A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.     

Multipoint Binding of the SLP-76 SH2 Domain to ADAP Is Critical for Oligomerization of SLP-76 Signaling Complexes in Stimulated T Cells

The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155–7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling.     

T cell-dendritic cell immunological synapses contain TCR-dependent CD28-CD80 clusters that recruit protein kinase C theta

Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca(2+) signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs.     

Synergistic assembly of linker for activation of T cells signaling protein complexes in T cell plasma membrane domains

Transmembrane adaptor molecule LAT (linker for activation of T cells) forms a central scaffold for signaling protein complexes that accumulate in the vicinity of activated T cell antigen receptors (TCR). Here we used biochemical analysis of immunoisolated plasma membrane domains and fluorescence imaging of green fluorescence protein-tagged signaling proteins to investigate the contributions of different tyrosine-based signaling protein docking sites of LAT to the formation of LAT signaling protein assemblies in TCR membrane domains. We found that the phospholipase C gamma docking site of LAT and different Grb2/Gads docking sites function in an interdependent fashion and synergize to accumulate LAT, Grb2, and phospholipase C gamma in TCR signaling assemblies. Two-dimensional gels showed that Grb2 is a predominant cytoplasmic adaptor in the isolated LAT signaling complexes, whereas Gads, Crk-1, and Grap are present in lower amounts. Taken together our data suggest a synergistic assembly of multimolecular TCR.LAT signal transduction complexes in T cell plasma membrane domains.     

Vav1 acidic region tyrosine 174 is required for the formation of T cell receptor-induced microclusters and is essential in T cell development and activation

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.     

Expression of CD45 lacking the catalytic protein tyrosine phosphatase domain modulates Lck phosphorylation and T cell activation

The function of the second protein tyrosine phosphatase domain (D2) in two-domain protein tyrosine phosphatases (PTP) is not well understood. In CD45, D2 can interact with the catalytic domain (D1) and stabilize its activity. Although D2 itself has no detectable catalytic activity, it can bind substrate and may influence the substrate specificity of CD45. To further explore the function of D2 in T cells, a full-length construct of CD45 lacking the D1 catalytic domain (CD45RABC-D2) was expressed in CD45+ and CD45- Jurkat T cells. In CD45- Jurkat T cells, CD45RABC-D2 associated with Lck but, unlike its active counterpart CD45RABC, did not restore the induction of tyrosine phosphorylation or CD69 expression upon T cell receptor (TCR) stimulation. Expression of CD45RABC-D2 in CD45+ Jurkat T cells resulted in its association with Lck, increased the phosphorylation state of Lck, and reduced T cell activation. TCR-induced tyrosine phosphorylation was delayed, and although MAPK phosphorylation and CD69 expression were not significantly affected, the calcium signal and IL2 production were severely reduced. This indicates that the non-catalytic domains of CD45 can interact with Lck in T cells. CD45RABC-D2 acts as a dominant negative resulting in an increase in Lck phosphorylation and a preferential loss of the calcium signaling pathway, but not the MAPK pathway, upon TCR signaling. This finding suggests that, in addition to their established roles in the initiation of TCR signaling, CD45 and Lck may also influence the type of TCR signal generated.     

Cutting edge: A role for inside-out signaling in TCR regulation of CD28 ligand binding

Efficient T cell activation depends on the engagement of both TCR and CD28, although the molecular mechanisms that control this signal integration are not fully understood. Using fluorescence resonance energy transfer, we show that T cell activation can drive a reorientation of the cytosolic tails of the CD28 dimer. However, this is not mediated through CD28 ligand binding. Rather, TCR signaling itself mediates this conformation change in CD28. We also show that TCR signaling can induce CD28-ligand interactions. Although the CD28 dimer appears to bind ligand monovalently in solution, we show that both ligand binding sites are required to efficiently recruit CD28 to the immunological synapse. These results suggest, that analogous to the cross-talk from TCR that regulates integrin activation, TCR-initiated inside-out signaling may induce a conformational change to the extracellular domains of CD28, enabling ligand binding and initiating CD28 signaling.     

Coordination of Receptor Signaling in Multiple Hematopoietic Cell Lineages by the Adaptor Protein SLP-76

The adaptor protein SLP-76 is expressed in multiple hematopoietic lineages including T cells, platelets, and neutrophils. SLP-76 mediated signaling is dependent on its multiple protein interaction domains, as it creates a scaffold on which key signaling complexes are built. SLP-76 is critical for supporting signaling downstream of both immunoreceptors and integrins. The signaling molecules used both upstream and downstream of SLP-76 are similar among these receptors and across cell types; however, important differences exist. Appreciating how SLP-76 coordinates signal transduction across different cell and receptor types provides insights into the complex interplay of pathways critical for activation of cells of the immune system that are essential for host defense.Adaptor proteins are an important component of many signaling systems both within and beyond the immune system. Unlike enzymes that catalyze the chemical reactions required for signal propagation, adaptor proteins are molecular platforms on which other proteins assemble. Although lacking enzymatic function, adaptor proteins regulate signaling by stabilizing or restricting molecular interactions required for proper enzyme activation and for localizing these key effector molecules appropriately within the cell.Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is an adaptor present in a number of hematopoietic cell lineages including T cells, platelets, neutrophils, mast cells, macrophages, and NK cells. There are two SLP-76 homologs: SLP-65/B cell linker protein (BLNK), and cytokine-dependent hematopoietic cell linker (CLNK), also known as mast cell immunoreceptor signal transducer (MIST). BLNK is expressed in B cells and monocytes, and CLNK is expressed in mast cells, activated T cells, and NK cells. SLP-76 was first identified in T cells as a substrate of T-cell receptor (TCR) stimulated protein tyrosine kinases (PTKs) through “pull down” experiments using immobilized growth factor receptor-bound protein 2 (Grb2) (Jackman et al. 1995). Since then, SLP-76 has been shown to be critically important in the development of T cells and for propagating signals downstream of not only the TCR but also additional receptors present on hematopoietic cells.     

Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies

Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.     

The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling.     

Genetic analysis of SH2D4A, a novel adapter protein related to T cell-specific adapter and adapter protein in lymphocytes of unknown function, reveals a redundant function in T cells

T cell-specific adapter (TSAd) protein and adapter protein in lymphocytes of unknown function (ALX) are two related Src homology 2 (SH2) domain-containing signaling adapter molecules that have both been shown to regulate TCR signal transduction in T cells. TSAd is required for normal TCR-induced synthesis of IL-2 and other cytokines in T cells and acts at least in part by promoting activation of the LCK protein tyrosine kinase at the outset of the TCR signaling cascade. By contrast, ALX functions as a negative-regulator of TCR-induced IL-2 synthesis through as yet undetermined mechanisms. In this study, we report a novel T cell-expressed adapter protein named SH2D4A that contains an SH2 domain that is highly homologous to the TSAd protein and ALX SH2 domains and that shares other structural features with these adapters. To examine the function of SH2D4A in T cells we produced SH2D4A-deficient mice by homologous recombination in embryonic stem cells. T cell development, homeostasis, proliferation, and function were all found to be normal in these mice. Furthermore, knockdown of SH2D4A expression in human T cells did not impact upon their function. We conclude that in contrast to TSAd and ALX proteins, SH2D4A is dispensable for TCR signal transduction in T cells.     

The TCR zeta-chain immunoreceptor tyrosine-based activation motifs are sufficient for the activation and differentiation of primary T lymphocytes

The TCR complex signals through a set of 10 intracytoplasmic motifs, termed immunoreceptor tyrosine-based activation motifs (ITAMs), contained within the gamma-, delta-, epsilon-, and zeta-chains. The need for this number of ITAMs is uncertain. Limited and contradictory studies have examined the ability of subsets of the TCR's ITAMs to signal into postthymic primary T lymphocytes. To study signaling by a restricted set of ITAMs, we expressed in transgenic mice a chimeric construct containing the IAs class II MHC extracellular and transmembrane domains linked to the cytoplasmic domain of the TCR zeta-chain. Tyrosine phosphorylation and receptor cocapping studies indicate that this chimeric receptor signals T cells independently of the remainder of the TCR. We show that CD4+ and CD8+ primary T cells, as well as naive and memory T cells, are fully responsive to stimulation through the IAs-zeta receptor. Further, IAs-zeta stimulation can induce primary T cell differentiation into CTL, Th1, and Th2 type cells. These results show that the zeta-chain ITAMs, in the absence of the gamma, delta, and epsilon ITAMs, are sufficient for the activation and functional maturation of primary T lymphocytes. It also supports the isolated use of the zeta-chain ITAMs in the development of surrogate TCRs for therapeutic purposes.     

SLP-76 binding to p56lck: a role for SLP-76 in CD4-induced desensitization of the TCR/CD3 signaling complex.

Nonreceptor protein tyrosine kinases and associated substrates play a pivotal role in Ag receptor stimulation of resting cells and in the initiation of activation-induced cell death (AICD) of preactivated T cells. CD4-associated p56lck has been implicated not only in the activation of primary T cells, but also in the inhibition of T cell responses. We have previously shown that CD4+ T cell clones can be rescued from AICD when surface CD4 is engaged before the TCR stimulus. In this study, we show that prevention of AICD is associated with a CD4-dependent inhibition of TCR-triggered tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and Vav. We provide evidence for a SLP-76 interaction with Src homology 3 domains of p56lck and identify amino acids 185-194 of SLP-76 as relevant docking site. In view of the multiple functions of p56lck and SLP-76/Vav in the initiation of TCR/CD3/CD4 signaling, we propose a model for the CD4-dependent inhibition of TCR signaling and AICD of preactivated T cells. Our data suggest that preformed activation complexes of adapter proteins and enzymes in the vicinity of the CD4/p56lck complex are no longer available for the TCR signal when CD4 receptors are engaged before TCR stimulation.     

Immunological synapse formation licenses CD40-CD40L accumulations at T-APC contact sites

The maintenance of tolerance is likely to rely on the ability of a T cell to polarize surface molecules providing "help" to only specific APCs. The formation of a mature immunological synapse leads to concentration of the TCR at the APC interface. In this study, we show that the CD40-CD154 receptor-ligand pair is also highly concentrated into a central region of the synapse on mouse lymphocytes only after the formation of the TCR/CD3 c-SMAC. Concentration of this ligand was strictly dependent on TCR recognition, the binding of ICAM-1 to T cell integrins and the presence of an intact cytoskeleton in the T cells. This may provide a novel explanation for the specificity of T cell help directing the help signal to the site of Ag receptor signal. It may also serve as a site for these molecular aggregates to coassociate and/or internalize alongside other signaling receptors.     

Deficiency of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) causes enrichment of CD4+CD25+ regulatory T cells

A subpopulation of T cells, named regulatory T cells (T(reg) cells), has been shown to play a key role in tolerance and the prevention of autoimmunity. It is not known how changes in TCR signal strength during thymic T cell development affect the generation of a T(reg) population. In this study, we took two different strategies to modulate the TCR signal strength: an intrinsic approach, where signaling was enhanced by the loss of a negative regulator, and an extrinsic approach, where signaling strength was altered through variations in the concentrations of the selecting peptide. The tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a known negative regulator of TCR-mediated signaling. motheaten mice, lacking expression of SHP-1, showed a 2- to 3-fold increase in the percentage of CD4(+)CD25(+) T(reg) cells within the CD4(+) T cells. Similarly, the percentage of T(reg) cells was heightened in fetal thymic organ cultures (FTOCs) derived from motheaten mice compared with wild-type FTOCs, thus establishing the thymic origin of these T(reg) cells. Using FTOCs derived from DO11.10 TCR transgenic mice, we demonstrated that exposure to increasing concentrations of the cognate OVA peptide favored the appearance of T(reg) cells. Our data suggest that the development of CD4(+)CD25(+) T(reg) cells is intrinsically different from non-T(reg) cells and that T(reg) cells are selectively enriched under conditions of enhanced negative selection. Our data also reveal a key role for the SHP-1-mediated regulation of TCR signal strength in influencing the ratio of T(reg) vs non-T(reg) cells.     

Proteomic characterization of plasma membrane-proximal T cell activation responses

Early downstream responses of T lymphocytes following T cell antigen receptor (TCR) activation are mediated by protein complexes that assemble in domains of the plasma membrane. Using stable isotope labeling with amino acids in cell culture and mass spectrometry, we quantitatively related the proteome of αCD3 immunoisolated native TCR signaling plasma membrane domains to that of control plasma membrane fragments not engaged in TCR signaling. Proteins were sorted according to their relative enrichment in isolated TCR signaling plasma membrane domains, identifying a complex protein network that is anchored in the vicinity of activated TCR. These networks harbor widespread mediators of plasma membrane-proximal T cell activities, including propagation, balancing, and attenuation of TCR signaling, immune synapse formation, as well as cytoskeletal arrangements relative to TCR activation clusters. These results highlight the unique potential of systematic characterizations of plasma membrane-proximal T cell activation proteome in the context of its native lipid bilayer platform.     

Analysis of serial engagement and peptide-MHC transport in T cell receptor microclusters

In experiments where T cells interact with antigen-presenting-cells or supported bilayers bearing specific peptide-major-histocompatibility-complex (pMHC) molecules, T cell receptors (TCR) have been shown to form stable micrometer-scale clusters that travel from the periphery to the center of the contact region. pMHC molecules bind TCR on the opposing surface but the pMHC-TCR bond is weak and therefore pMHC can be expected to serially bind and unbind from TCR within the contact region. Using a novel mathematical analysis, we examine serial engagement of mobile clustered TCR by a single pMHC molecule. We determine the time a pMHC can be expected to remain within a TCR cluster. This also allows us to estimate the number of clustered TCR that are serially bound, and the distance a pMHC is transported by the clustered TCR. We find that TCR-pMHC binding alone does not allow substantial serial engagement of TCR and that the pMHC molecules are usually not transported to the center of the contact region by a single TCR cluster. We show that the presence of TCR coreceptors such as CD4 and CD8, or pMHC dimerization on the antigen-presenting cells, can substantially increase serial engagement and directed transport of pMHC. Finally, we analyze the effects of multiple TCR microclusters, showing that the size of individual clusters only weakly affects our prediction of TCR serial engagement by pMHC. Throughout, we draw parameter estimates from published data.