Loretta Tuosto

Organization of plasma membrane functional rafts upon T cell activation.

Raft microdomains have been shown to play a key role in T cell activation. We found that in human T lymphocytes the formation of functional rafts at the plasma membrane was induced by T cell priming. In resting T cells from peripheral blood Lck and the raft glycosphingolipid GM1 resided in intracellular membranes. T cell activation induced synthesis of GM1 and effector cells showed very high levels of this lipid, which became predominantly plasma membrane associated. TCR triggering also induced targeting of the cytosolic Lck to the plasma membrane. Thus, effector cells acquire an improved signaling machinery by increasing the amount of rafts at the plasma membrane. The fact that, when compared with naive T cells, memory T cells showed higher GM1 levels suggests that raft lipid synthesis may be developmentally regulated and tune T cell responsiveness.

CD28 and Lipid Rafts Coordinate Recruitment of Lck to the Immunological Synapse of Human T Lymphocytes

In T lymphocytes, the Src family kinase Lck associates lipid rafts and accumulates at the immunological synapse (IS) during T cell stimulation by APCs. Using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS depends on CD4, whereas CD28 sustains Lck activation. However, in human resting T cells, CD28 is responsible for promoting recruitment of lipid rafts to the IS by an unknown mechanism. Thus, we performed a series of experiments to determine 1) whether Lck is recruited to the IS through lipid rafts; and 2) whether Lck recruitment to the IS of human resting T cells depends on CD4 or on CD28 engagement. We found that CD28, but not CD4, stimulation induced recruitment of Lck into detergent-resistant domains as well as its accumulation at the IS. We also found that Lck recruitment to the IS depends on the CD28 COOH-terminal PxxPP motif. Thus, the CD28-3A mutant, generated by substituting the prolines in positions 208, 211, and 212 with alanines, failed to induce Lck and lipid raft accumulation at the synapse. These results indicate that CD28 signaling orchestrates both Lck and lipid raft recruitment to the IS to amplify T cell activation.

CD28 affects the earliest signaling events generated by TCR engagement

The efficiency and magnitude of T cell responses are influenced by ligation of the co‐stimulatory receptor CD28 by B7 molecules expressed on antigen‐presenting cells (APC). In contrast to most previous studies in which agonistic anti‐TCR/CD3 and anti‐CD28 antibodies were employed, here we have investigated the contribution of CD28 to T cell activation under physiological conditions of antigen presentation. Jurkat T cells and primary T cells from TCR‐transgenic mice stimulated with superantigen and antigen, respectively, presented by B7‐expressing APC were utilized. In both systems we show that inhibiting CD28/B7 interaction resulted in impaired TCR‐induced tyrosine phosphorylation of the signal‐transducing ζ chain and ZAP‐70. Consistent with a blockade of TCR‐proximal signaling events, Jurkat cells stimulated in the absence of CD28 ligation were found to have strongly diminished tyrosine phosphorylation of cellular substrates and downstream signaling pathways such as Ca2+ /calcineurin, ERK/MAPK and JNK. Our results provide evidence for a role of CD28 in enhancing TCR signaling capacity during the earliest stages of T cell  :  APC interaction.

Fyn and ZAP-70 Are Required for Vav Phosphorylation in T Cells Stimulated by Antigen-presenting Cells

In T cells, triggering of the T cell antigen receptor or of the co-stimulatory receptor CD28 can direct tyrosine phosphorylation of the signaling protein Vav. We investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in these processes in a T cell hybridoma after physiological stimulation of the T cell receptor (TCR) and CD28. A dominant-negative mutant approach based on overexpression of catalytically inactive alleles of these kinases showed that CD28-induced Vav phosphorylation preferentially requires Fyn, whereas ZAP-70 had no role. Consistently, Vav was strongly phosphorylated in Lck-deficient JCAM-1 cells after CD28 ligation. In contrast, ZAP-70 appeared to control TCR-directed Vav phosphorylation. However, overexpression of ZAP-70 carrying a mutated Tyr315, contained within a motif previously suggested to be a Vav Src homology 2 domain binding site, had little or no effect. Immunoprecipitation assays showed that phosphorylated Vav associated with Fyn after CD28 triggering and that this interaction, likely to involve binding of Fyn Src homology 2 domain to Vav, was more strongly detectable after concomitant CD28 and TCR stimulation. These data suggest that Fyn plays a major role in controlling Vav phosphorylation upon T cell activation and that the mechanism implicating ZAP-70 in this process may be more complex than previously anticipated.

CD28 Utilizes Vav-1 to Enhance TCR-Proximal Signaling and NF-AT Activation

The mechanism through which CD28 costimulation potentiates TCR-driven gene expression is still not clearly defined. Vav-1, an exchange factor for Rho GTPases thought to regulate, mainly through Rac-1, various signaling components leading to cytokine gene expression, is tyrosine phosphorylated upon CD28 engagement. Here, we provide evidence for a key role of Vav-1 in CD28-mediated signaling. Overexpression of Vav-1 in Jurkat cells in combination with CD28 ligation strongly reduced the concentration of staphylococcus enterotoxin E/MHC required for TCR-induced NF-AT activation. Surprisingly, upon Vav-1 overexpression CD28 ligation sufficed to activate NF-AT in the absence of TCR engagement. This effect was not mediated by overexpression of ZAP-70 nor of SLP-76 but necessitated the intracellular tail of CD28, the intactness of the TCR-proximal signaling cascade, the Src-homology domain 2 (SH2) domain of Vav-1, and SLP-76 phosphorylation, an event which was favored by Vav-1 itself. Cells overexpressing Vav-1 formed lamellipodia and microspikes reminiscent of Rac-1 and Cdc42 activation, respectively, for which the SH2 domain of Vav-1 was dispensable. Together, these data suggest that CD28 engagement activates Vav-1 to boost TCR signals through a synergistic cooperation between Vav-1 and SLP-76 and probably via cortical actin changes to facilitate the organization of a signaling zone.

Vav cooperates with CD28 to induce NF-κB activation via a pathway involving Rac-1 and mitogen-activated kinase kinase 1

CD28‐delivered costimulatory signals are required to induce NF‐κB activation in response to TCR stimulation. We have recently demonstrated that the mitogen‐activated kinase kinase 1 (MEKK1), a kinase known to regulate the c‐jun N‐terminal kinase (JNK) pathway, is also involved in the CD28‐ and TCR‐induced inhibitor of κB factor (IκB) kinases (IKK) and NF‐κB activation. Searching for molecules that couple TCR and CD28 to MEKK1, we found that the guanine nucleotide exchange factor Vav synergized with CD28 stimulation in Jurkat cells to induce NF‐κB transcriptional activity through the activation of IKKα and IKKβ. Dominant negative mutants of Vav inhibited TCR‐ and CD28‐NF‐κB‐dependent transcription by interfering with the activation of the IKK complex. Blocking Rac signaling downstream of Vav by dominant negative RacN17 exerts similar effects on IKK and NF‐κB activation after TCR/CD28 stimulation. Finally, Vav‐induced NF‐κB activation in CD28 costimulated cells was inhibited by dominant negative MEKK(KM). These results identify Vav, Rac‐1 and MEKK1 as components of a common pathway regulating both NF‐κB and AP‐1 that contributes to full activation of the CD28 response element (CD28RE).

Mutation of Tyrosines 492/493 in the Kinase Domain of ZAP-70 Affects Multiple T-cell Receptor Signaling Pathways

The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56lck, in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR ζ chain and was able to bind p56lck. This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56lck with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56lck, which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation.

Transglutaminase Type II Is Involved in the Pathogenesis of Endotoxic Shock

The pathogenesis of sepsis is characterized by the inability of the host to regulate the inflammatory response, and as a consequence, dysregulated inflammatory processes induce organ dysfunctions and death. Altered transglutaminase type II (TG2) expression is associated with the development of many inflammatory diseases. Therefore, in this study, we questioned whether TG2 could also contribute to the pathological inflammatory dysregulation occurring in septic shock in vivo. To this aim, we used as an experimental model the TG2 knockout mice, in which the process of septic shock was elicited by treatment with LPS. Interestingly, our results demonstrated that TG2 ablation leads to partial resistance to experimental sepsis. The increased survival of TG2−/− mice was reflected in a drastic reduction of organ injury, highlighted by a limited infiltration of neutrophils in kidney and peritoneum and by a better homeostasis of the proinflammatory mediators as well as mitochondrial function. We also showed that in wild-type mice, the TG2 expression is increased during endotoxemia and, being directly involved in the mechanisms of NF-κB activation, it may cause a continuous activation cycle in the inflammatory process, thus contributing to development of sepsis pathogenesis. We propose that the inhibition of TG2 could represent a novel approach in the treatment of inflammatory processes associated with sepsis.

Trichostatin A up-regulates p73 and induces Bax-dependent apoptosis in cisplatin-resistant ovarian cancer cells

Several studies in the last years evidenced that deregulation of proapoptotic and antiapoptotic pathways are key players in the onset and maintenance of chemoresistance in advanced ovarian cancers. To characterize the signaling events and molecules involved in the acquisition of cisplatin resistance, we used the human ovarian cancer cell line A2780 and its derivative cisplatin-resistant subline A2780 CIS. We found that the mitochondrial intrinsic apoptotic pathway, induced by cis-dichlorodiammineplatinum (CDDP) in A2780 wild-type cells, was compromised in the resistant subline CIS. The analysis of expression of proteins involved in mitochondria-dependent apoptosis revealed a role of Bax and p73 but not p53. Indeed, we found that CDDP treatment induced the up-regulation of p53 in both sensitive and resistant A2780 cell lines. By contrast, p73 and Bax expressions were compromised in resistant cells. Pretreatment of resistant A2780 CIS cells with the histone deacetylase inhibitor trichostatin A overcomes apoptosis resistance to CDDP by restoring both p73 and Bax but not p53 expression. Altogether, these data indicate that p73, but not p53, is involved in the regulation of apoptosis susceptibility to cisplatin in A2780 ovarian cancer cells and evidence a key contribution of histone deacetylase activation in the acquisition of chemotherapy resistance in human ovarian cancer cells. [Mol Cancer Ther 2008;7(6):1410–9]

Vav-1 and the IKKα subunit of IκB kinase functionally associate to induce NF-κB activation in response to CD28 engagement

We have recently observed that CD28 engagement initiates a signaling pathway leading to the activation of IκB kinase (IKK) complex and, consequently, to NF-κB activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKKα in both Jurkat and primary CD4+ T cells. Vav-1/IKKα association is mediated by their helix-loop-helix domains, does not involve IKKβ, and is functionally relevant in that Vav-1-associated IKKα kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-κB activation is augmented by both IKKα and Vav-1, but not IKKβ. Confocal microscopy showed that endogenous Vav-1 and IKKα, but not IKKβ, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-κB pathway by targeting IKKα in the T cell membrane and favoring its activation in response to CD28 stimulation.