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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.

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.

SummaryOxidative 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.

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca(2+) mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

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 alpha-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.

The molecular mechanisms regulating recruitment of intracellular signaling proteins like growth factor receptor–bound protein 2 (Grb2), phospholipase Cγ1, or phosphatidylinositol 3-kinase (PI3-kinase) to the plasma membrane after stimulation of the T cell receptor (TCR)– CD3–ζ complex are not very well understood. We describe here purification, tandem mass spectrometry sequencing, molecular cloning, and biochemical characterization of a novel transmembrane adaptor protein which associates and comodulates with the TCR–CD3–ζ complex in human T lymphocytes and T cell lines. This protein was termed T cell receptor interacting molecule (TRIM). TRIM is a disulfide-linked homodimer which is comprised of a short extracellular domain of 8 amino acids, a 19–amino acid transmembrane region, and a 159–amino acid cytoplasmic tail. In its intracellular domain, TRIM contains several tyrosine-based signaling motifs that could be involved in SH2 domain–mediated protein–protein interactions. Indeed, after T cell activation, TRIM becomes rapidly phosphorylated on tyrosine residues and then associates with the 85-kD regulatory subunit of PI3-kinase via an YxxM motif. Thus, TRIM represents a TCR-associated transmembrane adaptor protein which is likely involved in targeting of intracellular signaling proteins to the plasma membrane after triggering of the TCR.

In human and mouse lymphocytes the protein tyrosine phosphatase CD45, a key molecule involved in T cell activation, non-covalently associates with the tyrosine kinase p56(lck) and lymphocyte phosphatase-associated phosphoprotein (LPAP), a 32 kDa phosphoprotein of unknown function. In order to gain insight into the function of LPAP we have generated an LPAP-deficient Jurkat variant by means of antisense strategies. Analysis of the CD45-p56(lck) molecular complex in this cell line revealed that loss of LPAP does not alter the expression or the enzymatic activity of CD45 or p56(lck). In addition, the association between CD45 and p56(lck) is not affected in LPAP-deficient T cells. These data suggest that LPAP does not regulate the enzymatic activity of CD45 or p56(lck) and is not required for the association between these two proteins. In order to identify polypeptides that preferentially associate with LPAP we established a Jurkat variant expressing a chimeric receptor which was composed of the extracellular portion of the human HLA-A2.1 molecule and the full-length LPAP protein. Comparative two-dimensional analysis of CD45 and HLA-A2 immunoprecipitates obtained from these cells following metabolic labeling resulted in the identification of a 43 kDa protein that preferentially associates with LPAP under mild detergent conditions.

Two-dimensional gel electrophoresis of in vitro phosphorylated proteins coprecipitated by CD2 monoclonal antibody (mAb) from Brij58 lysates of resting human T lymphocytes and natural killer (NK) cells resulted in the identification of a novel 29/30-kD disulfide-linked dimer (pp29/30). Comparative two-dimensional analysis of CD2, CD3, CD4, CD5, and CD8 immunoprecipitates revealed that pp29/30 associates with these signaling receptor complexes but not with CD18, CD27, and CD29 in human T lymphocytes. Analysis of CD2 immunoprecipitates prepared from T cell antigen receptor/CD3-modulated T lymphocytes indicated that pp29/30 preferentially associates and comodulates with the human T cell antigen receptor (TCR). Since tyrosine phosphorylated pp29/30 selectively interacts with the Src homology type 2 domains (SHZ) of the protein tyrosine kinases p56lck and p59fyn but not ZAP70 the present data suggest that pp29/30 represents a novel signaling receptor associated phosphoprotein likely involved in the activation of human T lymphocytes and NK cells.

CD2-associated molecules were identified by means of in vitro kinase assays of CD2 immunoprecipitates obtained from nontransformed human T lymphocytes lysed in the detergent brij 58. Under these conditions CD2 was found to be associated with the protein tyrosine kinases p56lck and p59fyn as well as two low molecular weight phosphoproteins. The latter were identified as the zeta and epsilon chains, the major signaling components of the CD/7 TcR complex. Importantly, induction of T cell unresponsiveness towards CD2-mediated stimuli by means of CD3/T cell receptor (TcR) modulation results in uncoupling of zeta and epsilon from the CD2 molecule, while its associations with p56lck and p59fyn remain unaffected. Moreover, despite the incapacity of T lymphocytes to undergo DNA synthesis in the CD3/TcR-modulated state, CD2 triggering still results in tyrosine phosphorylation of some unknown protein substrates. Thus, the same zeta and epsilon chains which are components of a functional TcR complex appear to also couple to the CD2 molecular complex. Moreover, dissociation of TcR and CD2 complexes in intact cells seems to block CD2-mediated T cell growth but does not result in complete abolishment of the signal transducing capacity of the CD2 receptor.

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.

In vitro protein kinase assays of CD45 immunoprecipitates prepared from digitonin lysates of resting human T lymphocytes resulted in exclusive tyrosine phosphorylation of a 32-kDa protein (pp32). Reprecipitation of the in vitro phosphorylated proteins and Western blot analysis of whole CD45 immunoprecipitates employing antisera specifically directed at different protein tyrosine kinases demonstrated that the p56lck protein tyrosine kinase was responsible for in vitro phosphorylation of pp32. Since in vitro kinase assays of p56lck immunoprecipitates also resulted in phosphorylation of pp32, the present data strongly suggest that a functional complex is formed between CD45, p56lck and pp32. Such a notion is supported by the findings that phosphorylation of pp32 by p56lck correlated with expression of the CD45 molecules and that in vitro phosphorylated pp32 was completely dephosphorylated by purified CD45.

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-gamma, 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".

Adhesion of leukocytes is an early step in the formation of adaptive or innate immunity. In chronic inflammatory pathologies like atherosclerosis, regulation of adhesiveness is pivotal for the accumulation of leukocytes within the vessel wall. Therefore, the quantification of adhesion is crucial for the understanding and monitoring of immune responses in patients. However, so far, functional analysis of leukocyte adhesion has been time consuming and required prior purification of cell populations from peripheral blood. This reduced the number of samples and cell populations that could be analysed from limited patient material. Here, we introduce a novel method involving rapid quantification of integrin-mediated leukocyte adhesion in human whole blood using flow cytometry. The quantification relies on soluble multivalent immunocomplexes and is thus called “ligand-complex-based adhesion assay” (LC-AA). LC-AA evaluates both integrin affinity and avidity in T-cells, NK-cells and monocytes from as little as 20 μl of whole blood. In marked contrast to T-cells and NK-cells, unstimulated monocytes show non-blockable background binding of the complexes. Therefore, for this subset only, the stimulation-induced integrin activation is measurable. With the LC-AA, for the first time, measurement of adhesiveness of extremely rare cell populations like CD34+ peripheral blood stem cells can be assessed in the absence of prior purification steps. Finally, the small blood volumes needed for adhesion analysis with the LC-AA allow the evaluation of multiple cell subpopulations in large sample collectives, e.g. required in clinical studies.

T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain–containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain–containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor– and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2.

A glutathione-S-transferase-src-homology domain 2 (GST-SH2) fusion protein was employed to identify molecules interacting with the protein tyrosine kinase p59fyn. Among several proteins which bound to the fyn SH2 domain in lysates of human Jurkat T lymphocytes, alpha- and beta-tubulin were identified by N-terminal sequencing. Further analysis established that alpha-tubulin exists as a tyrosine-phosphorylated protein in Jurkat cells, where it interacts with p59fyn, but not with p56lck. By contrast, in untransformed resting human T lymphocytes alpha-tubulin is not detectable as a tyrosine phosphorylated protein. However, following T cell activation, it becomes rapidly phosphorylated on tyrosine residues and subsequently associates with the SH2 domain of fyn. Interestingly, constitutively tyrosine-phosphorylated alpha-tubulin that is able to interact with the fyn-SH2 domain is expressed in peripheral blood T lymphoblasts isolated from leukemic patients in the absence of external stimulation.

In human T lymphocytes a functional complex is formed between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and a phospho-protein, pp32, a possible common substrate. Here we demonstrate that the previously described pp32 protein is composed of two distinct molecules (pp29 and pp32) in both resting human T lymphocytes and continuously proliferating T lymphoma lines. Importantly, T lymphocyte activation employing CD2 monoclonal antibodies (mAb), CD3 mAb or phorbol 12, 13 dibutyrate results in loss of pp29 and pp32 from the CD45/p56lck molecular complex and concomitant association of two distinct phosphoproteins with different molecular weights (pp30 and pp31). These events appear to be unrelated to clonal T cell growth but rather depend on receptor-mediated differentiation signals. Reprecipitation experiments employing an antiserum directed at a consensus sequence of GTP-binding proteins suggest that all four pp29-pp32 molecules might represent proteins with GTP-binding properties. Biochemical analysis of pp29-pp32 employing V8-protease digestion indicates that they differ in low-molecular weight fragments of 8, 5, 4.5, 4 and 3 kDa, respectively.