Heterogeneous, delayed-onset killing by multiple-hitting T cells: Stochastic simulations to assess methods for analysis of imaging data

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.

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti‐tumor activity in vivo

“…Consistently, we provide evidence for frequent delivery of sublethal hits in the TME. Our result implies that a high density of CTLs is most likely required for tumor regression, in order to increase the probability of killing (Deguine & Bousso, 2013;Halle et al, 2016;Hamieh et al, 2019;Beck et al, 2020). This has been indeed predicted by modeling approach (Budhu et al, 2010), and consistently, we had previously observed in two distinct models that tumor regression was associated with the progressive accumulation of cytotoxic effectors (Breart et al, 2008;Cazaux et al, 2019).…”

Fatty acid synthase (FASN) signalome: A molecular guide for precision oncology

“…FASN-centered priming of cancer cells could also be considered to enhance the short lifespan and persistence of ACIs [268][269][270]. Contacts between cytotoxic immune cells and tumor cells are generally not binary 'life/death' events, but rather the damage done to cancer cells accumulates with multiple sublethal contacts with cytotoxic lymphocytes to reach a lethal threshold over time [271][272][273]. Importantly, mitochondria appear to be essential amplifiers of the death signal delivered by NK-cells and cytotoxic T-cells [274].…”

Spatially Fractionated GRID radiation potentiates immune-mediated tumor control

“…Following attempted or successful migration by an immune cell 𝑝, it can interact with other cells. Recent studies have shown support for the "multiple hit" hypothesis, in which target cells require multiple contacts from cytotoxic lymphocytes before death (53). Recently, it has been shown that cells can recover from sublethal damage caused by interactions with cytotoxic lymphocytes, with those receiving two or more "hits" displaying accelerated apoptosis induction (54).…”