´╗┐Supplementary MaterialsSupplemental: Fig

´╗┐Supplementary MaterialsSupplemental: Fig. cells recognize their cognate antigen on the surface of antigen-presenting cells (APCs) through the T cell receptor, which results in the formation of a contact region (immune synapse) between the two cells and the activation of the T cells. Activated T cells proliferate and differentiate into effector T cells that secrete cytokines, provide help to B cells, and kill target cells. We asked whether the actin cytoskeleton governs differences in signaling in effector T cells versus na?ve (unstimulated) T cells. Using atomic pressure microscopy and quantitative confocal microscopy, we found that na?ve T cells had a mechanically stiffer cortical cytoskeleton than that of effector cells, which resulted in na?ve cells forming smaller immune synapses with APCs. This suggests that the cytoskeletal stiffness of the T cell before it undergoes antigen stimulation predicts its subsequent dynamic engagement with APCs and its activation potential. Cytoskeletal rigidity depended on the activity of the actin-severing enzyme cofilin through a pathway requiring the small guanosine triphosphatase RhoA and the kinases ROCK (Rho-activated kinase) and LIMK. These findings suggest that the baseline cytoskeletal state controls T cell responses and that the underlying pathway could be a therapeutic target for modulating adaptive immunity. INTRODUCTION T cells must maintain a threshold of activation that is low enough for them to rapidly scrutinize the body for sparse and low-affinity antigens, yet high enough to avoid triggering autoimmune responses. The cellular machinery that controls T cell activation also enables memory and effector T cells to be activated more readily than antigenically na?ve cells, facilitating rapid effector responses upon reactivation (1C3). Here, we demonstrate that cytoskeletal differences in na?ve and effector T cells affect their activation, and we define the molecular mechanisms underlying PTPRC these differences. When T cell receptors (TCRs) recognize their cognate antigens on the surface of an antigen-presenting cell (APC), a series of cytoskeleton-dependent events are stimulated in the T cells that are critical for full activation. At the cellular scale, T cells stop crawling, turn to reorient their microtubule-organizing center Brofaromine toward the Brofaromine interface between the T cell and the APC, expand lamellipodia over (4), and forcefully push into APCs (5C7), increasing the surface area of the cell-cell contact and enhancing TCR signaling. The nanoscale changes at the T cellCAPC interface result in the assembly of an immune synapse (8), starting with the gathering of TCRs into microclusters, the formation of which depends on actin polymerization (9). Thus, the actin cytoskeleton is usually important for forming and maintaining the cell-cell interface, as well as for gathering the key signaling molecules required for T cell activation. Whether differences in cytoskeletal regulation between na?ve and effector cells mediate Brofaromine their differential activation has not been investigated; however, activated T cells and memory cells have preformed nanoclusters of TCRs around the cell membrane (10). These nanoclusters, which do not occur in na?ve cells, facilitate signaling and are organized by cytoskeletal molecules (11). Furthermore, effector and memory T cells have increased amounts of transcripts encoding cytoskeletal molecules, including actin, talin, Arp2/3, and stathmin (12, 13). Here, we examined the cytoskeletal pathway downstream of the Rho family member RhoA (14). The active, guanosine triphosphate (GTP)Cbound form of RhoA binds to and activates members of the Rho kinase (ROCK) family of serine and threonine kinases (15). ROCK mediates T cell crawling and polarization (16) and phosphorylates multiple downstream cytoskeletal regulators, including LIM kinase (LIMK) (17). LIMK, Brofaromine in turn, inhibits cofilin, an actin-severing and actin-depolymerizing enzyme. Upon TCR stimulation, cofilin is activated by phosphatases, which enhances actin branching and actin polymerization and is necessary for immune synapse formation (18). Here, we tested the hypothesis that cytoskeletal pathways modulate immune synapse size and thus regulate T cell activation. Using atomic pressure microscopy (AFM) and confocal microscopy to assess the single-cell responses of T cells to stimulation, we found that na?ve mouse CD4+ T cells, at baseline, had increased cytomechanical stiffness compared to that of effector CD4+ T cells because.

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