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/CD3complex. 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. Key words: Actin cytoskeleton . Immunological synapse . T cells Supporting Information available online IntroductionDuring the activation of T cells the immune synapse (IS) is formed at the area of interaction between T cells and APC [1,2]. The IS is involved in enhancing, directing and terminating the T-cell immune response (for review, see [3][4][5][6][7]). Within the IS, surface receptors as well as intracellular signaling and scaffolding proteins are organized in distinct structures, which are called supramolecular activation clusters (SMAC). The inner cluster (central SMAC or cSMAC) contains PKCY and the TCR/CD3 complex. The outer cluster (peripheral SMAC or pSMAC) is composed of the integrin LFA-1 (CD11a/CD18) and Talin [8].It is clear that for the development of an IS the actin cytoskeleton is of special importance [2,[9][10][11]. For construction of an actin meshwork, as it is found in the IS, crosslinking and bundling of F-actin is indispensable to support F-actin rigidity. Here, we Its function for the formation of the IS, however, was so far unexplored. Plastins belongs to the fimbrin family. Plastins contain two tandemly organized actin-binding sites at the C-terminus, which enable them to form very tight actin bundles [12,13]. But despite having two actin-binding domains, it was reported that plastins bind only weakly to pre-existing actin filaments. An optimal binding occurs, if plastin binds during the process of actin polymerization [14,15]. Bundling of F-actin reduces the speed of the actin turnover, thereby making F-actin structures more stable -but not inflexible and stiff. In this regard, it was also reported that plastin binding can protect actin filaments from depolymerization by cofilin in vitro [16]. Thus, plastins control the length of actin fibers and the speed of G/F-actin turnover. Only very little is known about the regulation of plastins ...

Oxidative stress leads to T-cell hyporesponsiveness or death. The actin-binding protein cofilin is oxidized during oxidative stress, which provokes a stiff actin cytoskeleton and T-cell hyporesponsiveness. Here, we show that long-term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic-like programmed cell death (PCD) in human T cells. Notably, cofilin mutants that functionally mimic oxidation by a single mutation at oxidation-sensitive cysteins (Cys-39 or Cys-80) predominately localize within the mitochondria. The expression of these mutants alone ultimately leads to necrotic-like PCD in T cells. Accordingly, cofilin knockdown partially protects T cells from the fatal effects of long-term oxidative stress. Thus, we introduce the oxidation and mitochondrial localization of cofilin as the checkpoint for necrotic-like PCD upon oxidative stress as it occurs, for example, in tumor environments.

The activity and function of T-cells are influenced by the intra- and extracellular redox milieu. Oxidative stress induces hypo responsiveness of untransformed T-cells. Vice versa increased glutathione (GSH) levels or decreased levels of reactive oxygen species (ROS) prime T-cell metabolism for inflammation, e.g., in rheumatoid arthritis. Therefore, balancing the T-cell redox milieu may represent a promising new option for therapeutic immune modulation. Here we show that sulforaphane (SFN), a compound derived from plants of the Brassicaceae family, e.g., broccoli, induces a pro-oxidative state in untransformed human T-cells of healthy donors or RA patients. This manifested as an increase of intracellular ROS and a marked decrease of GSH. Consistently, increased global cysteine sulfenylation was detected. Importantly, a major target for SFN-mediated protein oxidation was STAT3, a transcription factor involved in the regulation of TH17-related genes. Accordingly, SFN significantly inhibited the activation of untransformed human T-cells derived from healthy donors or RA patients, and downregulated the expression of the transcription factor RORγt, and the TH17-related cytokines IL-17A, IL-17F, and IL-22, which play a major role within the pathophysiology of many chronic inflammatory/autoimmune diseases. The inhibitory effects of SFN could be abolished by exogenously supplied GSH and by the GSH replenishing antioxidant N-acetylcysteine (NAC). Together, our study provides mechanistic insights into the mode of action of the natural substance SFN. It specifically exerts TH17 prone immunosuppressive effects on untransformed human T-cells by decreasing GSH and accumulation of ROS. Thus, SFN may offer novel clinical options for the treatment of TH17 related chronic inflammatory/autoimmune diseases such as rheumatoid arthritis.

Activation of naïve T cells requires costimulation via TCR/CD3 plus accessory receptors, which enables the dynamic rearrangement of the actin cytoskeleton and immune synapse maturation. Signaling events induced following costimulation may thus be valuable targets for therapeutic immunosuppression. Phosphorylation of the actin-bundling protein L-plastin represents such a costimulatory signal in primary human T cells. Phosphorylated L-plastin has a higher affinity toward F-actin. However, the importance of the L-plastin phosphorylation for actin cytoskeleton regulation upon antigen recognition remained unclear. Here, we demonstrate that phosphorylation of L-plastin is important for immune synapse maturation. Thus, expression of nonphosphorylatable L-plastin in untransformed human peripheral blood T cells leads to reduced accumulation of LFA-1 in the immune synapse and to a diminished F-actin increase upon T-cell activation. Interestingly, L-plastin phosphorylation is inhibited by the glucocorticoid dexamethasone. In line with this finding, dexamethasone treatment leads to a reduced F-actin content in stimulated T cells and prevents maturation of the immune synapse. This inhibitory effect of dexamethasone could be reverted by expression of a phospho-mimicking L-plastin mutant. In conclusion, our data introduce costimulation-induced L-plastin phosphorylation as an important event for immune synapse formation and its inhibition by dexamethasone as a novel mode of function of this immunosuppressive glucocorticoid.Key words: Actin cytoskeleton . Costimulation . Immunosuppression . L-plastin Supporting Information available online IntroductionActivation and inactivation of antigen-specific T cells is the basis of a functional adaptive immune system. To become fully activated, primary T cells need at least one secondary stimulus besides the antigen-specific TCR/CD3 signal; a process called costimulation. The costimulatory signal is antigen nonspecific and provided through the interaction of accessory receptors on T cells, e.g. CD2 or CD28, with their ligands on APCs. During antigen-specific T-cell activation, these surface receptors, along with intracellular signaling or scaffolding proteins, organize in supramolecular activation clusters (SMACs) and form an immunological synapse [1,2]. Functionally, this immune synapse provides a stop signal on APCs for migrating T cells [3] important for enhancing, directing or terminating T-cell immunity [4]. Since the immune synapse has an important function in T-cell activation, sustained signaling, and effector functions [4,5], it is important to elucidate whether clinically used immunosuppressive drugs interfere with immune synapse formation or stabilization. Glucocorticoids are commonly used immunosuppressants in organ transplantation or the treatment of dermatitis, arthritis, or inflammatory bowel disease. The immunosuppressive action of glucocorticoids is thought to be mainly based on the inhibition of cytokine expression and dependent on the regulation of cytoplasmic glucocor...

BackgroundTumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking.MethodsWe investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells.ResultsKnock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed.ConclusionsOur data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable.