Oreste Acuto

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.

Constitutively Active Lck Kinase in T Cells Drives Antigen Receptor Signal Transduction

Summary T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to ∼40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-ζ phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.

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.

Tyrosine 319 in the Interdomain B of ZAP-70 Is a Binding Site for the Src Homology 2 Domain of Lck

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to 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.

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.