Effective immune responses against infectious agents and cancer require that immune cells traffic efficiently to the correct locations and then identify and specifically respond to threats by interacting with other cells. Lymphocytes are particularly well suited for these tasks because they can completely reorganize their cellular architecture in a matter of minutes in response to surface receptor stimulation. This remarkable structural plasticity enables them to function both rapidly and effectively in a wide variety of physiological contexts. We aim to understand the signaling mechanisms that underlie these morphological changes, which could aid in the development of strategies to better modulate immune function in vivo. We are particularly interested in:
- Polarized cytoskeletal remodeling at the immunological synapse. We study how signals from activating receptors shape synaptic architecture and how inhibitory stimulation antagonizes synapse formation.
- The role of cell polarity and cytoskeletal dynamics in specifying lymphocyte effector responses, with special emphasis on cytotoxic killing by T cells and natural killer (NK) cells.
- Developing photochemical methods for single-cell analysis of polarized signal transduction. Our efforts focus on using localized pulses of light to stimulate cell surface receptors during imaging experiments.
Signal transduction plays a central role in nearly every aspect of immune function, and has been the subject of intense biochemical and genetic analysis for a number of years. As a result, many of the cell surface receptors and intracellular proteins important for these processes have been identified and characterized. Despite this progress, however, our knowledge of how these molecules work together dynamically in the context of a complex cellular response is limited. To circumvent these issues, our lab uses a multidisciplinary approach that combines photochemistry with single cell live imaging. Specifically, we have developed photocaged protein reagents that cannot bind to their cognate receptors until they are irradiated ultraviolet light. Using these reagents, we can control precisely where and when cell surface receptors are engaged, and also monitor subsequent signaling responses with high resolution imaging modalities such as total internal reflection fluorescence (TIRF) microscopy.
We have applied this photostimulation and imaging approach to study T cell activation and the acquisition of cell polarity. Recognition by a T cell of cognate antigen on the surface of an antigen-presenting cell (APC) induces the formation of a specialized junction between the T cell and the APC known as an immunological synapse (IS). The IS plays an important role in maintaining the specificity of effector responses by channeling the secretion of cytokines and cytolytic factors directionally toward the APC. In this manner, it minimizes unwanted inflammation or damage in surrounding tissue. IS formation is characterized by reorientation of the T cell’s microtubule-organizing center (MTOC) to a position at the center of the contact site. Concomitantly, filamentous actin (F-actin) accumulates in a dense ring at the periphery. Our work has demonstrated that this stereotyped cytoskeletal architecture is dictated by two distinct lipid second messengers, diacylglycerol (DAG) and phosphatidylinositol trisphosphate (PIP3). DAG accumulates in the center of the IS, where it guides the polarization of the MTOC. Conversely, PIP3 localizes to the periphery, where it drives annular accumulation of F-actin. Mechanisms like this are particularly well suited for the rapid generation of polarized structures, and are likely to be of general importance for the patterning of dynamic cell-cell contacts in other cell types. Our current research efforts focus on how these lipid second messenger gradients are formed, how they coordinate downstream cytoskeletal machinery, and how they influence T cell effector responses such as target cell killing.
We have also used photostimulation and imaging to explore how NK cells resolve conflicting activating and inhibitory signals. NK cells play a central role in immune responses to viruses and cancer by recognizing and eliminating infected or transformed target cells. Their activity is governed by a diverse array of receptors, some which deliver activating stimulation, and others that inhibit responses. Inhibitory receptors play a particularly important role in this regime by preventing the killing of normal, healthy cells. We have shown that they do this by directly destabilizing the IS that forms between an NK cell and a potential target. This has the effect of locally blocking killing responses without affecting the overall cytotoxic potential of the NK cell. In this manner, signals from inhibitory receptors can actually help guide NK cells to bona fide targets within a tissue. We are currently pursuing the signaling mechanisms coupling inhibitory receptor stimulation to IS remodeling, and also the roles of inhibitory receptor signaling at different stages during NK cell development.
Our studies of IS formation and dissolution in lymphocytes have sparked a more general interest in the molecular mechanisms and functional consequences of polarized signal transduction. To this end, we are pursuing synthetic strategies for “universalizing” the photostimulation and imaging approach for use with other receptors in other cell types. Topics of interest include cell migration and the elaboration of specialized cellular projections. It is our hope that close mechanistic study of these processes will provide general insights into how cells coordinate and shape their behavior in complex, non-uniform environments.
Photoactivation of the T cell receptor
Above, a schematic of the process. Photoactivatable pMHC is nonstimulatory to the T cell receptor (TCR) until it is exposed to UV light. Below, a typical photoactivation experiment. T cells bearing fluorescent signaling probes are attached to glass surfaces containing photoactivatable pMHC, and a portion of the surface is irradiated with focused UV light during an imaging experiment. A timelapse montage is shown containing consecutive images of a single cell that expresses probes for diacylglycerol (DAG, green) and the MTOC (red). The time and region of UV irradiation is indicated by the purple and yellow circle. Accumulation of DAG at the irradiated region precedes the reorientation of the MTOC.