Transience in polarization of cytolytic effectors is required for efficient killing and controlled by Cdc42

Abstract

Cytolytic effectors polarize toward target cells for effective killing and IFN-γ secretion. The spatiotemporal features of this polarization and their importance for cytolysis have not been resolved. In cytotoxic T cells and natural killer (NK) cells, transient polarization was consistently associated with effective killing. Polarization was regulated by Cdc42, a small Rho family GTPase universally critical for cytoskeletal dynamics. Transient accumulation of active Cdc42 at the cytolytic effector/target cell interface and focus of such accumulation on the interface center were closely related to cytolysis. Surprisingly, however, the intensity of Cdc42 activation was not. We interfered with Cdc42 activation in NK cells such that sustained polarization in long lasting nonkilling cell couples was selectively blocked. Thus the proportion of the NK cell population displaying transient polarization was increased. As a consequence, cytolytic responder frequency and IFN-γ production were enhanced upon such interference with Cdc42 activation. These data support the notion that transience in polarization is critical for cytolytic effector function, likely by preventing cytolytic effectors from becoming trapped in nonproductive target cell interactions. Each can also secrete IFN-γ to modulate adaptive immune function. To acquire effector capability, cytolytic effectors need to be primed. CTL priming occurs as the consequence of naive CD8 T cell interactions with antigen-presenting dendritic cells. NK cell priming requires cytokines generated predominantly by dendritic cells. However, the nature of the cytokines involved is less certain. IL-12 and IL-18 enable NK cells to secrete IFN-γ (1, 2) and are synergistic (3). IL-15 can promote NK cell expansion (4, 5). IL-15 needs transpresentation by the IL-15 receptor α chain (6, 7), which is induced on dendritic cells by engagement of Toll-like receptors (7). High concentrations of IL-2 effectively prime NK cells in vitro, as often used in clinical settings (8, 9). However, in vivo, IL-2 is dispensable for NK cell priming (5). To understand the role of the polarization of cytolytic effector in their function, the effect of priming conditions on NK cell polarization needs to be taken into account.Target cell contact triggers the complex polarization of primed cytolytic effectors, recruitment of cytolytic granules to the interface (10, 11), cytoskeletal reorientation (12, 13), and receptor clustering (14,15). Cytolytic effector polarization is likely functionally important, as key regulators of cytoskeletal dynamics, Vav and WASP, play critical roles (16-19). However, WASP and Vav are complex molecules with functions that may or may not require cytoskeletal regulation (20,21). The Rho family GTPases Rac and Cdc42 are the central regulators of cytoskeletal dynamics in many cell types (22). Although it has been established that Rac is required for cell coupling and polarization of cytolytic granules (16), the role Cdc42 has not been addressed. Despite the established importance of ...

2B4 Engagement Mediates Rapid LFA-1 and Actin-Dependent NK Cell Adhesion to Tumor Cells as Measured by Single Cell Force Spectroscopy

“…This is in contrast to the early increase in NK cell adhesion, which was evident already after 90 s cell contact. NK cells may therefore be much faster than T cells in establishing cellular adhesion, which is in line with their quick responses during innate immune reactions and that has been suggested to be essential for their effective cytotoxic activity (27). In T cells maximal adhesion forces of ∼14 nN were measured after 30 min contact time (26).…”

Cytokine Secretion by CD4+ T Cells at the Immunological Synapse Requires Cdc42-Dependent Local Actin Remodeling but Not Microtubule Organizing Center Polarity

“…This dynamic remodeling of actin in the center of the IS would depend on Cdc42 in CD4 + T cells thus controlling cytokine release. This could be specific to CD4 + T cells or cytokine release, as Wülfing and colleagues (36) have shown that a dominant negative mutant of Cdc42 does not inhibit granule secretion by CD8 + cytotoxic T cells. What are the downstream regulators of Cdc42-dependent actin remodeling at the IS?…”

Tentative and transient natural killer cell polarization balances the requirements for discriminatory recognition and cytolytic efficacy

“…While active Cdc42 is enriched selectively at the center of the CTL/target cell interface, it is spread out over the NK cell/target cell interface. 5 This becomes obvious when the amount of interface accumulation of active Cdc42 is normalized to its area. With such relation to accumulation area, CTLs are much more effective in generating a small area of intense Cdc42 accumulation than NK cells (Fig.…”

“…Rapid execution of the killing interaction allows a cytolytic effector to kill several target cells sequentially, promoting effective population-wide killing. To maximize cytolytic efficacy, CTLs are fully polarized within seconds of target cell contact with accumulation of signaling intermediates, actin and the MTOC at the CTL/target cell interface, indicative of strong activating signaling 5,14 ( Fig. 1).…”

“…Using live cell imaging we have characterized NK cell polarization in time and space. 5 Both requirements for effective NK cell function, responsiveness to cytokines and discriminatory recognition, are reflected in the regulation of NK cell polarization.…”