Protein Tyrosine Kinase P56lck Research Articles

How the Discovery of the CD4/CD8-p56lck Complexes Changed Immunology and Immunotherapy.

The past 25 years have seen enormous progress in uncovering the receptors and signaling mechanisms on T-cells that activate their various effecter functions. Until the late 1980s, most studies on T-cells had focused on the influx of calcium and the levels of cAMP/GMP in T-cells. My laboratory then uncovered the interaction of CD4 and CD8 co-receptors with the protein-tyrosine kinase p56lck which are now widely accepted as the initiators of the tyrosine phosphorylation cascade leading to T-cell activation. The finding explained how immune recognition receptors expressed by many immune cells, which lack intrinsic catalytic activity, can transduce activation signals via non-covalent association with non-receptor tyrosine kinases. The discovery also established the concept that a protein tyrosine phosphorylation cascade operated in T-cells. In this vein, we and others then showed that the CD4- and CD8-p56lck complexes phosphorylate the TCR complexes which led to the identification of other protein-tyrosine kinases such as ZAP-70 and an array of substrates that are now central to studies in T-cell immunity. Other receptors such as B-cell receptor, Fc receptors and others were also subsequently found to use src kinases to control cell growth. In T-cells, p56lck driven phosphorylation targets include co-receptors such as CD28 and CTLA-4 and immune cell-specific adaptor proteins such as LAT and SLP-76 which act to integrate signals proximal to surface receptors. CD4/CD8-p56lck regulated events in T-cells include intracellular calcium mobilization, integrin activation and the induction of transcription factors for gene expression. Lastly, the identification of the targets of p56lck in the TCR and CD28 provided the framework for the development of chimeric antigen receptor (CAR) therapy in the treatment of cancer. In this review, I outline a history of the development of events that led to the development of the “TCR signaling paradigm” and its implications to immunology and immunotherapy.

Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

BackgroundUpon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood.MethodsKnock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies.ResultsCo-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation.ConclusionsTaken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR.Major findingsOur data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.FN7ZF7ULbDVsMsT6Sckwh1Video

Molecular characterization of cobia (Rachycentron canadum) CD4 homologues revealed the first evidence of soluble CD4 in fish

The co-receptor CD4 plays an important role in distinguishing between helper T-cell (Th) and cytotoxic T lymphocyte (CTL). In the present study, we investigated the molecular features of CD4-2 cDNA to facilitate understanding of their roles in cobia (Rachycentron canadum). Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. The cDNA of CD4-2A consists of a 993 bp ORF encoding 330 aa with long intracytoplasmic tail containing conserved protein tyrosine kinase p56Lck binding (C-X-C) motif, a transmembrane region, and two extracellular Ig-like (Ig-like) domains are predicted. Comparatively, the cDNA of cobia CD4-2B consists of a 990 bp ORF encoding 329 aa without a transmembrane domain as well as C-X-C motif, and three Ig-like domains are present. Homology comparison showed that the CD4-2A aa sequence of cobia showed high similarity and similar structural features to CD4-2 from other species, while the deduced CD4-2B protein shares higher structural similarity to CD4-1 group. Phylogenetic analysis indicated that cobia CD4-2A was closer with CD4-2 molecules in other fish species, distant from the clade formed by fish CD4-1 and mammalian CD4 sequences. However, cobia CD4-2B grouped with other known teleost CD4-1 sequences. The expression pattern of CD4-2A and CD4-2B mRNA during the embryonic development followed the trend of an initial increase after fertilized, providing evidence of maternal transfer of CD4-2 homologues to the developing cobia embryos and larvae. All of these results are useful for better understanding of cell-mediated immunity of cobia.

High thioredoxin-1 levels in rheumatoid arthritis patients diminish binding and signalling of the monoclonal antibody Tregalizumab.

The humanized non-depleting anti-CD4 monoclonal antibody Tregalizumab (BT-061) is able to selectively activate the suppressive function of regulatory T cells and has been investigated up to phase IIb in clinical trials in patients suffering from rheumatoid arthritis (RA). A pharmacokinetic–pharmacodynamic model based on clinical data from RA and healthy volunteers, which used the cell surface CD4 downmodulation as marker of activity, confirmed a stronger effect in healthy volunteers compared with RA patients. We tried to understand this phenomenon and evaluated the influence of the small oxidoreductase thioredoxin-1 (Trx1). To counteract oxidative stress that is strongly associated with RA pathophysiology, the organism employs Trx1. Therefore, increased expression and secretion of Trx1 is found in the synovial fluid and plasma of RA patients. Moreover, the binding site of Tregalizumab is in close proximity to a disulphide bond in domain 2 (D2) of CD4, which is a known target for a reduction by oxidoreductase Trx1. With the experiments reported herein, we demonstrated that specific reduction of the D2 disulphide bond by Trx1 led to diminished binding of Tregalizumab to recombinant human soluble CD4 and membrane-bound CD4 on T cells. Moreover, we showed that this caused changes in the Tregalizumab-induced CD4 signalling pathway via the lymphocyte-specific protein tyrosine kinase p56Lck and CD4 downmodulation. In summary, we provide evidence that high Trx1 levels in RA patients compared with healthy subjects are a potential reason for diminished binding of Tregalizumab to CD4-positive T cells and offer an explanation for the observed decreased CD4 downmodulation in RA patients in comparison to healthy subjects.

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.

A novel member of lymphocyte-specific protein tyrosine kinase protein identified in lamprey, Lampetra japonica.

Lymphocyte-specific protein tyrosine kinase p56 (Lck) is a member of the Src family of non-receptor protein tyrosine kinase. Lck plays an important role in mediating T-cell receptor (TCR) signal transduction and the development, differentiation, proliferation, and activation of T-cells [1]. Lck contains N-terminus of myristylation sequence, a unique aminoterminus region, followed by Src homology 3 (SH3) and SH2 domains and a C-terminus of tyrosine kinase catalytic domain [2]. Lck can associate with the inner face of the plasma membrane through its myristoyl glycine and palmitoyl cysteines in the amino-terminus [3].Following the myristylation sequence, there is the unique region, a short region of 80 amino acids. This unique region is involved in the interaction of Lck with specific cellular proteins [4,5]. Downstream of this unique region are SH3 and SH2 domains which are involved in protein–protein interactions [6]. The tyrosine kinase domain is the catalytic domain of Lck catalyzing the transfer of the gamma-phosphate from ATP to tyrosine residues in proteins. The catalytic domain of human Lck contains a site of autophosphorylation (Tyr-394), which plays an important role in regulating the protein kinase activity [7]. Agnathans, represented by lamprey and hagfish, are the oldest vertebrates currently identified possessing the adaptive immune defenses [8]. A recent study of jawless vertebrate has provided a clue for the origin of adaptive immune defense. Though TCR and B-cell receptor system do not exist in jawless vertebrates, lamprey has been confirmed to possess an alternative immune system that could specifically recognize and respond to external pathogens [9]. The handling of lamprey (Lampetra japonica) and all experimental procedures were approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University. Adult lampreys were purchased from Tongjiang section of the Heilongjiang River (Tongjiang, China) in December. Adult lampreys (200–220 g in weight) were divided into two groups (20 animals per group); one group of animals was immunized with 0.1 mg of lipopolysaccharide (LPS) (Sigma-Aldrich, St Louis, USA) in 0.1 ml phosphate-buffered saline (PBS), and the control animals were injected with 0.1 ml PBS only. The animals were immunized at 8-day intervals by four intraperitoneal injections. Based on the expressed sequence tag analysis of the cDNA library which was constructed with lamprey lymphocyte-like cells in our laboratory, a Lck homolog was found using Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI; http://www. ncbi.nlm.nih.gov/). Total RNA was isolated from lamprey lymphocyte-like cells [10] using RNAiso (TaKaRa Biotechnology, Dalian, China) reagent following the manufacturer instructions, and dissolved in Diethyl pyrocarbonate-treated water and stored at 2808C. First strand 30and 50-RACEcDNAs were synthesized from 5 mg of total RNA by Reverse Transcriptase M-MLV at 308C for 10 min, 428C for 30 min, 708C for 15 min, 958C for 5 min, 48C for 60 min with the 30-coding sequence primer and 50-coding sequence primer and Random 9-mers primer following the manufacturer instructions (TaKaRa Biotechnology). The 30and 50-end sequences of Lj-Lck were obtained by polymerase chain reaction (PCR) with outer primer, inner primer (TaKaRa Biotechnology), and specific primers (Supplementary Table S1). LA Taq DNA polymerase (TaKaRa Biotechnology) was used for amplification with the following cycling conditions: 948C for 3 min, followed by 40 amplification cycles at 948C for 30 s, 558C for 30 s, 728C for 2 min, and a final extension step at 728C for 10 min. Products were analyzed by electrophoresis in a 2% agarose gel stained with ethidium bromide. The target band of PCR product was isolated and purified, subcloned into Acta Biochim Biophys Sin 2014, 46: 820–825 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu066. Advance Access Publication 25 July 2014

P56(lck) kinase inhibitor studies: a 3D QSAR approach towards designing new drugs from flavonoid derivatives.

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) based on 3D-QSAR (3D-quantitative structure activity relationship) studies were carried out on 97 flavonoid derivatives as potent P56(lck) protein tyrosine kinase inhibitors. The best prediction was obtained with CoMFA standard model (q² = 0.838, r² = 0.948) using steric, electrostatic along with CoMSIA standard model (q² = 0.714, r² = 0.921) using steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields. Of the 97 molecules a training set of 76 compounds and the predictive ability of the QSAR model were assessed employing a test set of 21 compounds. The resulting CoMFA and CoMSIA contour maps were used to identify the structural features relevant to the biological activity in this series of flavonoid derivatives, based upon which we identified and designed 10 novel molecules that showed superior inhibitory activity against P56(lck) protein which shed new light on effective therapeutic agents against these classes of enzymes.

A transgenic mouse model of human T cell leukemia virus type 1-associated diseases

Human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia/lymphoma (ATLL) and several inflammatory diseases. Tax, the protein encoded by HTLV-1, may be responsible for the development of the diseases caused by this virus. To investigate the pathogenic role of Tax, several transgenic mouse strains expressing Tax have been developed in recent years. These mice develop various tumors including large granular lymphocytic leukemia, as well as inflammatory diseases such as arthritis. These results suggest that Tax expression alone is sufficient to cause both malignant neoplastic diseases and inflammatory diseases. However, until recently, there were no tax transgenic mice that develop T cell leukemia and lymphoma resembling ATLL. The first successful induction of leukemia in T cells was pre-T cell leukemia generated in transgenic mice in which a mouse lymphocyte-specific protein tyrosine kinase p56lck (lck)-proximal promoter was used to express the tax gene in immature T cells. Subsequently, transgenic mice were established in which the lck-distal promoter was used to express Tax in mature T cells; these mice developed mature T cell leukemia and lymphoma that more closely resembled ATLL than did earlier mouse models.

In vivo study of tyrosine protein kinase Lck inhibited by siRNA in T cells of asthmatic mice

Objective Using the technology of siRNA to inhibit the gene expression of no-receptor tyrosine protein kinase Lck in T cells of asthmatic mice,and to study the therapeutic effect of Lck specific siRNA in asthmatic mice.Methods Receptor tyrosine protein kinase Lck specific siRNA fragments were taken from chemosynthesis.In vivo-jetPEITM was used to transfect the siRNA into mice body through tail vein injection.The mice were killed 48 hours later,and the levels of IL-4,IL-17 in bronchoalveolar lavage fluid (BALF) were detected with respondent ELISA kits.The change of inflammatory histopathology in lung was observed with H.E.staining.The expression of Lck in lung was detected with immunohistochemistry (IHC),and the level of Lck in lung tissue homogenate was detected with Western Blot.Results Compared with asthmatic group[ (234.68 ± 11.15 ) pg/ml,( 96.76 ± 8.28 ) pg/ml],the levels of IL-4,IL-17 [ (234.68 ± 11.15)pg/ml,(96.76 ±8.28) pg/ml] in the BALF of siRNA interference group decreased, and the inflammation in the lung relieved.IHC indicated that the expression of Lck in lung decreased and the level of Lck in lung tissue homogenate decreased ( P ＜ 0.05 ).Conclusions Lck specific siRNA could reduce the level of IL-4,IL-17 in the lung tissues of asthmatic mice,and relieve the inflammatory reaction in lung. Key words: RNA, small interfering ; Asthma/TH/ME ; Lymphocyte specific protein tyrosine kinase p56(lck)/ME

Proteasome inhibitor MG132-induced apoptosis via ER stress-mediated apoptotic pathway and its potentiation by protein tyrosine kinase p56lck in human Jurkat T cells

Exposure of human Jurkat T cells to MG132 caused apoptosis along with upregulation of Grp78/BiP and CHOP/GADD153, activation of JNK and p38MAPK, activation of Bak, mitochondrial membrane potential (Δψm) loss, cytochrome c release, activation of caspase-12, -9, -3, -7, and -8, cleavage of Bid and PARP, and DNA fragmentation. However, these MG132-induced apoptotic events, with the exceptions of upregulation of Grp78/BiP and CHOP/GADD153 and activation of JNK and p38MAPK, were abrogated by overexpression of Bcl-xL. Pretreatment with the pan-caspase inhibitor z-VAD-fmk prevented MG132-induced apoptotic caspase cascade, but allowed upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of JNK and p38MAPK, Δψm loss, and cleavage of procaspase-9 (47kDa) to active form (35kDa). Further analysis using selective caspase inhibitors revealed that caspase-12 activation was required for activation of caspase-9 and -3 to the sufficient level for subsequent activation of caspase-7 and -8. MG132-induced cytotoxicity, apoptotic sub-G1 peak, Bak activation, and Δψm loss were markedly reduced by p38MAPK inhibitor, but not by JNK inhibitor. MG132-induced apoptotic changes, including upregulation of Grp78/BiP and CHOP/GADD153 levels, activation of caspase-12, p38MAPK and Bak, and mitochondria-dependent activation of caspase cascade were more significant in p56lck-stable transfectant JCaM1.6/lck than in p56lck-deficient JCaM1.6/vector. The cytotoxicity of MG132 toward p56lck-positive Jurkat T cell clone was not affected by the Src-like kinase inhibitor PP2. These results demonstrated that MG132-induced apoptosis was caused by ER stress and subsequent activation of mitochondria-dependent caspase cascade, and that the presence of p56lck enhances MG132-induced apoptosis by augmenting ER stress-mediated apoptotic events in Jurkat T cells.

Th2-Specific Immunity and Function of Peripheral T Cells Is Regulated by the p56Lck Src Homology 3 Domain

T cell activation and effector function is essential for robust immunity. Ag TCR signals are known to regulate T lymphocyte differentiation, but the mechanisms involved in this regulation remain unclear. Recent work has demonstrated that the Src family protein tyrosine kinase p56Lck specifically links TCR signaling to activation of the MAPK pathway through the function of its Src homology 3 (SH3) domain. The MAPK pathway is involved in T cell activation and has previously been implicated in Th2 immunity. We have used Lck SH3 mutant knockin mice (LckW97A) to investigate the potential role of this regulatory mechanism in T lymphocyte activation and effector function. Our results demonstrate that Lck SH3 domain function regulates activation of T lymphocytes as indicated by reduced IL-2 production, CD69 induction, and proliferation of LckW97A T cells following TCR stimulation. Biochemical studies confirm that activation of the MAPK pathway is selectively altered following TCR ligation in LckW97A T lymphocytes. Phospho-ERK induction is reduced, but phospho-phospholipase Cgamma1 induction and calcium mobilization are largely unaffected. Immunization with DNP-keyhole limpet hemocyanin, heat-killed Brucella abortus, or infection with Nippostrongylus brasiliensis demonstrates selectively impaired Th2 immunity with reduced serum levels of IgG1, IgE, and IL-4. In vitro studies show that LckW97A T cells can differentiate into Th2-type cells, but they form IFN-gamma-producing cells under conditions that normally favor Th2 development. These data indicate that the Lck SH3 domain controls T lymphocyte activation by regulating MAPK pathway induction and demonstrate a novel role for Lck in the regulation of Th2-type immunity.

QSAR study of p56(lck) protein tyrosine kinase inhibitory activity of flavonoid derivatives using MLR and GA-PLS.

Quantitative relationships between molecular structure and p56lck protein tyrosine kinase inhibitory activity of 50 flavonoid derivatives are discovered by MLR and GA-PLS methods. Different QSAR models revealed that substituent electronic descriptors (SED) parameters have significant impact on protein tyrosine kinase inhibitory activity of the compounds. Between the two statistical methods employed, GA-PLS gave superior results. The resultant GA-PLS model had a high statistical quality (R2 = 0.74 and Q2 = 0.61) for predicting the activity of the inhibitors. The models proposed in the present work are more useful in describing QSAR of flavonoid derivatives as p56lck protein tyrosine kinase inhibitors than those provided previously.

Genomic organization and expression of CD8α and CD8β genes in fugu Takifugu rubripes

We have cloned cDNAs encoding the α and β chains of CD8 from the tiger pufferfish (fugu), Takifugu rubripes. The cDNA sequences encode a putative leader peptide, extracellular immunoglobulin variable region-like domain, stalk region, transmembrane region, and cytoplasmic tail. A protein tyrosine kinase p56 lck binding motif was not found in the putative fugu CD8α cytoplasmic tail. O-linked glycosylation sites were found in the stalk of both CD8 chains, suggesting possible stalk formation. Phylogenetic analysis showed that fugu CD8α and CD8β chains cluster with other vertebrate CD8α and CD8β chains, respectively. The fugu CD8 genes comprise six exons separated by five introns. The genes are tandemly aligned 3.6 kb apart and are in the same transcription orientation. Quantitative RT-PCR analysis demonstrated that fugu CD8 is expressed predominantly in lymphoid tissues. In situ hybridization showed that fugu CD8 genes are expressed in thymocytes and lymphocytes within lymphoid organs. Molecular characterization of CD8 in fish provides the basis for development of specific antibodies to identify T-cell subsets, as well as potentially important insights into the evolution of CD8 and the adaptive immunity.

Decay-Accelerating Factor Ameliorates Systemic Autoimmune Disease in MRL/lpr Mice via Both Complement-Dependent and -Independent Mechanisms

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol-anchored membrane protein that restricts complement activation on autologous cells. Previous studies have established a significant protective activity of DAF in the MRL/lpr murine model of human systemic lupus erythematosus. To dissect the mechanism of protection by DAF in this disease model, we evaluated the effect of C3 gene ablation on disease development in MRL/lpr-Daf-1(-/-) mice. We found no significant difference in lymphadenopathy, splenomegaly, or anti-chromatin autoantibody titer between complement-sufficient and complement-deficient MRL/lpr-Daf-1(-/-) mice. On the other hand, complement deficiency strikingly reduced the incidence and severity of dermatitis in MRL/lpr-Daf-1(-/-) mice. To assess the contribution of DAF expression on lymphocytes versus local tissues in suppressing dermatitis, we generated BM chimeric mice between MRL/lpr-Daf-1(-/-) and MRL/lpr-Daf-1(+/+) mice. Compared with MRL/lpr-Daf-1(-/-) --> MRL/lpr-Daf-1(-/-) controls, MRL/lpr-Daf-1(-/-) --> MRL/lpr-Daf-1(+/+) chimeras developed significantly attenuated dermatitis, suggesting that the protective effect of DAF in suppressing dermatitis is primarily attributable to its local expression. We conclude that DAF works as a complement regulator in the skin to protect MRL/lpr mice from skin inflammation, whereas its inhibitory role in the induction phase of MRL/lpr autoimmunity is complement-independent. Together, these results reveal multiple mechanisms of action for DAF in ameliorating systemic autoimmunity.

QSAR Study of Benzothiazole Derivatives as p56lck Inhibitors

Protein tyrosine kinase (p56lck) is known for its role in T cells, in which deficiency of Lck is shown to impair cell activation via T-cell antigen receptor. Benzothiazoles have been reported to be the molecules of interest, with potent anticancer activity and they act by binding to ATP binding site of protein kinases. ATP binding site of protein kinases provided an extensive opportunity to design newer analogs. With this background, we report an attempt to discern the structural and physicochemical requirements for inhibition of p56lck. QSAR studies were performed on a set of 45 analogs of benzothiazoles using Molecular Operating Environment (MOE) software. The models developed gave good predictive correlation coefficient (r2pred) of 0.765. A training set of 35 compounds was developed with correlation coefficient (r2) of 0.983 and a significant cross validated correlation coefficient (q2) of 0.723. Various sets of descriptors to meet the orthogonality of model to ensure that each descriptor is encoding different properties from others were used. Subdivided surface area, water accessible surface area, partial charge and adjacency and distance matrix were the major contributing descriptors. Leave one out and $ Z-score values were used to optimize the model. The model developed to predict the structural features of benzothiazole, revealed useful information about the structural features required for the molecules to inhibit p56lck. Major contributing descriptors were subdivided surface area, partial charges and water accessible surface area. Keywords: CD4 molecules, cell regulatory processes, QSAR model, ATP binding, benzothiazole derivatives

Signalling during hypoxia in human T lymphocytes – critical role of the src protein tyrosine kinase p56Lck in the O2 sensitivity of Kv1.3 channels

T lymphocytes encounter hypoxia when they migrate to pathological sites such as tumours and wounds. The inability of T cells to provide an efficient defence at these sites can in part be explained by the hypoxic environment. Kv 1.3 channels, important components of the T cell activation process are inhibited by hypoxia and their inhibition accounts for a hypoxia-induced decrease in T cell proliferation. Although Kv 1.3 channels play a key role in T cell O(2) sensing, the signalling mechanisms mediating their response to hypoxia are still not understood. In this study, we show that the src-protein tyrosine kinase p56Lck (Lck) is required for Kv 1.3 channel response to hypoxia. Pre-exposure to the src inhibitor PP2 abolished the hypoxia-induced inhibition of Kv 1.3 channels in primary human T lymphocytes. Moreover, Kv 1.3 channel sensitivity to hypoxia was lost in Lck-deficient Jurkat T cells. Further studies with recombinant Kv 1.3 channels showed that Kv 1.3 channels lack intrinsic O(2) sensitivity, but delivery of Lck into the cells and transfection of a constitutively active Lck (Y505FLck) restored their sensitivity to hypoxia. Although Lck is necessary for the Kv 1.3 channel response to hypoxia, it does not directly inhibit Kv 1.3 channels. Indeed, under normal oxygen tension, delivery of active Lck into L929 cells and overexpression of Y505FLck did not decrease recombinant Kv 1.3 currents. On the contrary, activation of endogenous src kinases increased wild-type Kv 1.3 currents in T lymphocytes. Our findings indicate that Lck is required for the acute response to hypoxia of human T lymphocytes as it is necessary to confer O(2) sensitivity on Kv 1.3 channels.

Multiple populations of T lymphocytes are distinguished by the level of CD4 and CD8 coexpression and require individual consideration

Expression of CD4 or CD8 on T cells is pivotal in defining them as T helper or T cytotoxic cells, respectively. Aside from an intermediate stage during T cell maturation in the thymus, CD4 and CD8 molecules were thought to be mutually exclusive on mature T cells. However, accumulating evidence has proven that these two molecules can be coexpressed on mature T cells [1–7]. Populations of CD8 T cells re-expressing CD4 have been designated CD4CD8 or CD4CD8 T cells, and likewise, populations of CD4 T cells re-expressing CD8 have been designated CD4CD8 or CD4CD8 T cells. Much of what we know about CD4 CD8 T cells is based on literature that has concentrated on the CD4CD8 T cell population. These studies collectively established the importance of studying CD4 CD8 T cell populations through demonstrating that the CD4 molecule expressed on CD8 T cells is functional, as indicated by several lines of evidence, including the ability to coprecipitate CD4 with the Scr family protein tyrosine kinase p56lck [1] and the ability of CD4 on CD8 T cells to mediate chemotaxis in response to interleukin-16, a CD4-specific ligand [3]. We have shown that CD4CD8 T cells recognize viral [cytomegalovirus (CMV)] antigen more potently than CD8 T cells, which do not re-express CD4 on their surface (CD4CD8 T cells) [6]. Recent data suggest that CD4CD8 T cells function as CD8 effector cells, as assessed in the severe combined immunodeficient (SCID) model of lymphocytic choriomeningitis virus (LCMV) and allogenic immunity [8, 9] and in the chimpanzee model of hepatitis C virus immunity [7]. In analyzing the role of CD4 on CD8 T cells and the biologic significance of this cell population, it is critical that these cells are defined appropriately and that no contaminating populations are included in the analysis. Based on a limited number of markers shown to be shared by both populations, some investigators have combined the analysis of CD4CD8 and CD4CD8 T cells as one population, defining all double-positive (CD4 CD8 ) T cells [7]. Combining these populations to deduce functional potential is problematic. Our own analyses, when collecting a minimum of 100,000 events per sample for immunostaining and using CD4 antibodies conjugated to phycoerythrin (PE) or allophycocyanin (APC), demonstrate that multiple subpopulations of T cells, defined by the level of their expression of CD4 and CD8, exist within the CD4 CD8 double-positive T cell population. As seen in Figure 1A, based on the intensity of CD4 and CD8 expression, six different populations are apparent: the singlepositive populations include CD8 CD4 (denoted as P1) and CD4 CD8 (denoted as P6), and the double-positive populations include CD4CD8 (denoted as P2), CD4CD8 (denoted as P3), CD4 CD8 (denoted as P4), and CD4CD8 (denoted as P5). Given that NKT cells can be CD4 CD8 , CD4CD8, CD4CD8 , or CD4 CD8, it is possible that CD4 CD8 NKT cells may be present within the subpopulations of CD4 CD8 T cells to varying degrees. Therefore, we examined the frequency of NKT cells within the CD4 CD8 T cell subpopulations. We demonstrate that invariant NKT cells, defined by coexpression of CD3 and 6B11 [10], and non-invariant NKT cells, defined by coexpression of CD3 and CD16/56, are present within these CD4 CD8 T cell subpopulations at varying frequencies (Fig. 1, C and D). Expression of invariant NKT and non-invariant NKT markers is most evident in the CD4CD8 (P5) subpopulation, where up to 29% of the cells were found to be invariant CD3 6B11 NKT cells, and up to 26% of the cells were found to be non-invariant CD3 CD16/56 NKT cells. Our studies here show that in characterizing the role of cells coexpressing CD4 and CD8, it is critical that multiple CD4 CD8 T cell subpopulations (CD4CD8, CD4CD8, CD4 CD8 , and CD4CD8) are not evaluated together as one double-positive population. Combining these populations, which may not share similar characteristics and may express differentiation and activation surface markers to varying degrees, and not depleting certain populations of contaminating cells (i.e., NKT cells) will mask the lineage and functional potential analysis of each individual subpopulation of CD4 CD8 T cells.

Apoptosis of Antigen-Specific T Lymphocytes upon the Engagement of CD8 by Soluble HLA Class I Molecules Is Fas Ligand/Fas Mediated: Evidence for the Involvement of p56lck, Calcium Calmodulin Kinase II, and Calcium-Independent Protein Kinase C Signaling Pathways and for NF-κB and NF-AT Nuclear Translocation

The binding of soluble HLA class I (sHLA-I) molecules to CD8 on EBV-specific CTL induced up-regulation of Fas ligand (FasL) mRNA and consequent sFasL protein secretion. This, in turn, triggered CTL apoptosis by FasL/Fas interaction. Molecular analysis of the biochemical pathways responsible for FasL up-regulation showed that sHLA-I/CD8 interaction firstly induced the recruitment of src-like p56(lck) and syk-like Zap-70 protein tyrosine kinases (PTK). Interestingly, p59(fyn) was activated upon the engagement of CD3/TCR complex but not upon the interaction of sHLA-I with CD8. In addition, sHLA-I/CD8 interaction, which is different from signaling through the CD3/TCR complex, did not induce nuclear translocation of AP-1 protein complex. These findings suggest that CD8- and CD3/TCR-mediated activating stimuli can recruit different PTK and transcription factors. Indeed, the engagement of CD8 by sHLA-I led to the activation of Ca2+ calmodulin kinase II pathway, which eventually was responsible for the NF-AT nuclear translocation. In addition, we found that the ligation of sHLA-I to CD8 recruited protein kinase C, leading to NF-kappaB activation. Both NF-AT and NF-kappaB were responsible for the induction of FasL mRNA and consequent CTL apoptosis. Moreover, FasL up-regulation and CTL apoptotic death were down-regulated by pharmacological specific inhibitors of Ca2+/calmodulin/calcineurin and Ca2+-independent protein kinase C signaling pathways. These findings clarify the intracellular signaling pathways triggering FasL up-regulation and apoptosis in CTL upon sHLA-I/CD8 ligation and suggest that sHLA-I molecules can be proposed as therapeutic tools to modulate immune responses.

Docosahexaenoic acid changes lipid composition and interleukin-2 receptor signaling in membrane rafts

Polyunsaturated fatty acids, including docosahexaenoic acid (DHA, 22:6n-3), modulate immune responses and exert beneficial immunosuppressive effects, but the molecular mechanisms inhibiting T-cell activation are not yet elucidated. Lipid rafts have been shown to play an important role in the compartmentalization and modulation of cell signaling. We investigated the role of DHA in modulating the lipid composition in lipid rafts and membrane subdomain distribution of interleukin-2 (IL-2) receptor signaling molecules. We found that DHA altered lipid components of rafts and modified the IL-2-induced Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway by partially displacing IL-2 receptors from lipid rafts. We fractionated plasma membrane subcellular compartments and discovered that certain amounts of STAT5a and STAT5b existed in detergent-resistant plasma membrane fractions of T-cells. After DHA treatment, STAT5a and STAT5b were not detected in lipid raft fractions and were located in detergent-soluble fractions. These data demonstrate for the first time that DHA alters the lipid composition of membrane microdomains and suppresses IL-2 receptor signaling in T-cells. Thus, our data provide evidence for a functional modification in lipid rafts by DHA treatment and explain PUFA-mediated immunosuppressive effects.

Identification and characterization of a second CD4-like gene in teleost fish

In fish, T cell subdivision is not well studied, although CD8 and CD4 homologues have been reported. This study describes a second teleost CD4-like gene, CD4-like 2 ( CD4L-2). Two rainbow trout copies of this gene were found, -2a and -2b, encoding molecules sharing 81% aa identity. The 2a/ 2b duplication may be related to tetraploid ancestry of salmonid fishes. In the Fugu genome CD4L-2 lies head to tail with an earlier reported, very different CD4-like gene [Suetake, H., Araki, K., Suzuki, Y., 2004. Cloning, expression, and characterization of fugu CD4, the first ectothermic animal CD4. Immunogenetics 56, 368–374], which was designated CD4L-1 in the present article. The flanking genes of the Fugu CD4L-1 and CD4L-2 are reminiscent of the genes surrounding CD4 and LAG-3 in mammals. However, neither synteny nor phylogenetic analysis could decide between CD4 and LAG-3 identity for the fish CD4L genes. CD4L-1 and CD4L-2 share a tyrosine protein kinase p56 lck binding motif in the cytoplasmic tail with CD4 but not with LAG-3. Trout CD4L-2 expression is highest in the thymus, similar to mammalian and chicken CD4, whereas Fugu CD4L-1 expression was highest in the spleen. However, CD4L-2 encodes only two IG-like domains, whereas CD4L-1, CD4 and LAG-3 encode four. The CD4- like genes 1 and 2 in fish apparently went through an evolution different from that of LAG-3 and CD4 in higher vertebrates.