Abstract
Although quantitative insights into the killing behaviour of Cytotoxic T Lymphocytes (CTLs) are necessary for the rational design of immune-based therapies, CTL killing function remains insufficiently characterised. One established model of CTL killing treats CTL cytotoxicity as a Poisson process, based on the assumption that CTLs serially kill antigen-presenting target cells via delivery of lethal hits, each lethal hit corresponding to a single injection of cytotoxic proteins into the target cell cytoplasm. Contradicting this model, a recent in vitro study of individual CTLs killing targets over a 12-hour period found significantly greater heterogeneity in CTL killing performance than predicted by Poisson-based killing. The observed killing process was dynamic and varied between CTLs, with the best performing CTLs exhibiting a marked increase in killing during the final hours of the experiments, along with a "burst killing" kinetic. Despite a search for potential differences between CTLs, no mechanistic explanation for the heterogeneous killing kinetics was found. Here we have used stochastic simulations to assess whether target cells might require multiple hits from CTLs before undergoing apoptosis, in order to verify whether multiple-hitting could explain the late onset, burst killing dynamics observed in vitro. We found that multiple-hitting from CTLs was entirely consistent with the observed killing kinetics. Moreover, the number of available targets and the spatiotemporal kinetics of CTL:target interactions influenced the realised CTL killing rate. We subsequently used realistic, spatial simulations to assess methods for estimating the hitting rate and the number of hits required for target death, to be applied to microscopy data of individual CTLs killing targets. We found that measuring the cumulative duration of individual contacts that targets have with CTLs would substantially improve accuracy when estimating the killing kinetics of CTLs.
Highlights
Cytotoxic T Lymphocytes (CTLs) are key effectors in the adaptive immune response, CTL function—or lack thereof—is relevant in many pathologies
We show that extraction of killing parameters is substantially improved if the cumulative contact times of CTLs with both killed and surviving target cells are monitored over time, and we offer an approach to fit such data in the future
Estimates of CTL killing based on in vitro and in vivo CTL killing assays vary, with some variation explained by e.g. different susceptibility of target cells to CTL killing or the type of antigen expressed by the targets [1,2]
Summary
Cytotoxic T Lymphocytes (CTLs) are key effectors in the adaptive immune response, CTL function—or lack thereof—is relevant in many pathologies. Long-lasting (median: 80s) calcium fluxes linked with CTL killing of virally infected cells occurred, on average, 480s (median) after CTLs established contact with virally infected targets[7]. Such killing times of around 10 minutes are consistent with the duration of killing events that can be observed in various supplemental videos elsewhere [8,9]. Given this lower bound it is difficult to see how solely granule-mediated killing could plausibly lead to killing rates in excess of ~6 hour-1, even in optimal situations where CTLs are not limited in their supply of targets and do not require time to search for new targets between killing events
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