Natural killer (NK) cells are the innate immune system’s cytolytic effectors. Using germline-encoded receptors, NK cells perform surveillance for virally infected or tumorigenic cells and upon recognition of sensitive targets kill them using directed secretion of specialized secretory lysosomes. In addition, NK cells are important regulators of the innate immune system through the secretion of cytokines, particularly type I interferons. People with impaired or defective NK cell function suffer severe viral infections and malignancies, underscoring the need for effective NK cell activity. We use human NK cells – both ex vivo and cell lines – to better understand the biological processes required for NK cell function. In particular, we have done pioneering work in the field of NK cell immunodeficiency, using patients with NK cell functional defects to study the biological processes required for their effector functions.
Directed secretion for target cell lysis is a highly controlled process with discretely regulated steps. These steps include the formation of an immunological synapse, with actin reorganization, movement of the secretory lysosomes (cytolytic granules) to the microtubule organizing center, movement of the MTOC to the synapse, and subsequent release of granules onto the target . Research in our lab is focused on highly quantitative analysis of the NK cell immune synapse. In particular, we have been defining the role of the cytoskeleton in the process of granule polarization and exocytosis using multiple high resolution imaging techniques.
One interest in the lab is the structure of actin at the NK cell immunological synapse. Early images of the synapse, taken from the perspective of a target cell and compiled using serially collected images through the z-axis (Z stacks) via confocal microscopy, showed a thick ring of peripheral actin surrounding a central clearance, through which it was suggested that lytic granules would be secreted . However, work by us and others showed that the actin-associated motor protein myosin IIa is required for degranulation and is on lytic granules, suggesting that actin may be present throughout the synapse, but was simply undetectable by conventional fluorescence microscopy [3, 4].
With STED, we showed that actin is present throughout the synapse and defined minimally sized actin clearances formed as NK cells degranulate. In addition, we showed interaction of granules found in these actin hypodensities with actin filaments, suggesting a dynamic interaction between the two prior to (or during) degranulation .
These findings, published back to back with complementary work from Dr. Daniel Davis and colleagues  have defined a new paradigm for the architecture and role of actin at the immune synapse in NK cells. It will be exciting to see, using super-resolution technologies, if the pervasive actin network seen in the NK cell synapse is common to other immune effectors, such as cytolytic T cells, and other immune cell functions, such as directed secretion of cytokines. Alternatively it may represent a unique checkpoint employed by "pre-armed" NK cells, which likely require tighter control over their effector function than T cells .
Fig. 1: Visualization of lytic granules imaged by CW-STED and confocal on F-actin. NK92 cells were adhered to glass coated with antibody to activating (NKp30) and adhesion (CD18) receptor then fixed, permeabilized and stained for perforin and actin. Cells were imaged using CW-STED (actin, green) and either CW-STED or confocal (perforin, red). Shown is the same cell with granules detected by STED (A) or confocal (B). A region of interest is enlarged to show greater resolution of granules (center panel). (C) Full width half maximum (FWHM) measurements of confocal (green line) and STED images (red line). Horizontal dashed lines show half maxima, vertical dashed lines show width at half maxima. (D) Representative line profile of pixel intensities of actin (green line) and perforin (red line) taken from a line bisecting a single granule (shown in white in STED image enlargement). AU: arbitrary units.
In addition to our use of super-resolution in studies described above, we continue to focus on the interaction of lytic granules with the cytoskeleton. Using STED nanoscopy we visualized individual myosin IIa filaments wrapped around granules, both isolated and within whole cells . These images enabled us to discern between alternative models of myosin IIa-granule interactions suggested by biochemical studies. We also have applied the use of STED to study and define granule size , and have optimized dual channel STED for the imaging of both granules and actin in super-resolution .
Our research centrally incorporates STED into a toolbox of multiple high resolution imaging techniques that we use, including total internal reflection microscopy, spinning disk and laser scanning confocal microscopy, and platinum replica electron microscopy. In addition, we have the ability to manipulate human NK cells by introducing fluorescent proteins and silencing constructs, allowing us to evaluate the function and regulation of critical NK cell proteins. Finally, we have access to patients with rare NK cell immunodeficiencies, giving us the unique perspective of human "knockouts".
Challenges as we move forward include identification of fluorophores suitable for multiple channel STED and the development and optimization of fluorescent proteins durable enough to withstand depletion laser scanning (required for STED), which would allow for live cell super resolution imaging. As always, new technologies also result in new needs for data analysis and management, and we continue to develop quantitative algorithms for analysis of super-resolution images.
Recent advances allowing us to overcome the diffraction barrier have resulted in an explosion of nanoscopic technologies. The ability to "see" within the cell with such unprecedented resolution gives us the opportunity to visualize structures and proteins with previously unimaginable clarity. With this ability, we can continue to elucidate the mechanism behind NK cell processes, allowing us to further understand their role in human health and disease.
- Orange JS: Formation and function of the lytic NK-cell immunological synapse. Nat Rev Immunol 8 (2008) 713–725.
- Orange JS, Harris KE, Andzelm MM, Valter MM, Geha RS, Strominger JL: The mature activating natural killer cell immunologic synapse is formed in distinct stages. Proc Natl Acad Sci USA 100 (2003) 14151–14156.
- Andzelm MM, Chen X, Krzewski K, Orange JS, Strominger JL: Myosin IIA is required for cytolytic granule exocytosis in human NK cells. J Exp Med 204 (2007) 2285–2291.
- Sanborn KB, Rak GD, Maru SY et al.: Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse. J Immunol 182 (2009) 6969–6984.
- Rak GD, Mace EM, Banerjee PP, Svitkina T, Orange JS: Natural killer cell lytic granule secretion occurs through a pervasive actin network at the immune synapse. PLoS Biol 9: e1001151.
- Brown AC, Oddos S, Dobbie IM et al.: Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy. PLoS Biol 9: e1001152.
- Wulfing C, Purtic B, Klem J, Schatzle JD: Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing. Proc Natl Acad Sci USA 100 (2003) 7767–7772.
- Sanborn KB, Mace EM, Rak GD et al.: Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity. Blood 118: 5862–5871.
- Mace EM, Orange JS: Dual channel STED nanoscopy of lytic granules on actin filaments in natural killer cells. Communicative and Integrative Biology 5 (2012) 2.