Henning Kirchgessner

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.

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.

Immunoreactivity of monoclonal anti-melanoma antibodies in relation to the amount of radioactive iodine substituted to the antibody molecule.

The damage to monoclonal anti-melanoma antibodies caused by iodination was investigated by comparing the results obtained using the chloramine-T method and the 1,3,4,6-tetrachloro-3α,6α-diphenyl-glycoluril (IODOGEN) method at different levels of iodine substitution to the molecule. The level of substitution at which losses in immunoreactivity occurred was evaluated in each monoclonal antibody (MAb) studied. This phenomenon was not dependent on the method of substitution, provided that mild conditions of reaction were used. Lineweaver-Burk plots and — in cases of alterations in binding affinity — Scatchard plots were found to provide an adequate description of the binding behaviour of individual MAbs after labelling. Immunoreactivity was shown to be determined not only by the proportion of bona fide reactive MAb molecules, but also by a substitution-dependent decrease in affinity constants. The practical consequences of altered binding parameters were demonstrated by quantitating specific antibody accumulation in melanoma transplants in vivo.

L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells.

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.

Metastasis of prostate cancer and melanoma cells in a preclinical in vivo mouse model is enhanced by L-plastin expression and phosphorylation

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.

Molecular alterations of the Fyn-complex occur as late events of human T cell activation.

Two‐dimensional gel electrophoresis of anti‐p59fyn immunoprecipitates obtained from non‐transformed resting human T lymphocytes resulted in the identification of an oligomeric protein complex which is constitutively formed between Fyn and several additional phosphoproteins (pp43, pp72, pp85, the protein tyrosine kinase Pyk2, as well as the two recently cloned adaptor proteins, SKAP55 and SLAP‐130). With the exception of pp85, these proteins seem to preferentially interact with Fyn since they are not detectable in Lck immunoprecipitates prepared under the same experimental conditions. Among the individual members of the Fyn‐complex pp85, SKAP55 and pp43 are constitutively phosphorylated on tyrosine residue(s) in vivo and likely interact with Fyn via its src homology 2 (SH2)‐domain. In contrast to non‐transformed T lymphocytes, continuously proliferating transformed human T cell lines express an altered Fyn‐complex. Thus, despite normal expression and tyrosine phosphorylation, SKAP55 does not associate with Fyn in Jurkat cells and in other human T cell lines. Instead two novel proteins interact with Fyn among which one has previously been identified as α‐tubulin. Importantly, almost identical alterations of the Fyn‐complex as observed in Jurkat cells are induced in non‐transformed T lymphocytes following mitogenic stimulation. These data suggest that Fyn and its associated proteins could be involved in the control of human T cell proliferation. Moreover, the analogous constitutive alterations in transformed T cell lines could indicate that deregulation of the Fyn‐complex might be functionally associated with the malignant phenotype of these cells.

Ras/PI3kinase/cofilin-independent activation of human CD45RA+ and CD45RO+ T cells by superagonistic CD28 stimulation.

T cell activation requires costimulation of TCR/CD3 plus accessory receptors (e.g. CD28). A hallmark of costimulation is the dynamic reorganization of the actin cytoskeleton, important for receptor polarization in the immunological synapse. The classical model of T cell costimulation was challenged by the detection of superagonistic anti‐CD28 antibodies. These induce T cell proliferation and – as demonstrated here – production of IFN‐γ, CD25 and CD69 even in the absence of TCR/CD3 coligation. Here, we analyzed whether superagonistic CD28 stimulation induces costimulatory signaling events. Costimulation leads to phosphorylation of the actin‐bundling protein L‐plastin and dephosphorylation of the actin‐reorganizing protein cofilin. Cofilin binds to F‐actin only in its dephosphorylated form. Binding of cofilin to F‐actin leads to depolymerization or severing of F‐actin. The latter ends up in smaller F‐actin fragments, which can be elongated at the free barbed ends. This results in enhanced actin polymerization. Dephosphorylation of cofilin requires activation of Ras and PI3Kinase. Interestingly, superagonistic CD28 stimulation activates human peripheral blood T cells independently of Ras and PI3Kinase. Accordingly, it does not lead to cofilin dephosphorylation and receptor polarization. Likewise, L‐plastin is not phosphorylated. Thus, superagonistic CD28 stimulation does not mimic costimulation. Instead, it leads to a Ras/PI3Kinase/cofilin‐independent state of “unpolarized T cell activation”.

A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP2) Inhibition

Background: Cofilin is a key molecule for actin dynamics whose activity can be locally inhibited by PIP2. Results: Changes in the cofilin structure upon reduction render cofilin insensitive to PIP2 inhibition. Conclusion: Local effects of PIP2 on cofilin activity are determined by the redox microenvironment. Significance: We discovered a mechanism of spatio-microenvironmental control of actin dynamics by cofilin reduction at the plasma membrane. Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.

InFlow microscopy of human leukocytes: A tool for quantitative analysis of actin rearrangements in the immune synapse

The actin cytoskeleton is a main component to preserve the cell shape. It represents a cellular machinery that enables morphological changes and orchestrates important dynamic cellular functions. Thereby, it supports T-cell migration, immune synapse formation, activation and execution of effector functions. The analysis of actin rearrangements in T-cells is therefore an important field of basic and clinical research. Actin reorganization is traditionally performed using flow cytometry or confocal microscopy. However, while flow cytometry lacks spatial and structural information, confocal microscopy is time consuming and not feasible for the characterization of rare events or of un-purified primary cell populations. Here we describe a methodology to analyze actin rearrangements using InFlow microscopy, which is a hybrid technique consisting of flow cytometric and microscopic features. We show that InFlow microscopy is a valuable tool for quantification of the amount and distribution of F-actin in human T-cells after stimulation with chemokines or antigen-presenting cells.

The pro-oxidative drug WF-10 inhibits serial killing by primary human cytotoxic T-cells

Cytotoxic T-cells (CTLs) play an important role in many immune-mediated inflammatory diseases. Targeting cytotoxicity of CTLs would allow to interfere with immune-mediated tissue destruction. Here we demonstrate that WF-10, a pro-oxidative compound, inhibits CTL-mediated cytotoxicity. WF-10 did not influence early steps of target-cell killing, but impaired the ability of CTLs to detach from the initial target cell and to move to a second target cell. This reduced serial killing was accompanied by stronger enrichment of the adhesion molecule LFA-1 in the cytolytic immune synapse. LFA-1 clustering requires activation of the actin-bundling protein L-plastin and was accordingly diminished in L-plastin knockdown cells. Interestingly, WF-10 likely acts through regulating L-plastin: (I) It induced L-plastin activation through phosphorylation leading to enhanced LFA-1-mediated cell adhesion, and, importantly, (II) WF-10 lost its influence on target-cell killing in L-plastin knockdown cells. Finally, we demonstrate that WF-10 can improve immunosuppression by conventional drugs. Thus, while cyclosporine A alone had no significant effect on cytotoxicity of CTLs, a combination of cyclosporine A and WF-10 blocked target-cell killing synergistically. Together, our findings suggest that WF-10 – either alone or in combination with conventional immunosuppressive drugs – may be efficient to control progression of diseases, in which CTLs are crucially involved.