Ancestral T Cells in Fish Require mTORC1-Coupled Immune Signals and Metabolic Programming for Proper Activation and Function.

T cells suddenly appeared in jawed fish ∼450 million years ago. Biological studies of fish T cells may provide helpful evidence to understand evolution of adaptive immune systems. To this end, using a Nile tilapia (Oreochromis niloticus) model, we revealed the regulatory mechanism of adaptive immunity mediated by ancestral T cells in jawed fish. Nile tilapia T cells as well as a tightly regulated mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway participate in the cellular adaptive immune response during Streptococcus agalactiae infection. Blockade of mTORC1 signaling by rapamycin impairs T cell activation and Ag-induced proliferation in this early vertebrate. More critically, we show that signals from mTORC1 are indispensable for primordial effector T cells to eliminate infection by promoting the expression of proinflammatory cytokines, cytotoxic-related molecules, and proapoptotic genes. Mechanistically, teleost mTORC1 directs effector T cell function by coordinating multiple metabolic programs, including glycolysis, glutaminolysis, and lipogenesis through activating key transcription factors c-Myc, HIF-1α, and sterol regulatory element-binding proteins, and thus links immune signals to metabolic reprogramming in jawed fish. To our knowledge, these results represent the first description of the regulatory mechanism for T cell-mediated adaptive immunity in a fish species. From an evolutionary viewpoint, our study suggests that primordial T cells are armed with sophisticated regulatory strategies like those in modern T cells prior to the divergence of bony fish from the tetrapod lineage. Therefore, our findings fill in an important gap regarding evolution of the adaptive immune system.

Immunosuppression and Aberrant T Cell Development in the Absence of N-Myristoylation.

N-myristoylation refers to the attachment of myristic acid to the N-terminal glycine of proteins and substantially affects their intracellular targeting and functions. The thymus represents an organ with a prominent N-myristoylation activity. To elucidate the role of protein N-myristoylation for thymocyte development, we generated mice with a T cell lineage-specific deficiency in N-myristoyl transferase (Nmt)1 and 2. Depletion of Nmt activity in T cells led to a defective transmission of TCR signals, a developmental blockage of thymocytes at the transition from double-negative 3 to 4 stages, and a reduction of all the following stages. We could demonstrate that Lck and myristoylated alanine-rich C kinase substrate, two main myristoylated kinases in T cells, were mislocalized in the absence of Nmt activity. N-myristoylation was also indispensable for early and distal TCR signaling events such as CD3ζ, Zap70, and Erk activation and for release of cytokines such as IFN-γ and IL-2. As a consequence, the initiation and propagation of the TCR signaling cascade was severely impaired. Furthermore, we showed that the absence of myristoylation had an immunosuppressive effect on T cells in vivo after treatment with CpG and stimulation of the TCR with the staphylococcal enterotoxin B superantigen. Therefore, protein myristoylation is indispensable in T cell development and activation and its inhibition might offer a novel strategy to achieve immunosuppression.

T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells.

BACKGROUND: The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. RESULTS: To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr(280) (pTyr(280)) and pTyr(305). These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr(280) and pTyr(305) on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. CONCLUSIONS: TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells.

Method for co-cluster analysis in multichannel single-molecule localisation data.

We demonstrate a combined univariate and bivariate Getis and Franklin's local point pattern analysis method to investigate the co-clustering of membrane proteins in two-dimensional single-molecule localisation data. This method assesses the degree of clustering of each molecule relative to its own species and relative to a second species. Using simulated data, we show that this approach can quantify the degree of cluster overlap in multichannel point patterns. The method is validated using photo-activated localisation microscopy and direct stochastic optical reconstruction microscopy data of the proteins Lck and CD45 at the T cell immunological synapse. Analysing co-clustering in this manner is generalizable to higher numbers of fluorescent species and to three-dimensional or live cell data sets.

Stage I non-small cell lung cancer: the presence of the lymphocyte-specific protein tyrosin kinase in the tumour infiltrate is associated with a better long-term prognosis.

We studied the expression in the tumour infiltrate of a T-cell activation marker, the lymphocyte-specific protein tyrosin kinase (LCK), to assess if it could be associated with a better prognostic outcome in early stage non-small cell lung cancer (NSCLC) patients. This retrospective study included 25 patients undergoing lobectomy with systematic hilo-mediastinal lymphadenectomy for pathological stage I NSCLC between July 2003 and June 2005. The presence of LCK was detected in the tumour infiltrate by immunohistochemistry on the specimens of all patients. No patient received adjuvant therapy. Twelve patients resulted LCK-positive and 13 LCK-negative. The distribution of patients according to the T-stage was similar between the LCK-positive group (1 T1a, 5 T1b, 6 T2a) and the LCK-negative group (1 T1a, 5 T1b, 7 T2a). Median overall survival (OS) time was not reached in the LCK-positive group and 30 months in the LCK-negative group (P = 0.01). OS was longer than 40 months in 75% of the LCK-positive patients and in 31% of the LCK-negative patients (P = 0.01). Median time to relapse (TTR) was significantly longer in LCK-positive patients than in LCK-negative patients (not reached vs. 25 months; P < 0.001). In conclusion, LCK-positive tumour infiltrate has been found to be associated with a significantly longer OS and TTR in patients with radically resected stage I NSCLC.

Augmentation of T-cell immune responses and signal transduction proteins in oral cancer patients: potential for IL-2-mediated immunotherapy.

PURPOSE: Immune impairment is hypothesized to be one of the reasons for the dismal treatment response in oral cancers. This study evaluates the immune impairment in patients with primary squamous cell carcinoma of the oral cavity and the effect of IL-2 administration on restoration of the immune responses. METHODS: T-cell populations were enumerated by flow cytometry; T-cell function by MTS proliferation assay to PHA and anti-CD3, expression of T-cell signaling proteins ZAP-70, TCRζ, p(56)lck, PKC and CD-ε in T cells with and without activation by IL-2 using Western blot and statistical analysis using X (2) test and bivariate correlation analysis in 112 patients. RESULTS: Reduction in proportion of CD3(+) and CD4(+) T lymphocytes, decrease in the CD4(+)/CD8(+) T-cell ratios, reduced lymphocyte transformation to PHA and anti-CD3 and reduced production of interleukin-2(IL-2) were observed in the patient group. Lymphocyte proliferation to anti-CD3 could be augmented in 59.5% of non-responders by IL-2 (range 10-90%) along with significant increase in the expression of TCR-ζ and ZAP-70, CD3ε, p(56) LCK and PKC to varying degrees. The expression of ZAP-70 and TCR-ζ was found to be closely related to treatment response and could be augmented by IL-2 in terms of proliferation and IL-2 production. CONCLUSIONS: The results suggest IL-2 to augment T-cell responses in a proportion of oral cancer patients with poor response to conventional therapy. IL-2 immunotherapy can be thought of as a personalized adjuvant therapy for oral cancer following the in vitro identification of IL-2 responders using the expression of TCRζ and ZAP-70 as biomarkers.

[Neuronal isoforms of Src, Fyn and Lck tyrosine kinases: A specific role for p56lckN in neuron protection]. / Les isoformes neuronales des tyrosines kinases Src, Fyn et Lck : rôle particulier de la p56lckN dans la survie neuronale.

The two main tyrosine kinases (TK) in the brain are p60Src and p59Fyn, expressed as specific isoforms (p60SrcNI, p60SrcNI+NII and p59fynB). They play a pivotal role in some major processes such as neuronal growth and myelinisation. Another member of this TK family was then reported in brain, the p56lck. Its name Lck (lymphocyte cell kinase) indicates its cellular specificity observed initially, so its presence in the brain was intriguing. But no further studies were performed to understand its role in brain until recent clinical studies on Alzheimer patients' brains. One study reveals a decreased p56lck level in the brains of these patients while another study shows an association between one peculiar SNP (single nucleotide polymorphism) of the lck gene and some cases of the disease. These new data prompt us to reinvestigate the original biochemical data and to confront them with the present knowledge. This analysis suggests some hypothesis concerning both the Lck protein expressed in the brain (rather an isoform than the lymphocyte protein itself) and its role (to maintain the neuronal survival presumably by protecting them from inflammation, the main pathway that leads to neuron degeneracy).

PALM imaging and cluster analysis of protein heterogeneity at the cell surface.

The authors employed photoactivatable localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM) imaging and image analysis based on Ripley's K-function to quantify the distribution and heterogeneity of proteins at the cell plasma membrane. The membrane targeting sequence of the N-terminal region of the T cell receptor-pathway kinase Lck fused to the photo-convertible fluorescent protein tdEos (Lck(N10)-tdEos), clusters into sub-100 nm regions which cover approximately 7% of the cell surface. 2-channel PALM imaging of Lck(N10)-tdEos and the N-terminus of the kinase Src (Src(N15)-PS-CFP2) are demonstrated. Finally, T cell microclusters at the immune synapse are imaged at super-resolution using dSTORM, showing that conventional TIRF images contain unresolved, small clusters. These methods are generally applicable to other cell and fluorophore systems to quantify 2-D molecular clustering at nanometer scales.

Pertussis toxin utilizes proximal components of the T-cell receptor complex to initiate signal transduction events in T cells.

Pertussis toxin (PTx) is an AB(5) toxin produced by the human pathogen Bordetella pertussis. Previous work demonstrates that the five binding (B) subunits of PTx can have profound effects on T lymphocytes independent of the enzymatic activity of the A subunit. Stimulation of T cells with holotoxin (PTx) or the B subunit alone (PTxB) rapidly induces signaling events resulting in inositol phosphate accumulation, Ca(2+) mobilization, interleukin-2 (IL-2) production, and mitogenic cell growth. Although previous reports suggest the presence of PTx signaling receptors expressed on T cells, to date, the receptor(s) and membrane proximal signaling events utilized by PTx remain unknown. Here we genetically and biochemically define the membrane proximal components utilized by PTx to initiate signal transduction in T cells. Using mutants of the Jurkat T-cell line deficient for key components of the T-cell receptor (TCR) pathway, we have compared stimulation with PTx to that of anti-CD3 monoclonal antibody (MAb), which directly interacts with and activates the TCR complex. Our genetic data in combination with biochemical analysis show that PTx (via the B subunit) activates TCR signaling similar to that of anti-CD3 MAb, including activation of key signaling intermediates such as Lck, ZAP-70, and phospholipase C-gamma1. Moreover, the data indicate that costimulatory activity, as provided by CD28 ligation, is required for PTx to fully stimulate downstream indicators of T-cell activation such as IL-2 gene expression. By illuminating the signaling pathways that PTx activates in T cells, we provide a mechanistic understanding for how these signals deregulate immune system functions during B. pertussis infection.

Molecular and biochemical analysis of rainbow trout LCK suggests a conserved mechanism for T-cell signaling in gnathostomes.

Two genes were identified in rainbow trout that display high sequence identity to vertebrate Lck. Both of the trout Lck transcripts are associated with lymphoid tissues and were found to be highly expressed in IgM-negative lymphocytes. In vitro analysis of trout lymphocytes indicates that trout Lck mRNA is up-regulated by T-cell mitogens, supporting an evolutionarily conserved function for Lck in the signaling pathways of T-lymphocytes. Here, we describe the generation and characterization of a specific monoclonal antibody raised against the N-terminal domains of recombinant trout Lck that can recognize Lck protein(s) from trout thymocyte lysates that are similar in size ( approximately 57kDa) to mammalian Lck. This antibody also reacted with permeabilized lymphocytes during FACS analysis, indicating its potential usage for cellular analyses of trout lymphocytes, thus representing an important tool for investigations of salmonid T-cell function.

A correlation between function and selected measures of T cell avidity in influenza virus-specific CD8+ T cell responses.

Activation of mature CD8+ T cells requires recognition, via the T cell receptor (TCR), of peptide + MHC (pMHC) complexes with an avidity that exceeds a designated threshold. Multiple indicators of T cell avidity have been described that provide unique information on the characteristics of T cell interactions. However, these indicators are routinely used in isolation, and, consequently, little is known about correlations between these measures or which measure, if any, correlates with the quality of the T cell response. Following influenza virus infection of C57BL/6J mice, we analyzed the relative avidities of five epitope-specific CD8+ T cell populations using five different measures. We demonstrated that the quality of CD8+ T cell responses, in terms of cytokine profiles, correlates with TCR dissociation rate and CD8 dependence, but not with the sensitivity to tetramer binding or peptide stimulation. Thus, we propose that, despite significant differences in TCR dissociation rate, the stimulation threshold of influenza-specific CD8+ T cell populations may be equivalent due to compensatory mechanisms largely provided by the CD8 coreceptor. Furthermore, this study shows that different indicators of avidity do not necessarily provide similar information and should be used in combination to obtain an overall picture of the characteristics of TCR binding.

Single-cell analysis of siRNA-mediated gene silencing using multiparameter flow cytometry.

BACKGROUND: Use of synthetic short interfering RNAs (siRNAs) to study gene function has been limited by an inability to selectively analyze subsets of cells in complex populations, low and variable transfection efficiencies, and semiquantitative assays for measuring protein down-regulation. Intracellular flow cytometry can overcome these limitations by analyzing populations at the single-cell level in a high-throughput and quantitative fashion. Individual cells displaying a knockdown phenotype can be selectively interrogated for functional responses using multiparameter analysis. METHODS: Lck-specific siRNA was delivered into Jurkat T cells or peripheral blood mononuclear cells (PBMCs) to suppress endogenous Lck expression. Transfected cells were fluorescently stained for intracellular Lck and analyzed using multiparameter flow cytometry. The Lck(lo) Jurkat subpopulation was selectively analyzed for CD69 up-regulation and phospho-states of signaling proteins following T-cell receptor (TCR) stimulation. Surface expression levels of CD4 and CD8 on transfected CD3+ gated PBMCs were correlated with intracellular Lck levels. RESULTS: A subpopulation of Jurkat cells with reduced levels of Lck was clearly resolved from cells with wildtype levels of Lck. Both CD69 up-regulation and ZAP70 phosphorylation were suppressed in Lck(lo) cells when compared with those in Lck(hi) cells upon TCR stimulation. Knockdown of intracellular Lck in primary T lymphocytes reduced surface expression of CD4 in a dose-dependent manner. CONCLUSIONS: Multiparameter flow cytometry is a powerful technique for the quantitative analysis of siRNA-mediated protein knockdown in complex hard-to-transfect cell populations.

Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion.

Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl-/- thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process.

Altered lipid raft-associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by abnormalities in T lymphocyte receptor-mediated signal transduction pathways. Our previous studies have established that lymphocyte-specific protein tyrosine kinase (LCK) is reduced in T lymphocytes from patients with SLE and that this reduction is associated with disease activity and parallels an increase in LCK ubiquitination independent of T cell activation. This study investigated the expression of molecules that regulate LCK homeostasis, such as CD45, C-terminal Src kinase (CSK), and c-Cbl, in lipid raft domains from SLE T cells and investigated the localization of these proteins during T cell receptor (TCR) triggering. Our results indicate that the expression of raft-associated ganglioside, GM1, is increased in T cells from SLE patients and LCK may be differentially regulated due to an alteration in the association of CD45 with lipid raft domains. CD45 tyrosine phosphatase, which regulates LCK activity, was differentially expressed and its localization into lipid rafts was increased in T cells from patients with SLE. Furthermore, T cells allowed to "rest" in vitro showed a reversal of the changes in LCK, CD45, and GM1 expression. The results also revealed that alterations in the level of GM1 expression and lipid raft occupancy cannot be induced by serum factors from patients with SLE but indicated that cell-cell contact, activating aberrant proximal signaling pathways, may be important in influencing abnormalities in T cell signaling and, therefore, function in patients with SLE.

Signalling molecules and cytokine production in T cells of multiple myeloma-increased abnormalities with advancing stage.

T-cell immune dysfunction in patients with malignant tumours has been attributed to the altered expression of components of the T-cell receptor (TCR)/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four-colour flow cytometry was applied to study the surface bound molecules TCRalphabeta, CD28, CD152 and CD154 involved in T-cell signalling and the signal transduction molecules CD3zeta, p56lck, p59fyn, ZAP-70 and phosphatidyl-inositol-3 kinase (PI3-k) as well as the intracellular cytokines interferon-gamma (IFN-gamma), interleukin (IL)-4 and IL-2 as a functional read-out of non-stimulated and superantigen (staphylococcus enterotoxin B)-stimulated blood T cells of multiple myeloma (MM) patients at different stages of the disease. Multiple abnormalities were demonstrated in the CD4 and CD8 populations, both under non-stimulated and superantigen-stimulated conditions. There was a marked reduction, particular in advanced stage MM, in the proportion of CD4 and CD8 cells expressing CD28, CD152, CD3zeta, p56lck, ZAP-70 and PI3-k. The level of intracellular T-cell cytokines (IFN-gamma, IL-2 and IL-4) was normal or increased in non-stimulated cells but activation-induced cytokine production was impaired. These results illustrated profound and multiple T-cell signalling defects, from the surface and down-stream, consistent with involvement of a master T-cell function, especially in advanced stage MM. These data should be taken into consideration when developing immune-based therapeutic approaches and when applying new emerging technologies that aim to restore T-cell functions.

The flotillins are integral membrane proteins in lipid rafts that contain TCR-associated signaling components: implications for T-cell activation.

Lipid rafts play an important role in signal integration and cellular activation by the T-cell antigen receptor (TCR). We demonstrate that flotillin-1 and flotillin-2 are important structural raft components, which redistribute to the site of TCR engagement. An antibody to flotillin-1 was able to immobilize other TCR-associated raft components. Although rafts purified from unstimulated cells demonstrated abundant Lck but inabundant LAT, rafts from stimulated cells include an abundance of both components. This suggests dynamic changes in lipid raft composition during CD3/CD28 costimulation. Stimulation of primary human CD4(+) T cells leads to increased GM1 and flotillin-1 expression in the surface membrane, where these components colocalize. This may reconstitute new signaling complexes required for T-cell activation. Altered lipid raft composition and function may play a role in the decline of antigen responsiveness in senescent T cells. In this regard, we observed an increase in the raft-associated gangliolipid, GM1, in resting human CD4(+) and CD8(+) lymphocytes with aging.

Induction of cellular and humoral immune responses to tumor cells and peptides in HLA-A24 positive hormone-refractory prostate cancer patients by peptide vaccination.

BACKGROUND: To assess the safety and immune response of a peptide-based immunotherapy for patients with hormone-refractory prostate cancer, a phase I clinical trial was conducted. METHODS: This study first investigated whether cytotoxic T-lymphocyte (CTL) precursors reacting to peptide with vaccine candidates (14 peptides for HLA-A24 positive patients) were detectable in the pre-vaccination peripheral blood mononuclear cells (PBMCs) of ten patients with hormone-refractory prostate cancer. Patients were then vaccinated subcutaneously with only those peptides to which pre-vaccination PBMCs reacted (CTL precursor-oriented peptide vaccine) for up to four kinds of peptides. RESULTS: Overall vaccinations were generally well tolerated, but most patients (nine of ten) developed grade 1 local redness and swelling at the injection site. Increased CTL response to both peptides and cancer cells were observed in four of ten patients. Anti-peptide IgG antibodies were also detected in post-vaccination sera of seven of ten patients. One patient achieved a partial response with an 89% decrease in PSA. Stable disease was demonstrated in five of ten patients (50%) for the median duration of 2 months (range, 2-5 months). There were no objective responses of measurable lesions. CONCLUSIONS: Increase in cellular and humoral immune responses, and decrease in PSA level in some patients support further development of peptide-based immunotherapy for hormone refractory prostate cancer.

Pyrrolo[2,3-d]pyrimidines containing diverse N-7 substituents as potent inhibitors of Lck.

A series of pyrrolo[2,3-d]pyrimidines was synthesized and evaluated as inhibitors of Lck. Lck accommodates a diverse set of substituents at N-7. Altering the substituent at N-7 provided compound 13, an orally available lck inhibitor which inhibited TCR mediated IL-2 production after oral dosing.

Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction.

Lateral mobility and spatial organization of proteins within the plasma membrane are likely to mediate the initial events coordinating T cell activation. Lipid rafts, distinct cholesterol/sphingolipid-rich membrane microdomains, provide a mechanism for this regulation by concentrating or excluding signaling proteins. We demonstrate in peripheral blood T cell lymphoblasts that immediate early phosphotyrosine signal transduction through the TCR complex is functionally dependent on a distinct population of lipid rafts. Specifically, cholesterol extraction destabilizes the membrane microdomains containing Lck, while the rafts containing the adapter protein linker for activation of T cells remain intact. Heterogeneity in the partitioning of these proteins in resting cells was confirmed by immunoelectron microscopy. After T cell activation, both Lck and the linker for activation of T cells colocalize to 50-100 nm microdomains in the plasma membrane, indicating that sequestration of these proteins into distinct lipid rafts may function to regulate the initiation of T cell signal transduction.