TCR signal transduction in antigen-specific memory CD8 T cells

Memory T cells are more responsive to Ag than naive cells. To determine whether memory T cells also have more efficient TCR signaling, we compared naive, effector, and memory CD8 T cells of the same antigenic specificity. Surprisingly, initial CD3 signaling events are indistinguishable. However, memory T cells have more extensive lipid rafts with higher phosphoprotein content before TCR engagement. Upon activation in vivo, they more efficiently induce phosphorylation of-LAT (linker for activation of T cells), ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38. Thus, memory CD8 T cells do not increase their TCR sensitivity, but are better poised to augment downstream signals. We propose that this regulatory mechanism might increase signal transduction in memory T cells, while limiting TCR cross-reactivity and autoimmunity.     

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.     

TCR signaling antagonizes rapid IP-10-mediated transendothelial migration of effector memory CD4+ T cells

Human microvascular endothelial cells (ECs) constitutively express MHC class II in peripheral tissues, the function of which remains unknown. In vitro assays have established that the recognition of EC MHC class II can affect cytokine expression, proliferation, and delayed transendothelial migration of allogeneic memory, but not naive, CD4+ T cells. Previously, we have shown that effector memory CD4+ T cells will rapidly transmigrate in response to the inflammatory chemokine IFN-gamma-inducible protein-10 (IP-10) in a process contingent upon the application of venular levels of shear stress. Using two models that provide polyclonal TCR signaling by ECs in this flow system, we show that TCR engagement antagonizes the rapid chemokine-dependent transmigration of memory CD4+ T cells. Inhibitor studies suggest that TCR signaling downstream of Src family tyrosine kinase(s) but upstream of calcineurin activation causes memory CD4+ T cell arrest on the EC surface, preventing the transendothelial migration response to IP-10.     

Hematopoietic progenitor kinase 1 associates physically and functionally with the adaptor proteins B cell linker protein and SLP-76 in lymphocytes

B cell linker protein (BLNK) is a SLP-76-related adaptor protein essential for signal transduction from the BCR. To identify components of BLNK-associated signaling pathways, we performed a phosphorylation-dependent yeast two-hybrid analysis using BLNK probes. Here we report that the serine/threonine kinase hematopoietic progenitor kinase 1 (HPK1), which is activated upon antigen-receptor stimulation and which has been implicated in the regulation of MAP kinase pathways, interacts physically and functionally with BLNK in B cells and with SLP-76 in T cells. This interaction requires Tyr(379) of HPK1 and the Src homology 2 (SH2) domain of BLNK/SLP-76. Via homology modeling, we defined a consensus binding site within ligands for SLP family SH2 domains. We further demonstrate that the SH2 domain of SLP-76 participates in the regulation of AP-1 and NFAT activation in response to T cell receptor (TCR) stimulation and that HPK1 inhibits AP-1 activation in a manner partially dependent on its interaction with SLP-76. Our data are consistent with a model in which full activation of HPK1 requires its own phosphorylation on tyrosine and subsequent interaction with adaptors of the SLP family, providing a mechanistic basis for the integration of this kinase into antigen receptor signaling cascades.     

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.     

A biochemical signature for rapid recall of memory CD4 T cells

Mechanisms for the rapid recall response mediated by memory T cells remain unknown. In this study, we present a novel, multiparameter analysis of TCR-coupled signaling and function in resting and activated naive and memory CD4 T cells, revealing a biochemical basis for immunological recall. We identify a striking elevation in expression of the proximal tyrosine kinase Zap70 in resting Ag-specific and polyclonal mouse memory vs naive CD4 T cells that is stably maintained independent of protein synthesis. Elevated Zap70 protein levels control effector function as IFN-gamma production occurs exclusively from the Zap70(high) fraction of activated T cells in vitro and in vivo, and specific down-modulation of Zap70 expression in memory CD4 T cells by small interfering RNA or protein inhibition significantly reduces rapid IFN-gamma production. Downstream of Zap70, we show quantitative differences in distal phosphorylation associated with effector function in naive and memory subsets, with low accumulation of phosphorylation in memory T cells producing IFN-gamma at early time points, contrasting extensive phosphorylation associated with IFN-gamma production following sustained activation of naive T cells. Our results reveal a novel biochemical signature imparted to memory CD4 T cells enabling efficacious responses through increased Zap70 expression and reduced accumulation of downstream signaling events.     

The FcR gamma subunit and Syk kinase replace the CD3 zeta-chain and ZAP-70 kinase in the TCR signaling complex of human effector CD4 T cells

The TCR-mediated signals required to activate resting T cells have been well characterized; however, it is not known how TCR-coupled signals are transduced in differentiated effector T cells that coordinate ongoing immune responses. Here we demonstrate that human effector CD4 T cells up-regulate the expression of the CD3zeta-related FcRgamma signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3epsilon, but not CD3zeta. The TCR/CD3/FcRgamma complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases.     

Phospho-LAT-independent activation of the ras-mitogen-activated protein kinase pathway: a differential recruitment model of TCR partial agonist signaling.

Stimulation of mature T cells with agonist ligands of the Ag receptor (TCR) causes rapid phosphorylation of tyrosine-based activation motifs in the intracellular portion of TCR-zeta and CD3 and activation of several intracellular signaling cascades. Coordinate activation of these pathways is dependent on Lck- and ZAP-70-mediated tyrosine phosphorylation of a 36-kDa linker for activation of T cells and subsequent recruitment of phospholipase C-gamma1, Grb2-SOS, and SLP-76-vav. Here, we show that TCR partial agonist ligands can selectively activate one of these pathways, the Ras-mitogen-activated protein kinase pathway, by inducing recruitment of Grb2-SOS complexes to incompletely phosphorylated p21 phospho-TCR-zeta. This bypasses the need for activation of Lck and ZAP-70, and for phosphorylation of the linker for activation of T cells to activate Ras. We propose a general model in which differential recruitment of activating complexes away from transmembrane linker proteins may determine selective activation of a given signaling pathway.     

Defective T cell receptor-mediated signal transduction in memory CD4 T lymphocytes exposed to superantigen or anti-T cell receptor antibodies

Lymphocytes must promote protective immune responses while still maintaining self-tolerance. Stimulation through the T cell receptor (TCR) can lead to distinct responses in naive and memory CD4 T cells. Whereas peptide antigen stimulates both naive and memory T cells, soluble anti-CD3 antibodies and bacterial superantigens stimulate only naive T cells to proliferate and secrete cytokines. Further, superantigens, like staphylococcal enterotoxin B (SEB), cause memory T cells to become anergic while soluble anti-CD3 does not. In the present report, we show that signal transduction through the TCR is impaired in memory cells exposed to either anti-CD3 or SEB. A block in signaling leads to impaired activation of the kinase ZAP-70 so that downstream signals and cell proliferation do not occur. We further show that the signaling defect is unique to each agent. In anti-CD3-treated memory T cells, the src kinase Lck is only transiently activated and does not phosphorylate and activate ZAP-70. In SEB-treated memory T cells, ZAP-70 does not interact with the TCR/CD3 complex to become accessible to Lck. Finally, we provide evidence that alternative signaling pathways are initiated in SEB-treated memory cells. Altered signaling, indicated by an elevation in activity of the src kinase Fyn, may be responsible for memory cell anergy caused by SEB. Thus, differentiation of naive T cells into memory cells is accompanied by alterations in TCR-mediated signaling that can promote heightened recall immunity or specific tolerance.     

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.     

Functional complementation of BLNK by SLP-76 and LAT linker proteins

Recent studies have demonstrated a requirement for the SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activation of T cells) adaptor/linker proteins in T cell antigen receptor activation and T cell development as well as the BLNK (B cell linker) linker protein in B cell antigen receptor (BCR) signal transduction and B cell development. Whereas the SLP-76 and LAT adaptor proteins are expressed in T, natural killer, and myeloid cells and platelets, BLNK is preferentially expressed in B cells and monocytes. Although BLNK is structurally homologous to SLP-76, BLNK interacts with a variety of downstream signaling proteins that interact directly with both SLP-76 and LAT. Here, we demonstrate that neither SLP-76 nor LAT alone is sufficient to restore the signaling deficits observed in BLNK-deficient B cells. Conversely, the coexpression of SLP-76 and LAT together restored BCR-inducible calcium responses as well as activation of all three families of mitogen-activated protein kinases. Together, these data suggest functional complementation of SLP-76 and LAT in T cell antigen receptor function with BLNK in BCR function.     

Functional association between SLAP-130 and SLP-76 in Jurkat T cells

T cell antigen receptor (TCR) engagement results in protein-tyrosine kinase activation which initiates signaling cascades leading to induction of the interleukin-2 gene. Previous studies identified two substrates of the TCR-induced protein-tyrosine kinases, SH2 domain-containing leukocyte specific protein of 76 kDa (SLP-76) and SLP-76-associated phosphoprotein of 130 kDa (SLAP-130). While SLP-76 appears to couple the TCR with downstream signals, SLAP-130 may play a negative regulatory role in T cell activation. In this study, we demonstrate that consistent with its ability to abrogate the SLP-76 augmentation of TCR-induced activation of the NFAT/AP1 region of the interleukin-2 promoter, overexpression of SLAP-130 also interferes with the rescue of signaling in SLP-76-deficient Jurkat cells in co-transfection experiments. The effect of SLAP-130 on SLP-76 function is specific for regulating TCR-induced ERK activation, but not phospholipase Cgamma 1 phosphorylation. By generating both deletion and point mutants of SLAP-130, we identified tyrosine 559 as critical for the interaction between SLP-76 and SLAP-130. We show that mutation of this residue in context of full-length SLAP-130 diminishes the ability of SLAP-130 to abrogate SLP-76 function. These data suggest that the SLAP-130/SLP-76 association is important for the negative regulatory role that SLAP-130 appears to play in T cell signaling.     

Attenuation of T Cell Receptor Signaling by Serine Phosphorylation-mediated Lysine 30 Ubiquitination of SLP-76 Protein

SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) is an adaptor protein that is essential for T cell development and T cell receptor (TCR) signaling activation. Previous studies have identified an important negative feedback regulation of SLP-76 by HPK1 (hematopoietic progenitor kinase 1; MAP4K1)-induced Ser-376 phosphorylation. Ser-376 phosphorylation of SLP-76 mediates 14-3-3 binding, resulting in the attenuation of SLP-76 activation and downstream signaling; however, the underlying mechanism of this action remains unknown. Here, we report that phosphorylated SLP-76 is ubiquitinated and targeted for proteasomal degradation during TCR signaling. SLP-76 ubiquitination is mediated by Ser-376 phosphorylation. Furthermore, Lys-30 is identified as a ubiquitination site of SLP-76. Loss of Lys-30 ubiquitination of SLP-76 results in enhanced anti-CD3 antibody-induced ERK and JNK activation. These results reveal a novel regulation mechanism of SLP-76 by ubiquitination and proteasomal degradation of activated SLP-76, which is mediated by Ser-376 phosphorylation, leading to down-regulation of TCR signaling.     

Role of two adaptor molecules SLP-76 and LAT in the PI3K signaling pathway in activated T cells

Previously, we identified p85, a subunit of PI3K, as one of the molecules that interacts with the N-terminal region of Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76). We also demonstrated that tyrosine phosphorylation either at the 113 and/or 128 position is sufficient for the association of SLP-76 with the Src homology 2 domain near the N terminus of p85. The present study further examines the role of the association of these two molecules on the activation of PI3K signaling cascade. Experiments were done to determine the role of SLP-76, either wild-type, tyrosine mutants, or membrane-targeted forms of various SLP-76 constructs, on the membrane localization and phosphorylation of Akt, which is an event downstream of PI3K activation. Reconstitution studies with these various SLP-76 constructs in a Jurkat variant cell line that lacks SLP-76 or linker for activation of T cells (LAT) show that the activation of PI3K pathway following TCR ligation requires both SLP-76 and LAT adaptor proteins. The results suggest that SLP-76 associates with p85 after T cell activation and that LAT recruits this complex to the membrane, leading to Akt activation.     

A population of in vivo anergized T cells with a lower activation threshold for the induction of CD25 exhibit differential requirements in mobilization of intracellular calcium and mitogen-activated protein kinase activation

Chronic exposure of mature T cells with specificity for self-Ags can lead to the induction of a nonfunctional state which is referred to as T cell anergy. It is unclear whether anergic T cells are destined for cell death and thereby harmless or whether they can contribute to the induction of autoimmunity and/or regulation of anti-self reactivity. We have begun to address this issue. In a recent study, we showed that a population of mature CD4-CD8- T cells that express a transgenic TCR specific for the Ld MHC class I molecule are rendered anergic in Ld-expressing mice. In this study, we show that this population of anergic T cells possess a lower activation threshold for the induction of CD25 and CD69 in response to stimulation by antigenic ligands. Furthermore, these anergic T cells undergo extensive proliferation when stimulated with a low-affinity ligand in the presence of an exogenous source of IL-2. Biochemical analysis of the early intracellular signaling events of these in vivo anergized T cells showed that they have a signaling defect at the level of ZAP-70 and linker for the activation of T cell (LAT) phosphorylation. They also exhibit a defect in mobilization of intracellular calcium in response to TCR signaling. However, these anergic T cells demonstrate no defect in SLP-76 phosphorylation and extracellular signal-regulated kinase 1/2 activation. These biochemical characteristics of the anergic T cells were associated with an elevated level of Fyn, but not Lck expression. The potential contributions of these anergic T cells in the induction and/or regulation of autoimmune responses are discussed.     

A pool of central memory-like CD4 T cells contains effector memory precursors

The L51S mutation in the D10.G4.1 TCR alpha-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the beta-chain of I-A(k), and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44(high)CD62L(neg)CD45RB(neg) CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R alpha only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.     

HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76-Signaling Microclusters following TCR Stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.