Consideraciones generales

Although the APC side of the synapse has long been assumed to play a passive role, a growing body of evidence indicates the DC F-actin network plays a critical role in T cell activation. There are already some hints that APCs, DCs in particular, play an active role in regulating IS formation and structure (209). T cells responding to B cells or stimulatory supported lipid bilayers form the classical mature synapse with a characteristic pSMAC and cSMAC pattern (210, 211). This shows that in the absence of barriers to receptor mobility, TCR and LFA-1 microclusters will be driven towards the IS center in a T cell autonomous fashion. The DC / T cell IS is not characterized by this symmetric pattern and is instead characterized by multiple patches of variable protein overlap, and even at late time points, fails to form a central accumulation of CD3 (212). Therefore, it is highly likely that the DC forms barriers to the free diffusion of T cell ligands that are either cytoskeletal or topological in nature. Importantly, these two possibilities are not mutually exclusive and the lateral mobility of some proteins could be regulated through linkage to the DC actin cytoskeleton while others are restricted through topological barriers. In either case, the DC actin cytoskeleton would necessarily play a crucial role in defining and maintaining this diffusive barrier. The creation of such a barrier has important implications for the mechanical regulation of signaling at the IS. While the T cell actin-cytoskeleton could activate mechano-sensitive molecules at the IS by applying force to the receptor – ligand bond, barriers to diffusion provided by the DC actin-cytoskeleton could provide a retention, or counter force on the ligand, thereby increasing tension at the molecular level. Furthermore, regulation of ligand mobility may prevent microcluster centralization and deactivation, thereby enhancing T cell activation. Following upregulation of T cell ligands, control of molecular mobility would thus

function as a second stage at which DCs could modulate their T cell stimulatory capacity.

The DC cytoskeleton plays a critical role in T cell priming

At the functional level, the DC F-actin network polarizes towards a cognate T cell in an MHC dependent manner (213), and treatment of DCs with actin depolymerizing agents impairs the DCs ability to prime T cell responses (214). Polarization of the F- actin network is mediated by Rho family GTPases, and is required for proper conjugate formation, IL-2 production, and T cell proliferation (215-217). Similarly, the ARP2/3 complex activator WASp promotes the maintenance of T cell – DC interactions; WASp- deficient DCs exhibit fewer and shorter-lived contacts with cognate T cells, and a diminished ability to prime T cell proliferation (218, 219). While it is clear that the F- actin network on the DC side of the synapse plays a key role in T cell conjugate formation and T cell activation, how these changes are regulated, and the role in IS mediated signal transduction are open questions in the field.

The DC actin network undergoes specific changes during maturation

Following recognition of danger signals through pattern recognition receptors, DCs undergo a maturation process that increases their stimulatory potential as APCs. As such, changes in actin-regulatory proteins during maturation are of key interest in understanding the regulation of the DC F-actin network at the IS. Several actin regulatory proteins have been documented to increase in response to inflammatory stimuli including robust upregulation of the actin bundling protein fascin (220, 221) and activation of the actin severing protein cofilin (222). These proteins may be acting together to create a more dynamic F-actin network (223), and are associated with an

increase in F-actin content in mature DCs and increased plasma membrane ruffling. Interestingly, fascin polarizes, along with F-actin, to the site of T cell engagement on DCs. Additionally the protein plexin-A1 is upregulated during maturation by CIITA, the master regulator of MHCII expression, and is required for Rho GTPase activation, F- actin polarization to the IS, and T cell activation (217, 224). While many of these changes to the DC F-actin network are likely to be necessary for the migration of DCs from tissues to the lymphnodes, they clearly also play a key role in increasing the stimulatory potential of DCs. Concerted changes in the DC F-actin network could provide the physical reorganization of the DC membrane or provide cytoskeletal tethers that could both limit the lateral mobility of T cell ligands. In the following sections I describe regulated changes in F-actin binding proteins of the Actinin and Ezrin / Radixin / Moesin (ERM) families. Furthermore, I show that these changes are required for the control of the lateral mobility of specific T cell ligands on the surface of DCs.

Control of MHC lateral mobility

While there is little evidence in the literature for APC control of lateral mobility affecting T cell activation, there is significant evidence that the modulation of ligand mobility on artificial APCs can enhance responding T cell activation. Physically trapping TCR microclusters in the periphery of the IS by adding a barrier to diffusion of the αCD3

stimulatory antibody in the artificial APC causes increased localized phosphorylation and increased cellular activation (68). Interestingly, limiting the forward mobility of TCR microclusters causes local deformation of the F-actin network (22, 67, 225). At sites of TCR confinement, molecular forces between the TCR and the viscoelastic actin network

are therefore at their maximum, as these actin deformations are not seen with mobile microclusters. Thus, the increased signaling witnessed at such confinement sites may be due to increased mechanical activation of the TCR signaling complex. Though MHC class II lateral mobility is not constrained by the F-actin network in B cells (56), the lateral mobility of MHC class I is restricted through interactions with the F-actin network (226, 227). To fully understand mechanotransduction at the IS it is essential to know if the regulated changes in the DC actin cytoskeleton correlate with changes in MHC class I or class II mobility, and if such changes in mobility result in enhanced T cell stimulation. In later sections I will discuss the regulation of, or lack-there-of, MHC II lateral mobility on the surface of DCs and the implications for T cell signaling.

Control of integrin ligand lateral mobility

As mentioned earlier integrins are known to be mechanosensitive proteins. In addition to the forces applied on the receptor integrins can respond to the physical properties of their environment. As part of this sensitivity to the environmental factors, proper integrin activation requires sufficient retention forces on the integrin ligand. In fact, integrin mediated cell spreading does not occur unless the ligand can withstand roughly 40pN of applied force (207). In line with this idea, stiffness of the extracellular matrix correlates with outside in signaling (228), and surface immobilization of ICAM-1 is required for TCR induced LFA-1 conformational change (229). Importantly the regulation of the lateral mobility of ICAM-1 can have dramatic affects on immune cell function. In particular, NK cells adhere firmly to target cells in which ICAM-1 lateral mobility is low. Increasing the lateral mobility of ICAM-1, and thereby decreasing the tension on the ICAM-1 / LFA-1 bond, decreases the efficiency of conjugate formation

and granule polarization (230). This suggests that restriction of the lateral mobility of integrin ligands is critical for reaching the perceived force barrier of 40pN. In endothelial cells, members of the actinin and ERM family of actin binding proteins limit the lateral mobility of ICAM-1 through interactions with a concerted polybasic region on the ICAM-1 cytoplasmic tail. Importantly, the constrained lateral mobility of ICAM-1 greatly increases the efficiency of T cell diapedesis suggesting that this is a critical determining factor of LFA-1 adhesiveness (231, 232). This suggests that one way cell - cell interactions can be regulated is through the control of integrin ligand clustering and lateral mobility. As with the control of MHC lateral mobility we will describe in detail how DCs can modulate ICAM-1 lateral mobility for maximal T cell activation.

Control of CD80/86 lateral mobility

If CD28 signaling is in-fact mechanosensitive, then the control of the lateral mobility of the main stimulatory ligands CD80 and CD86 on the surface of APCs may also be essential for proper APC function. The cytoplasmic domains of CD80 and CD86 have been shown to mediate protein clustering and cytoskeletal interactions through a highly conserved poly-basic motif (233, 234). The cytoplasmic tail of each protein is critical in regulating their costimulatory potential. Additionally, the CD80 cytoplasmic tail mediates the separation of CD28 microclusters from TCR microclusters at the immunological synapse (235). The polybasic domains in the CD80 and CD86 cytoplasmic tails are similar to known ERM binding sites in other proteins, including ICAM-1(236) (232). As we have discussed for ICAM-1 the connection to the underlying actin cytoskeleton and limitation of CD80 / CD86 lateral mobility may be important for their functions. Given the importance of F-actin linkage and reorganization to CD28

function, it will be interesting to see if DCs can modulate the costimulatory signal through changes in the lateral mobility of CD80 and 86, and the mechanism by which they do so.

VI. T cells and DCs coordinately regulate the mechanical activation of