Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.     

The Src-homology domain 2-bearing protein tyrosine phosphatase-1 inhibits antigen receptor-induced apoptosis of activated peripheral T cells.

Restimulation of Ag receptors on peripheral T lymphocytes induces tyrosine phosphorylation-based signaling cascades that evoke Fas ligand expression and induction of Fas-mediated programmed cell death. In view of the role for the Src homology domain 2-bearing protein tyrosine phosphatase-1 (SHP-1) in modulating TCR signaling, we investigated the influence of SHP-1 on TCR-mediated apoptosis by assaying the sensitivity of peripheral T cells from SHP-1-deficient viable motheaten (mev) mice to cell death following TCR restimulation. The results of these studies revealed mev peripheral T cells to be markedly more sensitive than wild-type cells to induction of cell death following TCR stimulation. By contrast, PMA/ionophore and anti-Fas Ab-induced apoptotic responses were no different in mev compared with wild-type activated cells. Enhanced apoptosis of TCR-restimulated mev lymphocytes was associated with marked increases in Fas ligand expression as compared with wild-type cells, but was almost abrogated in both mev and wild-type cells by Fas-Fc treatment. Thus, the increased sensitivity of mev T cells to apoptosis following TCR restimulation appears to reflect a TCR-driven phenomenon mediated through up-regulation of Fas-Fas ligand interaction and induction of the Fas signaling cascade. These findings, together with the hyperproliferative responses of mev peripheral T cells to initial TCR stimulation, indicate that SHP-1 modulation of TCR signaling translates to the inhibition of both T cell proliferation and activation and, as such, is likely to play a pivotal role in regulating the expansion of Ag-stimulated T cells during an immune response.     

A response calculus for immobilized T cell receptor ligands

To address the molecular mechanism of T cell receptor (TCR) signaling, we have formulated a model for T cell activation, termed the 2D-affinity model, in which the density of TCR on the T cell surface, the density of ligand on the presenting surface, and their corresponding two-dimensional affinity determine the level of T cell activation. When fitted to T cell responses against purified ligands immobilized on plastic surfaces, the 2D-affinity model adequately simulated changes in cellular activation as a result of varying ligand affinity and ligand density. These observations further demonstrated the importance of receptor cross-linking density in determining TCR signaling. Moreover, it was found that the functional two-dimensional affinity of TCR ligands was affected by the chemical composition of the ligand-presenting surface. This makes it possible that cell-bound TCR ligands, despite their low affinity in solution, are of optimal two-dimensional affinity thereby allowing effective TCR binding under physiological conditions, i.e. at low ligand densities in cellular interfaces.     

Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection.

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.     

Stochastic effects and bistability in T cell receptor signaling

The stochastic dynamics of T cell receptor (TCR) signaling are studied using a mathematical model intended to capture kinetic proofreading (sensitivity to ligand-receptor binding kinetics) and negative and positive feedback regulation mediated, respectively, by the phosphatase SHP1 and the MAP kinase ERK. The model incorporates protein-protein interactions involved in initiating TCR-mediated cellular responses and reproduces several experimental observations about the behavior of TCR signaling, including robust responses to as few as a handful of ligands (agonist peptide-MHC complexes on an antigen-presenting cell), distinct responses to ligands that bind TCR with different lifetimes, and antagonism. Analysis of the model indicates that TCR signaling dynamics are marked by significant stochastic fluctuations and bistability, which is caused by the competition between the positive and negative feedbacks. Stochastic fluctuations are such that single-cell trajectories differ qualitatively from the trajectory predicted in the deterministic approximation of the dynamics. Because of bistability, the average of single-cell trajectories differs markedly from the deterministic trajectory. Bistability combined with stochastic fluctuations allows for switch-like responses to signals, which may aid T cells in making committed cell-fate decisions.     

CD22 is a functional ligand for SH2 domain-containing protein-tyrosine phosphatase-1 in primary T cells

The intracellular Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (SHP-1) has been characterized as a negative regulator of T cell function, contributing to the definition of T cell receptor signaling thresholds in developing and peripheral mouse T lymphocytes. The activation of SHP-1 is achieved through the engagement of its tandem SH2 domains by tyrosine-phosphorylated proteins; however, the identity of the activating ligand(s) for SHP-1, within mouse primary T cells, is presently unresolved. The identification of SHP-1 ligand(s) in primary T cells would provide crucial insight into the molecular mechanisms by which SHP-1 contributes to in vivo thresholds for T cell activation. Here we present a combination of biochemical and yeast genetic analyses indicating CD22 to be a T cell ligand for the SHP-1 SH2 domains. Based on these observations we have confirmed that CD22 is indeed expressed on mouse primary T cells and capable of associating with SHP-1. Significantly, CD22-deficient T cells demonstrate enhanced proliferation in response to anti-CD3 or allogeneic stimulation. Furthermore, the co-engagement of CD3 and CD22 results in a raising of TCR signaling thresholds hence demonstrating a previously unsuspected functional role for CD22 in primary T cells.     

PD-L1 signaling on human memory CD4+ T cells induces a regulatory phenotype

Programmed cell death protein 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. The fact that PD-L1 itself is also expressed on T cells upon activation has been largely neglected. Here, we demonstrate that PD-L1 ligation on human CD25-depleted CD4⁺ T cells, combined with CD3/TCR stimulation, induces their conversion into highly suppressive T cells. Furthermore, this effect was most prominent in memory (CD45RA⁻CD45RO⁺) T cells. PD-L1 engagement on T cells resulted in reduced ERK phosphorylation and decreased AKT/mTOR/S6 signaling. Importantly, T cells from rheumatoid arthritis patients exhibited high basal levels of phosphorylated ERK and following PD-L1 cross-linking both ERK signaling and the AKT/mTOR/S6 pathway failed to be down modulated, making them refractory to the acquisition of a regulatory phenotype. Altogether, our results suggest that PD-L1 signaling on memory T cells could play an important role in resolving inflammatory responses; maintaining a tolerogenic environment and its failure could contribute to ongoing autoimmunity.

This study shows that programmed death cell receptor ligand 1 (PD-L1) signaling in memory CD4+ T cells from healthy individuals induces a regulatory phenotype; this mechanism seems to be defective in equivalent T cells from rheumatoid arthritis patients and could be in part responsible for the pathology.     

ERK Positive Feedback Regulates a Widespread Network of Tyrosine Phosphorylation Sites across Canonical T Cell Signaling and Actin Cytoskeletal Proteins in Jurkat T Cells

Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor activation by creating an ultrasensitive, bistable switch to selectively enhance responses to foreign ligands while suppressing signals from self peptides. In response to T cell receptor agonist engagement, ERK is activated to positively regulate T cell receptor signaling through phosphorylation of Ser⁵⁹ Lck. To obtain a wide-scale view of the role of ERK in propagating T cell receptor signaling, a quantitative phosphoproteomic analysis of 322 tyrosine phosphorylation sites by mass spectrometry was performed on the human Jurkat T cell line in the presence of U0126, an inhibitor of ERK activation. Relative to controls, U0126-treated cells showed constitutive decreases in phosphorylation through a T cell receptor stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (VAV1, PLCγ1, Itk, NCK1). Additional constitutive decreases in phosphorylation were found on the majority of identified proteins implicated in the regulation of actin cytoskeleton pathway. Although the majority of identified sites on T cell receptor signaling proteins showed decreases in phosphorylation, Tyr⁵⁹⁸ of ZAP-70 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of this site via ERK feedback. These findings shed new light on ERK’s role in positive feedback in T cell receptor signaling and reveal novel signaling events that are regulated by this kinase, which may fine tune T cell receptor activation.     

Priming with very low-affinity peptide ligands gives rise to CD8<Superscript>+</Superscript> T-cell effectors with enhanced function but with greater susceptibility to transforming growth factor (TGF)β-mediated suppression

While the effects of TCR affinity and TGFβ on CD8⁺ T-cell function have been studied individually, the manner in which TCR affinity dictates susceptibility to TGFβ-mediated suppression remains unknown. To address this issue, we utilized OVA altered peptide ligands (APLs) of different affinities in the OT-I model. We demonstrate that while decreased TCR ligand affinity initially results in weakened responses, such interactions prime the resultant effector cells to respond more strongly to cognate antigen upon secondary exposure. Despite this, responses by CD8⁺ T cells primed with lower-affinity TCR ligands are more effectively regulated by TGFβ. Susceptibility to TGFβ-mediated suppression is associated with downregulation of RGS3, a recently recognized negative regulator of TGFβ signaling, but not expression of TGFβ receptors I/II. These results suggest a novel tolerance mechanism whereby CD8⁺ T cells are discriminately regulated by TGFβ according to the affinity of the ligand on which they were initially primed. In addition, because of the major role played by TGFβ in tumor-induced immune suppression, these results identify the affinity of the priming ligand as a primary concern in CD8⁺ T-cell-mediated cancer immunotherapeutic strategies.     

TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1

An examination of thymocytes and peripheral T cells from SHP-1-deficient motheaten mice possessing a transgenic MHC class I-restricted TCR has implicated SHP-1 in regulating TCR signaling thresholds at three checkpoints in T cell development and activation. First, in the population of CD4-CD8- double negative thymocytes, SHP-1 appears capable of regulating signals from TCR complexes that control the maturation and proliferation of double negative thymocytes. Second, the loss of SHP-1 increased the number of CD4+CD8+ double positive thymocytes capable of maturing as TCRhigh single positive thymocytes. Third, the loss of SHP-1 altered the basal level of activation of naive lymph node T cells. Accordingly, SHP-1-deficient lymph node T cells bearing the transgenic TCR demonstrated a hyperresponsiveness to stimulation with cognate peptide. However, the loss of SHP-1 did not alter the cytolytic ability of mature effector cytotoxic T lymphocytes. Together these results suggest that SHP-1 contributes to establishing thresholds for TCR signaling in thymocytes and naive peripheral T cells.     

CD3γ-independent pathways in TCR-mediated signaling in mature T and iNKT lymphocytes

Antigen recognition by T-lymphocytes through the T-cell antigen receptor, TCR–CD3, is a central event in the initiation of an immune response. CD3 proteins may have redundant as well as specific contributions to the intracellular propagation of TCR-mediated signals. However, to date, the relative role that each CD3 chain plays in signaling is controversial. In order to examine the roles of CD3γ chain in TCR signaling, we analyzed proximal and distal signaling events in human CD3γ^−/− primary and Herpesvirus saimiri (HVS)-transformed T cells. Following TCR–CD3 engagement, certain early TCR signaling pathways (ZAP-70, ERK, p38 and mTORC2 phosphorylation, and actin polymerization) were comparable with control HVS-transformed T cells. However, other signaling pathways were affected, such TCRζ phosphorylation, indicating that the CD3γ chain contributes to improve TCR signaling efficiency and survival. On the other hand, CD3γ^−/− primary invariant NKT cells (iNKT cells) showed a normal expansion in response to alpha-galactosylceramide (α-GalCer) and TCRVβ11^bright iNKT cells were preferentially selected in this in vitro culture system, perhaps as a consequence of selective events in the thymus. Our results collectively indicate that a TCR lacking CD3γ can propagate a number of signals through the remaining invariant chains, likely the homologous CD3δ chain, which replaces it at the mutant TCR.     

Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3 (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency of the SEC3 variants correlated with enhanced binding without any optimum in the binding range covered by native TCR ligands. Comparable studies using anti-TCR antibodies of known affinity confirmed these observations. By comparing the biological potency of the two sets of ligands, we found a significant correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength.     

Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family

Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the presence of both the FGF agonist ligand and heparan sulfate glycosaminoglycan. FGFR1 homodimers in turn trigger a variety of downstream signaling cascades via autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR1. By means of Bioluminescence Energy Resonance Transfer (BRET) as a sign of FGFR1 homodimerization, we evaluated in HEK293T cells the effects of all known FGF agonist ligands on homodimer formation. A significant correlation between BRET² signaling and ERK1/2 phosphorylation was observed, leading to a further characterization of the binding and signaling properties of the FGF subfamilies. FGF agonist ligand-FGFR1 binding interactions appear as the main mechanism for the control of FGFR1 homodimerization and MAPK signaling which demonstrated a high correlation. The bioinformatic analysis demonstrates the interface of the two pro-triplets SSS (Ser–Ser–Ser) and YGS (Tyr–Gly–Ser) located in the extracellular and intracellular domain of the FGFR1. These pro-triplets are postulated participate in the FGFR1 homodimerization interface interaction. The findings also reveal that FGF agonist ligands within the same subfamily of the FGF gene family produced similar increases in FGFR1 homodimer formation and MAPK signaling. Thus, the evolutionary relationship within this gene family appears to have a distinct functional relevance.     

Elucidation of T cell signalling models

A potential mechanism that allows T cells to reliably discriminate pMHC ligands involves an interplay between kinetic proofreading, negative feedback and a destruction of this negative feedback. We analyse a detailed model of these mechanisms which involves the TCR, SHP1 and ERK. We discover that the behaviour of pSHP1 negative feedback is of primary importance, and particularly the influence of a kinetic proofreading base negative feedback state on pSHP1 dynamics. The CD8 co-receptor is shown to benefit from a kinetic proofreading locking mechanism and is able to overcome pSHP1 negative influences to sensitise a T cell.     

TGF-beta inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector/memory T cells in the presence of sustained TCR signal transduction

TGF-beta signaling is critical for controlling naive T cell homeostasis and differentiation; however, the biological and biochemical changes induced by TGF-beta in effector/memory T cells are poorly defined. We show that although TGF-beta inhibits effector/memory peripheral blood T lymphoblast proliferation and IL-2 production, the intensity and kinetics for TCR-induced global tyrosine phosphorylation are markedly increased compared with that in untreated cells or naive T cells. After TCR ligation, tyrosine phosphorylation of proximal tyrosine kinases and docking proteins like linker for activation of T cells is maintained for >30 min in TGF-beta-primed cells compared with untreated cells where phosphorylation of these targets returned to basal levels by 10 min. Extended phosphorylation of linker for activation of T cells in treated peripheral blood T selectively prolongs ERK 1/2 signaling and phospholipase C-gamma1 activation leading to increased Ca(2+) flux. A kinase/phosphatase imbalance could not account for extended phosphorylation as CD45R, SHP-1, and SHP-2 expression remains unaltered. The contradiction between prolonged signal transduction and inhibition of proliferation is partially explained by the observation that TGF-beta priming results in ERK 1/2-independent p21 induction and decreased cyclin D1 expression leading to accumulation of T cells in G(0)/G(1) phases of the cell cycle and cell cycle arrest. Despite inhibition of T cell function by TGF-beta priming, TCR and cytokine signaling pathways are intact and selectively extended, suggesting that suppression in the effector/memory T cell is mediated by reprogramming signal transduction, rather than its inhibition as in the naive T cell.     

Computational analysis of T cell receptor signaling and ligand discrimination--past, present, and future

Signaling through the T cell receptor for antigen (TCR) has been studied for years by conventional biochemical means. More recently, attempts have been made to develop computational models of signaling through this receptor, with a specific focus on understanding how this recognition system discriminates between closely related (self and non-self) ligands. Here we discuss recent advances centered on the role of feedback regulation, especially the key finding that a combination of digital and analog control circuits is fundamental to the discrimination properties of the TCR. We end by pointing to future, more biologically accurate models that incorporate spatial aspects of molecular organization in antigen-engaged T lymphocytes with this underlying biochemistry.     

Height,but not binding epitope,affects the potency of synthetic TCR agonists

Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRβ Fab′ antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab′-DNA ligand efficiently triggers TCR as a monomer when membrane associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for efficient TCR triggering and cellular activation by Fab′-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex but that length of the DNA tether is important. Increasing, the intermembrane distance spanned by Fab′-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for constructing synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.     

Localization of Src homology 2 domain-containing phosphatase 1 (SHP-1) to lipid rafts in T lymphocytes: functional implications and a role for the SHP-1 carboxyl terminus

The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) has previously been shown to be a negative regulator of signaling mediated via the TCR. A growing body of evidence indicates that the regulated localization of proteins within certain membrane subdomains, referred to as lipid rafts, is important for the successful transduction of signaling events downstream of the TCR. However, considerably less is known about the localization of negative regulators during these lipid raft-dependent signaling events. In this study we have investigated the subcellular localization of SHP-1 and its role in regulation of TCR-mediated signaling. Our studies demonstrate that in a murine T cell hybridoma as well as in primary murine thymocytes, a fraction of SHP-1 localizes to the lipid rafts, both basally and after TCR stimulation. Interestingly, although SHP-1 localized in the nonraft fractions is tyrosine phosphorylated, the SHP-1 isolated from the lipid rafts lacks the TCR-induced tyrosine phosphorylation, suggesting physical and/or functional differences between these two subpopulations. We identify a requirement for the C-terminal residues of SHP-1 in optimal localization to the lipid rafts. Although expression of SHP-1 that localizes to lipid rafts potently inhibits TCR-mediated early signaling events and IL-2 production, the expression of lipid raft-excluded SHP-1 mutants fails to elicit any of the inhibitory effects. Taken together these studies reveal a key role for lipid raft localization of SHP-1 in mediating the inhibitory effects on T cell signaling events.