Oxidation of cofilin mediates T cell hyporesponsiveness under oxidative stress conditions.

Oxidative stress leads to impaired T cell activation. A central integrator of T cell activation is the actin-remodelling protein cofilin. Cofilin is activated through dephosphorylation at Ser3. Activated cofilin enables actin dynamics through severing and depolymerization of F-actin. Binding of cofilin to actin is required for formation of the immune synapse and T cell activation. Here, we showed that oxidatively stressed human T cells were impaired in chemotaxis- and costimulation-induced F-actin modulation. Although cofilin was dephosphorylated, steady-state F-actin levels increased under oxidative stress conditions. Mass spectrometry revealed that cofilin itself was a target for oxidation. Cofilin oxidation induced formation of an intramolecular disulfide bridge and loss of its Ser3 phosphorylation. Importantly, dephosphorylated oxidized cofilin, although still able to bind to F-actin, did not mediate F-actin depolymerization. Impairing actin dynamics through oxidation of cofilin provides a molecular explanation for the T cell hyporesponsiveness caused by oxidative stress.

An MEK-cofilin signalling module controls migration of human T cells in 3D but not 2D environments.

T cells infiltrate peripheral tissues to execute immunosurveillance and effector functions. For this purpose, T cells first migrate on the two-dimensional (2D) surface of endothelial cells to undergo transendothelial migration. Then they change their mode of movement to undergo migration within the three-dimensional (3D)-extracellular matrix of the infiltrated tissue. As yet, no molecular mechanisms are known, which control migration exclusively in either 2D or 3D environments. Here, we describe a signalling module that controls T-cell chemotaxis specifically in 3D environments. In chemotaxing T cells, Ras activity is spatially restricted to the lamellipodium. There, Ras initiates activation of MEK, which in turn inhibits LIM-kinase 1 activity, thereby allowing dephosphorylation of the F-actin-remodelling protein cofilin. Interference with this MEK-cofilin module by either inhibition of MEK or by knockdown of cofilin reduces speed and directionality of chemotactic migration in 3D-extracellular matrices, but not on 2D substrates. This MEK-cofilin module may have an important function in the tissue positioning of T cells during an immune response.

Sustained LFA-1 cluster formation in the immune synapse requires the combined activities of L-plastin and calmodulin.

Formation of immune synapses (IS) between T cells and APC requires multiple rearrangements in the actin cytoskeleton and selective receptor accumulation in supramolecular activation clusters (SMAC). The inner cluster (central SMAC) contains the TCR/CD3 complex. The outer cluster (peripheral SMAC) contains the integrin LFA-1 and Talin. Molecular mechanisms selectively stabilizing receptors in the IS remained largely unknown. Here, we demonstrate that sustained LFA-1 clustering in the IS is a consequence of the combined activities of the actin-bundling protein L-plastin (LPL) and calmodulin. Thus, upon antigen-recognition of T cells, LPL accumulated predominantly in the peripheral SMAC. siRNA-mediated knock-down of LPL led to a failure of LFA-1 and Talin redistribution - however, not TCR/CD3 relocalization - into the IS. As a result of this LPL knock-down, the T-cell/APC interface became smaller over time and T-cell proliferation was inhibited. Importantly, binding of calmodulin to LPL was required for the maintenance of LPL in the IS and consequently inhibition of calmodulin also prevented stable accumulation of LFA-1 and Talin, but not CD3, in the IS.

Actin cytoskeletal dynamics in T lymphocyte activation and migration.

Dynamic rearrangements of the actin cytoskeleton are crucial for the function of numerous cellular elements including T lymphocytes. They are required for migration of T lymphocytes through the body to scan for the presence of antigens, as well as for the formation and stabilization of the immunological synapse at the interface between antigen-presenting cells and T lymphocytes. Supramolecular activation clusters within the immunological synapse play an important role for the initiation of T cell responses and for the execution of T cell effector functions. In addition to the T cell receptor/CD3 induced actin nucleation via Wasp/Arp2/3-activation, signals through accessory receptors of the T cell (i.e., costimulation) regulate actin cytoskeletal dynamics. In this regard, the actin-binding proteins cofilin and L-plastin represent prominent candidates linking accessory receptor stimulation to the rearrangement of the actin cytoskeleton. Cofilin enhances actin polymerization via its actin-severing activity, and as a long-lasting effect, cofilin generates novel actin monomers through F-actin depolymerization. L-plastin stabilizes actin filament structures by means of its actin-bundling activity.

High affinity interaction of integrin alpha4beta1 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) enhances migration of human melanoma cells across activated endothelial cell layers.

The capacity of tumor cells to form metastatic foci correlates with their ability to interact with and migrate through endothelial cell layers. This process involves multiple adhesive interactions between tumor cells and the endothelium. Only little is known about the molecular nature of these interactions during extravasation of tumor cells. In human melanoma cells, the integrin alphavbeta3 is involved in transendothelial migration and its expression correlates with metastasis. However, many human melanoma cells do not express beta3 integrins. Therefore, it remained unclear how these cells undergo transendothelial migration. In this study we show that human melanoma cells with different metastatic potency, which do not express beta2 or beta3 integrins, express the VCAM-1 receptor alpha4beta1. VCAM-1 is up-regulated on activated endothelial cells and is known to promote transendothelial migration of leukocytes. Interestingly, despite comparable cell surface levels of alpha4beta1, only the highly metastatic melanoma cell lines MV3 and BLM, but not the low metastatic cell lines IF6 and 530, bind VCAM-1 with high affinity without further stimulation, and are therefore able to adhere to and migrate on isolated VCAM-1. Moreover, we demonstrate that function-blocking antibodies against the integrin alpha4beta1, as well as siRNA-mediated knock-down of the alpha4 subunit in these highly metastatic human melanoma cells reduce their transendothelial migration. These data imply that only high affinity interactions between the integrin alpha4beta1 on melanoma cells and VCAM-1 on activated endothelial cells may enhance the metastatic capacity of human beta2/beta3-negative melanoma cells.

Costimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25.

Rearrangements in the actin cytoskeleton play a pivotal role for costimulation-induced formation of the immunological synapse and T cell activation. Yet, little is known about the actin-binding proteins that link costimulation to rearrangements in the actin cytoskeleton. Here we demonstrate that phosphorylation of the actin bundling protein L-plastin in response to costimulation through TCR/CD3 plus CD2 or CD28, respectively, is important for the activation of human peripheral blood T lymphocytes (PBT). Mass spectrometry and site-directed mutagenesis revealed that Ser5 represents the only phospho-acceptor site of L-plastin in PBT. Wild-type L-plastin (wt-LPL) and a non-phosphorylatable 5A-L-plastin (5A-LPL) equally relocalized to the immunological synapse between PBT and APC. Yet importantly, cells expressing 5A-LPL showed a significantly lower expression of the T cell activation molecules CD25 and CD69 on the cell surface than cells expressing wt-LPL. This effect is due to a failure in the transport of CD25 and CD69 to the cell surface since the total amount of these proteins within the cells remained unchanged. In conclusion, phosphorylation of the actin bundling protein L-plastin represents a so-far-unknown mechanism by which costimulation controls the transport of activation receptors to the T cell surface.

Phosphorylation of ectopically expressed L-plastin enhances invasiveness of human melanoma cells.

The leukocyte specific actin-binding protein L-plastin is aberrantly expressed in several nonhematopoetic malignant tumors. However, little is known about the functional consequences of L-plastin expression. Here, we investigated the function of L-plastin in human malignant melanoma cells. Knock-down of endogenous L-plastin by siRNA treatment reduced migration of the melanoma cell line IF6. However, in melanoma patients, no correlation existed between L-plastin expression and tumor stages. This implied that additional factors such as phosphorylation of L-plastin may influence its function in tumor cells. To investigate this further, EGFP-tagged wild-type L-plastin (wt-LPL-EGFP) and a mutated, nonphosphorylatable L-plastin protein (5A7A-LPL-EGFP), were expressed in the L-plastin negative melanoma cell line MV3. Biochemical analysis revealed that wt-LPL-EGFP is phosphorylated in MV3 cells while 5A7A-LPL-EGFP is not. Although both wt-LPL-EGFP and 5A7A-LPL-EGFP were targeted to, and promote the formation of, vinculin-containing adhesion sites, static adhesion to either Matrigel or isolated extracellular matrix molecules was neither influenced by expression of wt-LPL-EGFP nor by expression of 5A7A-LPL-EGFP when compared with EGFP expressing control cells. In contrast, haptotactic, but not chemotactic, migration of melanoma cells towards either Matrigel or isolated extracellular matrix molecules was similarly enhanced, if either 5A7A-LPL-EGFP or wt-LPL-EGFP were expressed in MV3 cells. Interestingly, only cells expressing the phosphorylatable wt-LPL-EGFP protein showed enhanced invasion into Matrigel. In line with these findings the in vivo metastatic capacity of mouse B16 melanoma cells correlates with expression and phosphorylation of L-plastin. These data show that an increase in melanoma cell invasiveness requires not only expression but also phosphorylation of L-plastin.

Phosphatidylinositol 3-kinase functions as a Ras effector in the signaling cascade that regulates dephosphorylation of the actin-remodeling protein cofilin after costimulation of untransformed human T lymphocytes.

The activity of cofilin, an actin-remodeling protein, is required for T lymphocyte activation with regard to formation of the immunological synapse, cytokine production, and proliferation. In unstimulated T PBL (PB-T), cofilin is present in its Ser3-phosphorylated inactive form. Costimulation of TCR/CD3 and CD28 induces dephosphorylation and, thus, activation of cofilin. In this study we characterized the signaling cascades leading to cofilin activation in untransformed human PB-T. We show that a Ras-PI3K cascade regulates dephosphorylation of cofilin in PB-T. The GTPase Ras is a central mediator of this pathway; transient expression of an activated form of H-Ras in PB-T triggered the dephosphorylation of cofilin. Inhibition of either MAPK/ERK kinase or PI3K blocked both Ras-induced and costimulation-induced cofilin dephosphorylation in PB-T, showing that the combined activities of both signaling proteins are required to activate cofilin. That Ras functions as a central regulator of cofilin dephosphorylation after costimulation through CD3 x CD28 was finally proven by transient expression of a dominant negative form of H-Ras in primary human PB-T. It clearly inhibited costimulation-induced cofilin dephosphorylation, and likewise, activation of PI3K was diminished. Our data, in addition, demonstrate that regarding the downstream effectors of Ras, a clear difference exists between untransformed human PB-T and the T lymphoma line Jurkat. Thus, in PB-T the Ras signaling cascade is able to activate PI3K, whereas in Jurkat cells this is not the case. In addition to the insights into the regulation of cofilin, this finding discloses a to date unrecognized possibility of PI3K activation in T lymphocytes.