A Story of Kinases and Adaptors: The Role of Lck, ZAP-70 and LAT in Switch Panel Governing T-Cell Development and Activation.

Specific antigen recognition is one of the immune system's features that allows it to mount intense yet controlled responses to an infinity of potential threats. T cells play a relevant role in the host defense and the clearance of pathogens by means of the specific recognition of peptide antigens presented by antigen-presenting cells (APCs), and, to do so, they are equipped with a clonally distributed antigen receptor called the T-cell receptor (TCR). Upon the specific engagement of the TCR, multiple intracellular signals are triggered, which lead to the activation, proliferation and differentiation of T lymphocytes into effector cells. In addition, this signaling cascade also operates during T-cell development, allowing for the generation of cells that can be helpful in the defense against threats, as well as preventing the generation of autoreactive cells. Early TCR signals include phosphorylation events in which the tyrosine kinases Lck and ZAP70 are involved. The sequential activation of these kinases leads to the phosphorylation of the transmembrane adaptor LAT, which constitutes a signaling hub for the generation of a signalosome, finally resulting in T-cell activation. These early signals play a relevant role in triggering the development, activation, proliferation and apoptosis of T cells, and the negative regulation of these signals is key to avoid aberrant processes that could generate inappropriate cellular responses and disease. In this review, we will examine and discuss the roles of the tyrosine kinases Lck and ZAP70 and the membrane adaptor LAT in these cellular processes.

Expression of Non-T Cell Activation Linker (NTAL) in Jurkat Cells Negatively Regulates TCR Signaling: Potential Role in Rheumatoid Arthritis.

T lymphocytes are key players in adaptive immune responses through the recognition of peptide antigens through the T Cell Receptor (TCR). After TCR engagement, a signaling cascade is activated, leading to T cell activation, proliferation, and differentiation into effector cells. Delicate control of activation signals coupled to the TCR is needed to avoid uncontrolled immune responses involving T cells. It has been previously shown that mice deficient in the expression of the adaptor NTAL (Non-T cell activation linker), a molecule structurally and evolutionarily related to the transmembrane adaptor LAT (Linker for the Activation of T cells), develop an autoimmune syndrome characterized by the presence of autoantibodies and enlarged spleens. In the present work we intended to deepen investigation into the negative regulatory functions of the NTAL adaptor in T cells and its potential relationship with autoimmune disorders. For this purpose, in this work we used Jurkat cells as a T cell model, and we lentivirally transfected them to express the NTAL adaptor in order to analyze the effect on intracellular signals associated with the TCR. In addition, we analyzed the expression of NTAL in primary CD4+ T cells from healthy donors and Rheumatoid Arthritis (RA) patients. Our results showed that NTAL expression in Jurkat cells decreased calcium fluxes and PLC-γ1 activation upon stimulation through the TCR complex. Moreover, we showed that NTAL was also expressed in activated human CD4+ T cells, and that the increase of its expression was reduced in CD4+ T cells from RA patients. Our results, together with previous reports, suggest a relevant role for the NTAL adaptor as a negative regulator of early intracellular TCR signaling, with a potential implication in RA.

Mutation of the glycine residue preceding the sixth tyrosine of the LAT adaptor severely alters T cell development and activation.

The LAT transmembrane adaptor is essential to transduce intracellular signals triggered by the TCR. Phosphorylation of its four C-terminal tyrosine residues (136, 175, 195, and 235 in mouse LAT) recruits several proteins resulting in the assembly of the LAT signalosome. Among those tyrosine residues, the one found at position 136 of mouse LAT plays a critical role for T cell development and activation. The kinetics of phosphorylation of this residue is delayed as compared to the three other C-terminal tyrosines due to a conserved glycine residue found at position 135. Mutation of this glycine into an aspartate residue (denoted LATG135D) increased TCR signaling and altered antigen recognition in human Jurkat T cells and ex vivo mouse T cells. Here, using a strain of LATG135D knockin mice, we showed that the LATG135D mutation modifies thymic development, causing an increase in the percentage of CD4+CD8+ double-positive cells, and a reduction in the percentage of CD4+ and CD8+ single-positive cells. Interestingly, the LATG135D mutation alters thymic development even in a heterozygous state. In the periphery, the LATG135D mutation reduces the percentage of CD8+ T cells and results in a small increment of γδ T cells. Remarkably, the LATG135D mutation dramatically increases the percentage of central memory CD8+ T cells. Finally, analysis of the proliferation and activation of T lymphocytes shows increased responses of T cells from mutant mice. Altogether, our results reinforce the view that the residue preceding Tyr136 of LAT constitutes a crucial checkpoint in T cell development and activation.

Activation of T-bet, FOXP3, and EOMES in Target Organs From Piglets Infected With the Virulent PRRSV-1 Lena Strain.

Transcription factors (TFs) modulate genes involved in cell-type-specific proliferative and migratory properties, metabolic features, and effector functions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogen agents in the porcine industry; however, TFs have been poorly studied during the course of this disease. Therefore, we aimed to evaluate the expressions of the TFs T-bet, GATA3, FOXP3, and Eomesodermin (EOMES) in target organs (the lung, tracheobronchial lymph node, and thymus) and those of different effector cytokines (IFNG, TNFA, and IL10) and the Fas ligand (FASL) during the early phase of infection with PRRSV-1 strains of different virulence. Target organs from mock-, virulent Lena-, and low virulent 3249-infected animals humanely euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi) were collected to analyze the PRRSV viral load, histopathological lesions, and relative quantification through reverse transcription quantitative PCR (RT-qPCR) of the TFs and cytokines. Animals belonging to both infected groups, but mainly those infected with the virulent Lena strain, showed upregulation of the TFs T-bet, EOMES, and FOXP3, together with an increase of the cytokine IFN-γ in target organs at the end of the study (approximately 2 weeks post-infection). These results are suggestive of a stronger polarization to Th1 cells and regulatory T cells (Tregs), but also CD4+ cytotoxic T lymphocytes (CTLs), effector CD8+ T cells, and γδT cells in virulent PRRSV-1-infected animals; however, their biological functionality should be the object of further studies.

A Novel, LAT/Lck Double Deficient T Cell Subline J.CaM1.7 for Combined Analysis of Early TCR Signaling.

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex.

Increased Protein Stability and Interleukin-2 Production of a LATG131D Variant With Possible Implications for T Cell Anergy.

The adaptor LAT plays a crucial role in the transduction of signals coming from the TCR/CD3 complex. Phosphorylation of some of its tyrosines generates recruitment sites for other cytosolic signaling molecules. Tyrosine 132 in human LAT is essential for PLC-γ activation and calcium influx generation. It has been recently reported that a conserved glycine residue preceding tyrosine 132 decreases its phosphorylation kinetics, which constitutes a mechanism for ligand discrimination. Here we confirm that a LAT mutant in which glycine 131 has been substituted by an aspartate (LATG131D) increases phosphorylation of Tyr132, PLC-γ activation and calcium influx generation. Interestingly, the LATG131D mutant has a slower protein turnover while being equally sensitive to Fas-mediated protein cleavage by caspases. Moreover, J.CaM2 cells expressing LATG131D secrete greater amounts of interleukin-2 (IL-2) in response to CD3/CD28 engagement. However, despite this increased IL-2 secretion, J.CaM2 cells expressing the LATG131D mutant are more sensitive to inhibition of IL-2 production by pre-treatment with anti-CD3, which points to a possible role of this residue in the generation of anergy. Our results suggest that the increased kinetics of LAT Tyr132 phosphorylation could contribute to the establishment of T cell anergy, and thus constitutes an earliest known intracellular event responsible for the induction of peripheral tolerance.

Activation of the extrinsic apoptotic pathway in the thymus of piglets infected with PRRSV-1 strains of different virulence.

In the last decade, the outbreaks caused by virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains from both PRRSV-1 and PRRSV-2 have considerably increased. PRRSV is able to modulate the host's immune response through the induction of apoptosis of cells in lymphoid organs like thymus, increasing the susceptibility to secondary infectious agents. The present study aimed to compare the impact of two PRRSV-1 strains, a field low virulent strain (3249 strain) and a virulent strain (Lena strain), in the thymus of infected pigs, focusing on clinical signs, histological analysis, viraemia, thymus viral load and the study of the different routes of apoptosis phenomena by immunohistochemistry. Sera and thymus samples were collected from infected animals with 3249 strain, Lena strain and mock-infected animals at 1, 3, 6, 8 and 13 days post-infection (dpi). Lena-infected animals showed severe clinical disease, high sera and thymus viral loads with evident thymic atrophy since 6 dpi, matching with PRRSV-N protein, TUNEL and cCasp3 expression in the thymic cortex. In both infected groups, there was an increase in the number of cells expressing molecules related to the extrinsic pathway of apoptosis (cCasp8 and Fas) in cortex and medulla, showing an important role in the apoptosis induction produced in thymus of PRRSV-infected piglets. The extensive apoptosis in the thymus through this pathway would lead to a decrease in the number of mature T lymphocytes and the sustained release of viral particles, which may explain the greater severity of the clinical signs observed in Lena-infected pigs.

Kinetics of the expression of CD163 and CD107a in the lung and tonsil of pigs after infection with PRRSV-1 strains of different virulence.

The emergence of virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV), causing atypical and severe outbreaks, has been notified worldwide. This study assesses the expression, distribution and kinetics of PRRSV N-protein, CD163 and CD107a in the lung and tonsil from experimentally-infected piglets with three different PRRSV-1 strains: a virulent PRRSV-1 subtype 3 strain (SU1-bel) and two low-virulent subtype 1 strains, Lelystad virus (LV) and 215-06. SU1-bel replicated more efficiently in the lungs and tonsils. The number of CD163+ cells decreased in both tissues from all infected groups at 7 dpi, followed by an increase at the end of the study, highlighting a negative correlation with the number of N-protein+-infected cells. A significant increase in CD107a was observed in all infected groups at 35 dpi but no differences were observed among them. Whereas the initial decrease of CD163+ cells appears to be associated to virus replication and cell death, the later recovery of the CD163+ population may be due to either the induction of CD163 in immature cells, the recruitment of CD163+ cells in the area of infection, or both. These results highlight the ability of macrophage subpopulations in infected animals to recover and restore their potential biological functions at one-month post-infection, with the greatest improvement observed in SU1-bel-infected animals.

A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling.

The adaptor protein linker for activation of T cells (LAT) has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT-Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL) in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance (p = 0.051). Nevertheless, downstream signals such as Ca2+ influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT-Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex.

Activation of extrinsic- and Daxx-mediated pathways in lymphoid tissue of PRRSV-infected pigs.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a major infectious pathogen in pigs leading to huge economical losses worldwide. PRRSV is able to escape from host immunity and causes transient infections. In the present study, expression of different apoptotic markers and its connection with PRRSV were assessed in tonsil and mediastinal lymph node from PRRSV-infected pigs. Cleaved caspase (CCasp)8, CCasp9, Fas, Daxx, CCasp3 and PRRSV expression were analyzed by immunohistochemistry. An up-regulation of CCasp8, Fas and CCasp3 expression in lymphocytes and macrophages from both organs was found during PRRSV infection, indicating the activation of the extrinsic-mediated pathway of apoptosis. Moreover, Daxx expression was also enhanced in macrophages of both organs, suggesting a simultaneous caspase-independent pathway of apoptosis. A correlation between the expression of the different apoptotic markers and IL-10, IL-6 and TGF-ß but not with PRRSV antigen was found in our study, which supports the hypothesis of an indirect mechanism in PRRSV-induced apoptosis.

Enhanced expression of TGFß protein in lymphoid organs and lung, but not in serum, of pigs infected with a European field isolate of porcine reproductive and respiratory syndrome virus.

Transforming growth factor ß (TGFß) is an immunomodulatory cytokine which is able to modulate the host immune response eliciting an inefficient response against pathogens. In this sense, the role of this cytokine in porcine reproductive and respiratory syndrome (PRRS) has been poorly studied and the reported results are contradictory. Thus, in the present study, the expression of TGFß was analysed both at tissue (lymphoid organs and lung) and serum level to study its correlation with the expression of PRRS virus (PRRSV). To carry out this study, 32 pigs were inoculated with the European PRRSV field isolate 2982 and sequentially killed from 0 dpi to the end of the study (24 dpi). Blood and tissue samples were collected to determine the expression of PRRSV and TGFß. PRRSV was detected in inoculated animals from 3 dpi until the end of the study, however TGFß was not detected in sera from inoculated animals. Contrary, an increase of TGFß antigen was observed both in the lymphoid organs and in the lung of PRRSV-inoculated pigs when compared with control group. Since TGFß play a role as an immunomodulatory cytokine of the immune response and also in the differentiation of regulatory T cells (Tregs), the upregulation of the TGFß at tissue level may play a role in the impairment of the host immune response observed during PRRS, being observed a significant correlation between PRRSV and TGFß expression at lung level.

Immunohistochemical detection of extrinsic and intrinsic mediators of apoptosis in porcine paraffin-embedded tissues.

Apoptosis is a strictly regulated mechanism of cell death that involves a complex network of biochemical pathways. Whether a cell undergoes apoptosis or not depends on a delicate balance of anti- and pro-apoptotic stimuli. This phenomenon can be induced by two different pathways: intrinsic and extrinsic pathways. The main aim of this study was to determine the ideal fixative and antigen retrieval method in porcine paraffin embedded tissues for the immunohistochemical detection of apoptosis mediators, from both extrinsic and intrinsic pathways. Tonsil, retropharyngeal lymph node and lung tissue samples were fixed in 10% neutral buffered formalin, Bouin solution and zinc salts fixative (ZSF) and different unmasking methods were carried out. Both 10% neutral buffered formalin and ZSF resulted as the fixatives of election to study apoptosis phenomena. Tween 20 (0.01% in PBS), citrate buffer (microwave, pH 6.0) and/or protease type XIV were the antigen retrieval methods which displayed better labelling. Our results allow to deep in the knowledge of apoptosis and its role in the pathogenesis of porcine diseases.

The integrated action of oestrogen receptor isoforms and sites with progesterone receptor in the gonadotrope modulates LH secretion: evidence from tamoxifen-treated ovariectomized rats.

The specific role of each oestrogen receptor (ER) isoform (alpha and beta ) and site (nucleus and plasma membrane) in LH release was determined in ovariectomized (OVX) rats injected over 6 days (days 15-20 after OVX) with a saturating dose (3 mg/day) of tamoxifen (TX), a selective ER modulator with nuclear ERalpha agonist actions in the absence of oestrogen. This pharmacological effect of TX was demonstrated by the fact that it was blocked by the selective ERalpha antagonist methyl-piperidinopyrazole. Over the past 3 days of the 6-day TX treatment, rats received either 25 microg/day oestradiol benzoate (EB), 1.5 mg/day selective ERalpha agonist propylpyrazole triol (PPT) and the selective ERbeta agonist diarylpropionitrile (DPN), or a single 3 mg injection of the antiprogestin onapristone (ZK299) administered on day 20. Blood samples were taken to determine basal and progesterone receptor (PR)-dependent LH-releasing hormone (LHRH)-stimulated LH secretion and to evaluate LHRH self-priming, the property of LHRH that increases gonadotrope responsiveness to itself. Blood LH concentration was determined by RIA and gonadotrope PR expression by immunohistochemistry. Results showed that i) EB and DPN potentiated the negative feedback of TX on basal LH release; ii) DPN reduced TX-induced PR expression; iii) EB and PPT blocked TX-elicited LHRH self-priming and iv) ZK299 reduced LHRH-stimulated LH secretion and blocked LHRH self-priming. These observations suggest that oestrogen action on LH secretion in the rat is exerted at the classic ERalpha pool and that this action might be modulated by both ERbeta and membrane ERalpha through their effects on PR expression and action respectively.

Oestradiol-17beta inhibits tamoxifen-induced LHRH self-priming blocking hormone-dependent and ligand-independent activation of the gonadotrope progesterone receptor in the rat.

In the rat, administration of tamoxifen (TX) in the absence of oestrogen (E) induces LHRH self-priming, the progesterone receptor (PR)-dependent property of LHRH that increases gonadotrope responsiveness to itself. The oestrogen-dependent PR can be phosphorylated/activated by progesterone (P4) and, in the absence of the cognate ligand, by intracellular LHRH signals, particularly cAMP/protein kinase A. We have recently found that oestradiol-17beta (E2), acting on a putative membrane estrogen receptor-alpha in the gonadotrope, inhibits this agonist action of TX. This study investigated the mechanism by which E2 inhibits TX-elicited LHRH self-priming using both incubated pituitaries from TX-treated ovariectomized (OVX) rats and anterior pituitary cells from OVX rats cultured with TX. It was found that (1) in addition to the inhibitory effect on TX-elicited LHRH self-priming, E2 blocked P4 and adenylyl cyclase activator forskolin augmentation of LHRH-stimulated LH secretion, and (2) E2 did not affect the increasing action of TX on gonadotrope PR expression or pituitary cAMP content. Furthermore, inhibition of protein phosphatases with okadaic acid suppressed E2 inhibition of TX-elicited LHRH-induced LH secretion, while stimulation of protein phosphatases with ceramide blocked TX-induced LHRH self-priming. Together, these results indicated that membrane ER-mediated E2 inhibition of the TX-stimulated LHRH self-priming pathway involves a blockade of gonadotrope PR phosphorylation/activation, but not a deficient response of PR to phosphorylases. Results also suggested that the inhibitory effect of E2 on TX-induced LHRH self-priming is exerted through modulation of cellular protein phosphatase activity in the gonadotrope.