Mechanobiology of the immune synapse: signal integration via actin dynamics

ABSTRACT
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.

Grant Number: 5R01AI147118-09
NIH Institute/Center: NIH
Principal Investigator: Janis Burkhardt