolution (part 1) or apoptosis induction (part 2). Arrowheads indicate DNA repair foci. Time, hours:min. Field size 60 × 60 μm (part 1), 80 × 80 μm (part 2).
Supplementary movie 7. Serial engagements of multiple OT1 CTL (green) with B16F10/OVA target cell expressing the Ca2+ sensor GCaMP6s (Fire LUT) followed by target cell apoptosis. Time, hours:min:sec. Field size 140 × 85 μm.
Supplementary movie 8. Dynamic sublethal conjugations of tissue invading B16F10/OVA cells expressing Histone-2b-mCherry (Red) by OT1 CTL (dsRed2, green). Perfused blood vessels (Al750, blue), Collagen fiber (SHG, red). hours:min. Field size 220 × 90 μm.
Supplementary movie 9. Serial engagements of multiple OT1 CTL (dsRed2, yellow) with invading B16F10/OVA target cells (H2B-mCherry nuclei, red) followed by apoptosis of several target cells after multiple CTL contacts. Perfused blood vessels (Al750, blue), Collagen fiber (SHG, cyan). Arrow heads indicate nuclear condensation as first sign of apoptosis. hours:min. Field size 220 × 170 μm.
Supplementary movie 10. B16F10/OVA cells expressing the Ca2+ sensor GCaMP6s (Cyan/ Fire LUT) in the tumor rim, infiltrated by OT1 CTL (dsRed2, green). CTL conjugation cause sublethal hits in few tumor cells. Perfused blood vessels (Al750, blue), Collagen fiber (SHG, red). Circles indicate Ca2+ events. hours:min:sec. Field size 330 × 330 μm.
Supplementary movie 11. Tissue invading B16F10/OVA cells expressing the Ca2+ sensor GCaMP6s (Cyan/ Fire LUT) serially contacted by OT1 CTL (dsRed2, green). Dynamic CTL conjugations cause repetitive Ca2+ events in a high percentage of tumor cells. Perfused blood vessels (Al750, blue), Collagen fiber (SHG, red). Circles indicate Ca2+ events. hours:min:sec. Field size 160 × 120 μm.
Acknowledgements
This work was supported by the Dutch Cancer Foundation (KWF 2008-4031) to C.G.F.. and P.F., NWO-Rubicon (019.162LW.020) to BW, a personal KWF grant to AdB), the FP7 of the European Union (ENCITE HEALTH TH-15-2008-208142), NWO-VICI (918.11.626), the European Research Council (617430-DEEPINSIGHT), and the Cancer Genomics Cancer, The Netherlands to P.F.. Time-lapse confocal microscopy was enabled by an NWO investment grant (834.13.003). We thank Stephen P. Schoenberger for providing the MEC-1/OVA cell line.