Design Parameters for Granzyme-Mediated Cytotoxic Lymphocyte Target-Cell Killing and Specificity

Abstract

Cytotoxic lymphocytes are key elements of the immune system that are primarily responsible for targeting cells infected with intracellular pathogens, or cells that have become malignantly transformed. Target cells are killed mainly via lymphocyte exocytosis of specialized lysosomes containing perforin, a pore-forming protein, and granzymes, which are proteases that induce apoptosis. Due to its central role in lymphocyte biology, as well as its implication in a host of pathologies from cancer to autoimmunity, the granzyme-perforin pathway has been the subject of extensive investigation. Nevertheless, the details of exactly how granzyme and perforin cooperate to induce target-cell death remain controversial. To further investigate this system, we developed a biophysical model of the immunological synapse between a cytotoxic lymphocyte and a target cell using a spatial stochastic simulation algorithm. We used this model to calculate the spatiotemporal evolution of granzyme B and perforin from the time of their exocytosis to granzyme internalization by the target cell. We used a metric of granzyme internalization to delineate which biological processes were critical for successful target-cell lysis. We found that the high aspect ratio of the immunological synapse was insufficient in this regard, and that molecular crowding within the synapse is critical to preserve sufficient concentrations of perforin and granzyme for consistent pore formation and granzyme transfer to target cells. However, even when pore formation occurs in our model, a large amount of both granzyme and perforin still escape from the synapse. We argue that a tight seal between the cytotoxic lymphocyte and its target cell is not required to avoid bystander killing. Instead, we propose that the requirement for spatiotemporal colocalization of granzyme and perforin acts as an effective bimolecular filter to ensure target specificity.

Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing

“…It has been proposed that the IS enhances the intensity and specificity of intercellular communication by restricting the diffusion of secreted factors (Huse et al, 2006; Stinchcombe and Griffiths, 2007; Woodsworth et al, 2015). Studies of cytokine-mediated communication, however, indicate that soluble proteins escape the T cell-target cell interface very quickly (Feinerman et al, 2010; Muller et al, 2012; Sanderson et al, 2012).…”

Targeted Cell-to-Cell Delivery of Protein Payloads via the Granzyme-Perforin Pathway

“… 27 Importantly, recent work has shown that following release into the immune synapse, the trafficking of granzyme B to the target cell membrane and entry into the target cell via perforin pores is likely a result of passive diffusion only. 28 , 29 , 33 …”

“…This interpretation is predicated on the assumption that entry into the target cell via perforin pores is passive, which, while historically controversial, is supported by most recent experimental and theoretical data. 26 , 28 , 29 , 33 , 40 …”

“…The diffusivity of the fusion protein is important to consider, as this dictates the propensity for the granzyme fusion protein to both spread throughout the synapse, translocate through the pore, and escape the synapse. 33 While empirical measurement of the fusion protein diffusivity would be a substantial undertaking, we can at least make estimates regarding the change in diffusivity using the Stokes-Einstein relationship, in which the diffusivity D , is proportional to the inverse of the radius r of the molecule. (The exact relationship is D = K B T /(6 πηr ), where K B T is the thermal energy and η is the solvent viscosity.…”

“…Importantly, surrounding bystander cells typically do not receive appreciable quantities of granzymes. 31 , 32 , 33 In summary, surface receptor-mediated target cell recognition results in specific, cytoplasm-to-cytoplasm intercellular transfer of granzymes to that same target cell. More broadly, cytotoxic lymphocytes have exceptional utility as a cellular chassis because they are targetable, expandable, and amenable to genetic manipulation.…”