Frédérique Michel

CD28-mediated co-stimulation: a quantitative support for TCR signalling

The ability of naive T cells to clonally expand and acquire effector functions depends on the strength of signals received by the T-cell receptor (TCR) and by an array of co-stimulatory receptors — the most prominent of which is CD28. In this review, we discuss recent genetic, biochemical and biophysical data that indicate a modified view of the molecular mechanism by which ligation of CD28 amplifies TCR-mediated T-cell activation. These studies indicate that the commonly held notion of a qualitative signalling role of CD28 in T-cell activation should be revised.

The Membrane-Microfilament Linker Ezrin Is Involved in the Formation of the Immunological Synapse and in T Cell Activation

Dynamic interactions between membrane and cytoskeleton components are crucial for T cell antigen recognition and subsequent cellular activation. We report here that the membrane-microfilament linker ezrin plays an important role in these processes. First, ezrin relocalizes to the contact area between T cells and stimulatory antigen-presenting cells (APCs), accumulating in F-actin-rich membrane protrusions at the periphery of the immunological synapse. Second, T cell receptor (TCR)-mediated intracellular signals are sufficient to induce ezrin relocalization, indicating that this protein is an effector of TCR signaling. Third, overexpression of the membrane binding domain of ezrin perturbs T cell receptor clustering in the T cell-APC contact area and inhibits the activation of nuclear factor for activated T cells (NF-AT).

Tailoring T-cell receptor signals by proximal negative feedback mechanisms.

The T-cell receptor (TCR) signalling machinery is central in determining the response of a T cell (establishing immunity or tolerance) following exposure to antigen. This process is made difficult by the narrow margin of self and non-self discrimination, and by the complexity of the genetic programmes that are induced for each outcome. Recent studies have identified novel negative feedback mechanisms that are rapidly induced by TCR engagement and that have key roles in the regulation of signal triggering and propagation. In vitro and in vivo data suggest that they are important in determining ligand discrimination by the TCR and in regulating signal output in response to antigen.

CD28 as a molecular amplifier extending TCR ligation and signaling capabilities.

Evidence has gathered that CD28 costimulation facilitates T cell activation by potentiating TCR intrinsic-signaling. However, the underlying molecular mechanism is largely unknown. Here we show that, by enhancing T cell/APC close contacts, CD28 facilitates TCR signal transduction. Moreover, the signal supplied by CD28 does not lead to increased Zap-70 and Lat phosphorylation, but amplifies PLCgamma1 activation and Ca(2+) response. We provide evidence that the PTK Itk controls the latter function. Our data suggest that CD28 binding to B7 contributes to setting the level of TCR-induced phosphorylated Lat for recruiting signaling complexes, whereas the CD28 signal boosts multiple pathways by facilitating PLCgamma1 activation. These results should provide a conceptual framework for understanding quantitative and qualitative aspects of CD28-mediated costimulation.

CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling

The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR ζ-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.

Tyrosine 319, a Newly Identified Phosphorylation Site of ZAP-70, Plays a Critical Role in T Cell Antigen Receptor Signaling

Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivocapacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.

T cell receptor for antigen induces linker for activation of T cell–dependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2

Adaptor proteins positively or negatively regulate the T cell receptor for antigen (TCR) signaling cascade. We report that after TCR stimulation, the inhibitory adaptor downstream of kinase (Dok)-2 and its homologue Dok-1 are involved in a multimolecular complex including the lipid phosphatase Src homology 2 domain–containing inositol polyphosphate 5′-phosphatase (SHIP)-1 and Grb-2 which interacts with the membrane signaling scaffold linker for activation of T cells (LAT). Knockdown of LAT and SHIP-1 expression indicated that SHIP-1 favored recruitment of Dok-2 to LAT. Knockdown of Dok-2 and Dok-1 revealed their negative control on Akt and, unexpectedly, on Zap-70 activation. Our findings support the view that Dok-1 and -2 are critical elements of a LAT-dependent negative feedback loop that attenuates early TCR signal. Dok-1 and -2 may therefore exert a critical role in shaping the immune response and as gatekeepers for T cell tolerance.

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.

Molecular modifiers of T cell antigen receptor triggering threshold: the mechanism of CD28 costimulatory receptor

CD28 was thought to represent a prototypic membrane receptor responsible for delivering the classically defined ‘second signal’ needed to avoid T cell paralysis when recognizing antigen presented by appropriate antigen presenting cells (APCs). Almost two decades after its molecular identification, the mechanism by which this ‘second receptor’ facilitates clonal expansion and differentiation upon antigen encounter is still not fully elucidated. There may be at least two reasons for this partially gray picture: the use of nonphysiological experimental conditions to study it and the fact that the action of CD28 may be partly masked by the presence of additional T cell surface receptors that also provide some costimulatory signals, although not equivalent to the one delivered through CD28. Thus, instead of aging, the study of CD28 is still a topical subject. What is appearing through work of recent years is that far from being purely qualitative, the CD28 signal provides a key quantitative contribution to potently boost the T cell antigen receptor (TCR) signal. In other words, CD28 is in part a signaling ‘sosia’ of the TCR. Also, it is clear now that CD28 operates via multiple molecular effects. Still, what we do not understand is the ‘qualitative’ part of this signal, perhaps due to lack of identification of unique signaling components and/or pathways activated by CD28 only. Here we review a series of recent findings pointing towards novel avenues to better understand the molecular basis of CD28 function.