Abstract
Cytotoxic lymphocytes (CTL) have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment – on average 21 hours – which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.
Highlights
The skin is a very tolerant organ
We developed a live-cell assay to study the kinetics of effector cell travel, attachment, and killing
We find that the process of killing of primary keratinocytes presenting physiological levels of non-self antigen is very slow, in contrast to tumor models and cell lines where killing has been reported within minutes to hours [19]
Summary
The skin is a very tolerant organ. It forms a primary barrier against environmental insults and is colonized by a large array of microorganisms against which it does not mount an immune response. Cytotoxic CD8+ T cells are highly dynamic once activated They have been shown to rapidly traffic in tissue in response to cytokine with a primary function to find and kill target cells expressing cognate antigen on their surface. These cells are certainly capable of killing KC targets in vitro [1,2] and have been shown to effect skin graft rejection in vivo [3]. Activated T cells were shown in vivo to traffic to inflamed skin, even in the absence of cognate antigen [4]. We use the term kinematics to to refer to the local motility and dynamic behavior of CD8 T cells as distinct from T cell traffic into and out of tissues
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