Mechanical force is essential for T cell activation. It activates TCR signaling, and allows the T cell to sample the quality of TCR-pMHC interactions. This greatly expands the dynamic range of TCR responses and permits antigen discrimination during thymic selection, T cell priming, and effector responses. Our understanding of how force influences TCR-pMHC interactions has advanced significantly, thanks to biophysical studies at the single molecule level. However, there are large gaps in knowledge at the cell biological level. This project seeks to identify the biochemical and mechanical circuits within the TCR signal transduction network that permit the rapid translation of small differences in the physical characteristics of the TCR?pMHC interactions into distinct cellular responses. During the first project period, we showed that the T cell actin network exerts force on the integrin LFA-1 as well as the TCR, supporting mechanical crosstalk that influences the activation of both molecules. Interestingly, this process is sensitive to the biophysical features of the stimulatory surface, including ligand mobility and stiffness. These parameters are physiologically relevant, as they are regulated during DC maturation to optimize T cell priming. Further analysis reveals that this mechanobiology also impacts cytoplasmic signaling molecules that interact with the actin cytoskeleton. In particular, we find that T cell stiffness responses involve phosphorylation of the stretch-sensitive adapter protein CasL. On the basis of these findings, we hypothesize that TCR-induced actin polymerization allows the cell to sense biophysical cues provided by the interacting APC, initiating mechanical feedback loops that modulate force-dependent signaling of cell surface receptors and intracellular signaling molecules that interact with the actin cytoskeleton. To test this hypothesis, we will carry out 3 specific aims. First, we will determine how ligand mobility influences actin dynamics and TCR signaling. Using stimulatory glass coverslips, planar bilayers with different mobility properties, and mixed mobility patterned surfaces, we will ask how the agonist strength and mobility of pMHC complexes and integrin ligands influences actin dynamics and TCR signaling. As part of this analysis, we will use TCR tension probes to define how altering the mobility of TCR and integrin ligands influences the forces experienced by the TCR. Next, we will carry out similar studies to understand how substrate stiffness influences T cell activation. We will stimulate T cells on hydrogels of varying stiffness, and analyze the effects on actin dynamics, TCR tension, and TCR signaling events needed for full T cell activation. Finally, we will investigate the role of CasL, a prototypic force-sensitive signaling intermediate. Using T cells lacking CasL, we will study the function of CasL during T cell responses to changes in ligand mobility and substrate stiffness. In addition, we will probe the signaling pathways leading to CasL phosphorylation during stiffness responses, and use mass spectrometry to identify relevant binding partners.
Activation of T cells by dendritic cells is essential for the initiation of adaptive immunity to pathogens. This proposal addresses a poorly understood aspect of this process, the mechanisms through which forces exerted by cytoskeletal elements coordinate signaling events at the cell-cell contact site. The knowledge gained from these studies will be important for optimizing vaccine development, and for modulating immune responses in patients with immunodeficiency and autoimmune disease.
