Cell Antigen Receptor Complex Research Articles

Human inborn errors of immunity caused by defects of receptor and proteins of cellular membrane.

Inborn errors of immunity are diseases of the immune system resulting from mutations that alter the expression of encoded proteins or molecules. Total updated number of these disorders is currently 406, with 430 different identified gene defects involved. Studies of the underlying mechanisms have contributed in better understanding the pathophysiology of the diseases, but also the complexity of the biology of innate and adaptive immune system and its interaction with microbes. In this review we present and briefly discuss Inborn Errors of Immunity caused by defects in genes encoding for receptors and protein of cellular membrane, including cytokine receptors, T cell antigen receptor (TCR) complex, cellular surface receptors or receptors signaling causing predominantly antibody deficiencies, co-stimulatory receptors and others. These alterations impact many biological processes of immune-system cells, including development, proliferation, activation and down-regulation of the immunological response, and result in a variety of diseases that present with distinct clinical features or with overlapping signs and symptoms.

CD45 functions as a signaling gatekeeper in T cells.

T cells require the protein tyrosine phosphatase CD45 to detect and respond to antigen because it activates the Src family kinase Lck, which phosphorylates the T cell antigen receptor (TCR) complex. CD45 activates Lck by opposing the negative regulatory kinase Csk. Paradoxically, CD45 has also been implicated in suppressing TCR signaling by dephosphorylating the same signaling motifs within the TCR complex upon which Lck acts. We sought to reconcile these observations using chemical and genetic perturbations of the Csk/CD45 regulatory axis incorporated with computational analyses. Specifically, we titrated the activities of Csk and CD45 and assessed their influence on Lck activation, TCR-associated ζ-chain phosphorylation, and more downstream signaling events. Acute inhibition of Csk revealed that CD45 suppressed ζ-chain phosphorylation and was necessary for a regulatable pool of active Lck, thereby interconnecting the activating and suppressive roles of CD45 that tune antigen discrimination. CD45 suppressed signaling events that were antigen independent or induced by low-affinity antigen but not those initiated by high-affinity antigen. Together, our findings reveal that CD45 acts as a signaling "gatekeeper," enabling graded signaling outputs while filtering weak or spurious signaling events.

Abstract A074: A Zap70-dependent negative feedback loop governs Lck activation by CD45

Abstract The kinase Lck phosphorylates the T cell antigen receptor (TCR) complex when antigen is encountered and therefore its activity sets a critical threshold for T cell activation. Yet how Lck activity is regulated in resting or stimulated cells remains poorly understood. We have identified that a cryptic phosphosite within Lck, Y192, profoundly affects Lck activity when mutated. Notably, mutation of Y192 blocks critical TCR proximal signaling events including phosphorylation of the effector kinase Zap70 and subsequent calcium response. Accordingly, thymocyte development in retrogenic mice harboring a Y192 mutation is severely impaired. These mice are unable to produce mature T cells and accumulate double negative and double positive (CD4/CD8) thymocytes. We determined that these defects are caused by hyperphosphorylation of the canonical inhibitory phosphorylation site (Y505) within Lck's C-terminus. Lck's C-terminus is phosphorylated by the inhibitory kinase Csk and we have employed a regulatable reconstituted system to acutely inhibit Csk activity. Upon Csk inhibition, the phosphatase CD45 dephosphorylates Lck's C-terminus (Y505). Interestingly, mutation of Y192 disrupts CD45's ability to dephosphorylate Y505 and activate Lck. Because Y192 phosphorylation decreases in response to small molecule inhibition of Zap70, an Lck substrate, our findings support a mechanism of negative feedback. We predict that tonic or basal signaling events that occur in the absence of TCR ligand use negative feedback, including Y192 phosphorylation, to tune Lck activity. Because modulation of Lck activity impacts TCR sensitivity, feedback mechanisms are likely critical to establishing a threshold for T cell activation. Citation Format: Adam H. Courtney, Theresa A. Kadlecek, Byron Au-Yeung, Arthur Weiss. A Zap70-dependent negative feedback loop governs Lck activation by CD45 [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A074.

Characterization and expression analysis of B Cell receptor accessory molecule CD79 gene in humphead snapper (Lutjanus sanguineus)

CD79, a key component of the B cell antigen receptor complex, is composed of CD79α(Igα) and CD79β(Igβ) encoded by mb-1 and B29 respectively, and plays an important role in B cell signaling. In this study, we isolated and characterized mb-1 and B29 from humphead snapper (Lutjanus sanguineus). Their tissue distribution and expression profiles after stimulations in vitro and in vivo were also investigated. The humphead snapper mb-1 and B29 contain open reading frames of 684 bp and 606 bp, encoding 227 amino acids and 201 amino acids, respectively. Both CD79α and CD79β possess signal peptide, extracellular Ig domain, transmembrane region and immunoreceptor tyrosine kinase activation motif (ITAM). Mb-1 is highly expressed in lymphoid organs (thymus, posterior kidney and spleen) and mucosal-associated lymphoid tissues (gill and intestine), while B29 is mainly detected in posterior kidney, spleen, gill and skin. Furthermore, transcription of mb-1 and B29 in head kidney leucocytes was up-regulated following lipopolysaccharide (LPS), pokeweed mitogen (PWM), and polyinosinic-polycytidylic acid (PolyI:C) stimulation, respectively, and their expression level in anterior kidney and spleen was also increased after challenged with formalin-inactived Vibrio harveyi. These results indicated that humphead snapper CD79 molecule might play an important role in immune response to pathogen infection.

SHARPIN controls regulatory T cells by negatively modulating the T cell antigen receptor complex.

SHARPIN forms a linear-ubiquitin-chain-assembly complex that promotes signaling via the transcription factor NF-κB. SHARPIN deficiency leads to progressive multi-organ inflammation and immune system malfunction, but how SHARPIN regulates T cell responses is unclear. Here we found that SHARPIN deficiency resulted in a substantial reduction in the number of and defective function of regulatory T cells (Treg cells). Transfer of SHARPIN-sufficient Treg cells into SHARPIN-deficient mice considerably alleviated their systemic inflammation. SHARPIN-deficient T cells displayed enhanced proximal signaling via the T cell antigen receptor (TCR) without an effect on the activation of NF-κB. SHARPIN conjugated with Lys63 (K63)-linked ubiquitin chains, which led to inhibition of the association of TCRζ with the signaling kinase Zap70; this affected the generation of Treg cells. Our study therefore identifies a role for SHARPIN in TCR signaling whereby it maintains immunological homeostasis and tolerance by regulating Treg cells.

Abstract B042: Regulation of Lck activity by a cryptic phosphosite

Abstract Src family kinases (SFKs) are initiators of receptor-mediated signaling events, enabling cells to perceive and interact with their environment. For immune cells SFKs are especially crucial because they couple antigen recognition to intracellular signaling pathways. In T cells, the SFK Lck phosphorylates the T cell antigen receptor (TCR) complex upon antigen encounter. Because Lck phosphorylates the TCR complex, its activity sets a critical threshold for T cell activation; yet how Lck activity is regulated in resting or stimulated cells is poorly understood. Through quantitative mass spectrometry (SILAC), we recently identified a cryptic Lck phosphosite (Y192) as sensitive to inhibition of the effector kinase Zap70. Because Zap70 is an Lck substrate, and dramatic reduction in phosphorylation of Y192 occurred upon Zap70 inhibition, we reasoned this site might function to regulate Lck in response to changes in Zap70 activity. To test this hypothesis we reconstituted Lck-deficient Jurkat T cells with Lck Y192 variants. Mutation of Y192 reveals that this site has a profound impact on Lck activity. Cells reconstituted with Y192E or Y192A show a marked reduction in Lck activity, characterized by hyperphosphorylation on Lck's inhibitory C-terminus (Y505) and a reduction in Lck autophosphorylation within the activation loop (Y394). Consistent with a defect in Lck activity, cells that express Lck Y192E/A variants, when assessed for hallmarks of T cell activation, reveal striking defects in TCR signaling. Interestingly, these defects are reminiscent of CD45-deficient T cells. Preliminary findings suggest that mutation of Y192 disrupts the ability of the phosphatase CD45 to dephosphorylate Lck's inhibitory C-terminus (Y505). Because of the profound effect that Y192 modification exerts on Lck activity, and therefore TCR signaling, we believe that phosphorylation of Y192 comprises a Zap70-dependent negative feedback mechanism that tunes Lck activity. Such a mechanism predicts that increases in Zap70 activity cause phosphorylation of Y192 within Lck's SH2 domain, favouring Lck inactivation by preventing CD45-mediated dephosphorylation of the inhibitory C-terminus. Citation Format: Adam H. Courtney, Hanna E. Sjolin-Goodfellow, Qinqin Ji, Theresa A. Kadlecek, Arthur R. Salomon, Arthur Weiss. Regulation of Lck activity by a cryptic phosphosite. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B042.

Modulation of Igβ is essential for the B cell selection in germinal center.

The positive and negative selection of antigen-reactive B cells take place in the germinal center (GC) during an immune responses. However, the precise molecular mechanisms underlying these selection machineries, including the involvement of antigen receptor signaling molecules, remain to be elucidated. We found that expression levels of Igα and Igβ, which are the essential components of B cell antigen-receptor complex, were differentially regulated in GC B cells and that the expression of Igβ was more prominently down-regulated in a portion of GC B cells. The suppression of Igβ down-regulation reduced the number of GL7+GC B cells and the affinity maturation in T-dependent responses was markedly impaired. In addition, the disease phenotypes in autoimmune-prone mice were ameliorated by blocking of Igβ down-regulation. These results suggest that Igβ down-regulation is involved in the normal positive selection in GC and the accumulation of autoreactive B cells in autoimmune-prone mice.

Suppression of rheumatoid arthritis B cells by XmAb5871, an anti-CD19 antibody that coengages B cell antigen receptor complex and Fcγ receptor IIb inhibitory receptor.

Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. The aim of this study was to characterize B cell immunosuppression mediated by the Fc-engineered antibody, XmAb5871, which coengages FcγRIIb with the B cell antigen receptor (BCR) complex and that is currently in clinical development for the treatment of rheumatoid arthritis (RA). Because rheumatoid factor (RF) might interfere with the binding of XmAb5871 to FcγRIIb, we correlated RF titers with the potency of XmAb5871. We analyzed the expression of CD19, FcγRIIb, and CD86 on naive and memory B cells from 50 patients with RA and 66 healthy donors, quantified XmAb5871-induced promotion of FcγRIIb phosphorylation and suppression of calcium flux in activated B cells, measured CD86 inhibition in whole blood, and correlated RF and anti-citrullinated protein antibody (ACPA) levels with drug potency. We engrafted RA peripheral blood mononuclear cells (PBMCs) into SCID mice, treated them with XmAb5871, and quantified human total IgG, total IgM, and anti-tetanus IgG antibody levels in vivo. B cells from all donors expressed CD19 and FcγRIIb, and the expression of FcγRIIb was higher on naive, but not memory, B cells from donors with RA compared with healthy donors. BCR-mediated calcium flux was suppressed by XmAb5871 and was associated with FcγRIIb phosphorylation. XmAb5871 inhibited CD86 induction, and the levels of RF and ACPAs did not affect efficacy. XmAb5871 suppressed B cell activation regardless of disease severity. In SCID mice engrafted with PBMCs from a patient with RA, XmAb5871 suppressed humoral responses. Coengagement of the BCR complex and FcγRIIb by XmAb5871 inhibits B cell activation and function. The similar potency in patients with RA and healthy donors and the absence of autoantibody interference suggest that XmAb5871 may represent a new therapeutic strategy to suppress autoreactive B cells in RA.

Involvement of natural immunoglobulin M secreted by epithelial cells in innate immune response (P4155)

Abstract It is well known that B-1 B cells are the main cell type which is responsible for the production of natural immunoglobulin M (IgM) and can respond to infection by increasing IgM secretion. However, in this study, we reported that some epithelial cells also can secrete natural IgM that has natural IgM characteristics. 1. Epithelial cell-derived IgM revealed germline-encoded and restricted VDJ/VJ rearrangement patterns. 2. Epithelial cells can spontaneously secrete IgM, however, TLR9 agonists, CpG, can increase the secretion of IgM in epithelial cancer cells. 3. The CD79A and CD79B, two molecules that are physically linked to membranous IgM on the surface of B cells to form the B cell antigen receptor (BCR) complex, were also expressed on the cell surface of epithelial cancer cells and co-located with IgM to form BCR-like complex. 4. The epithelial cancer cell-derived IgM showed oligo-reactivity, which can recognize single-stranded DNA, double-stranded DNA, lipopolysaccharide, and some autoantigens. Our findings further expand the understanding for the significance of epithelial cells in the innate immune response, especially in the anti-bacterial immunity via secretion of IgM. In addition, this finding suggested that B-1 B cells may be not the main cell type that is responsible for the production of natural IgM.

B cell receptor accessory molecule CD79α: Characterisation and expression analysis in a cartilaginous fish, the spiny dogfish (Squalus acanthias)

CD79α (also known as Igα) is a component of the B cell antigen receptor complex and plays an important role in B cell signalling. The CD79α protein is present on the surface of B cells throughout their life cycle, and is absent on all other healthy cells, making it a highly reliable marker for B cells in mammals. In this study the spiny dogfish (Squalus acanthias) CD79α (SaCD79α) is described and its expression studied under constitutive and stimulated conditions. The spiny dogfish CD79α cDNA contains an open reading frame of 618 bp, encoding a protein of 205 amino acids. Comparison of the SaCD79α gene with that of other species shows that the gross structure (number of exons, exon/intron boundaries, etc.) is highly conserved across phylogeny. Additionally, analysis of the 5′ flanking region shows SaCD79α lacks a TATA box and possesses binding sites for multiple transcription factors implicated in its B cell-specific gene transcription in other species. Spiny dogfish CD79α is most highly expressed in immune tissues, such as spleen, epigonal and Leydig organ, and its transcript level significantly correlates with those of spiny dogfish immunoglobulin heavy chains. Additionally, CD79α transcription is up-regulated, to a small but significant degree, in peripheral blood cells following stimulation with pokeweed mitogen. These results strongly indicate that, as in mammals, spiny dogfish CD79α is expressed by shark B cells where it associates with surface-bound immunoglobulin to form a fully functional BCR, and thus may serve as a pan-B cell marker in future shark immunological studies.

IL4-induced IgE class switching is regulated by NFkB alternative pathway

Event Abstract Back to Event IL4-induced IgE class switching is regulated by NFkB alternative pathway Kano Tanabe1, Hitomi Sakamoto1 and Seiji Inui1* 1 Graduate School of Health Sciences, Kumamoto University, Department of Immunology and Hematology, Japan Alpha4 was identified as a signal transduction molecule which associated with Ig-alpha, a component of B cell antigen receptor (BCR) complex. Alpha4 was ubiquitously expressed and gene targeting experiment revealed that alpha4 was essential for cell survival. B cell specific gene targeting confirmed that alpha4 was not only essential for BCR signal transduction but also for CD40, IL4 and LPS signals. At the same time, we found that alpha4 associated with a protein phosphatase PP2A catalytic subunit and regulated its enzymatic activity. Neuron specific gene targeting of alpha4 revealed that alpha4/PP2Ac associated with CaMKII to regulate learning and memory. In this study, we show that IL4 receptor signaling activates alternative as well as canonical NFkB signal transduction pathway. IL4 induced the serine and threonine phosphorylation of alpha4. Both alternative NFkB pathway and threonine phosphorylation of alpha4 were inhibited by a potent CaMKII inhibitor KN93. These results suggest that alpha4/CaMKII molecules are involved in IL4 receptor-induced NFkB alternative pathway. These molecules also regulate IgE class switching induced by IL4 and CD40. Establishment of M12 B cell transfectant which inducibly express TRAF3 will be also discussed. Keywords: class switching, TNF Receptor-Associated Factor 3, NF-kappa B, alpha4, IgE Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Tanabe K, Sakamoto H and Inui S (2013). IL4-induced IgE class switching is regulated by NFkB alternative pathway. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00611 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Seiji Inui, Graduate School of Health Sciences, Kumamoto University, Department of Immunology and Hematology, Kumamoto, Kumamoto, 862-0976, Japan, inui@kumamoto-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kano Tanabe Hitomi Sakamoto Seiji Inui Google Kano Tanabe Hitomi Sakamoto Seiji Inui Google Scholar Kano Tanabe Hitomi Sakamoto Seiji Inui PubMed Kano Tanabe Hitomi Sakamoto Seiji Inui Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells

It is well known that B-1 B cells are the main cell type that is responsible for the production of natural immunoglobulin M (IgM) and can respond to infection by increasing IgM secretion. However, we unexpectedly found that some epithelial cells also can express rearranged IgM transcript that has natural IgM characteristics, such as germline-encoded and restricted rearrangement patterns. Here we studied IgM expression in human non-B cells and found that IgM was frequently expressed by many human epithelial cancer cells as well as non-cancer epithelial cells. Moreover, CD79A and CD79B, two molecules that are physically linked to membranous IgM on the surface of B cells to form the B cell antigen receptor complex, were also expressed on the cell surface of epithelial cancer cells and co-located with IgM. Like the natural IgM, the epithelial cancer cell-derived IgM recognized a series of microbial antigens, such as single-stranded DNA, double-stranded DNA, lipopolysaccharide, and the HEp-2 cell antigen. More important, stimulation of the toll-like receptor 9 (TLR9), which mimics bacterial infection, substantially increased the secretion of IgM in human epithelial cancer cells. These findings indicate that human epithelial cancer cells as well as non-cancer epithelial cells can spontaneously produce IgM with natural antibody activity.

Wide-scale and quantitative phosphoproteomic method for analysis of bidirectional signaling within interacting B and T cells (58.11)

Abstract Physiological interactions between T cells and antigen presenting cells (APC) induce the formation of the immunological synapse. The subsequent reversible tyrosine phosphorylation of a variety signaling proteins plays a pivotal role in regulation of T cell activation. Novel phosphoproteomic methods in mass spectrometry can facilitate and accelerate elucidation of cell signaling networks by providing a global picture of cellular phosphorylation. In this study a B and T cell line were labeled with stable isotopes of amino acids in cell culture, using the SILAC method. This labeling helped to differentiate and quantitate phosphopeptides from either type of cell through a timecourse of their interaction. Quantitative phosphoproteomic analysis revealed a wide array of newly discovered phosphorylation sites that changed significantly during B/T cell interaction. Phosphorylation sites were observed and quantified on the earliest upstream signaling components and the following downstream target proteins in both T cell and B cells (T cells: TCR-CD3 subunits, CD28, Erk, Fyn, Lck, P38, PI3K, PLCγ2, Zap-70 et al; B cells: B cell antigen receptor complex, liner protein, CD19, Fyn, Jnk, Lck, Lyn, P38, PI3K, PLCγ2, et al) .

Toward a molecular understanding of adaptive immunity: a chronology – part II

By 1980 it was obvious that to more fully understand adaptive immunity, one needed to somehow reduce the tremendous complexity of antigen recognition by T cell populations. Thus, there were two developments that resulted in a paradigm shift in immunology, one being the generation of monoclonal antibodies (MoAbs), and the other the development of monoclonal functional antigen-specific T cell lines. For the first time, the cellular reagents became available to ask new questions as to how individual cells comprising the complex cell populations recognize and respond to changes in their molecular environments. The first successful generation of monoclonal T cells depended upon the understanding that antigen renders cells responsive to the antigen non-specific T cell growth factor that came to be termed interleukin-2 (IL-2), which could then be used in propagating large numbers of the progeny of single cells, which in turn could then be used for molecular analyses. Monoclonal functional human T cells were used to immunize mice to generate clone-specific (clonotypic) MoAbs, which then permitted the first biochemical characterizations of the antigen recognition elements of the T cell antigen receptor (TCR) complex. Moreover, the use of monoclonal cytolytic and helper/inducer human T cell clones essentially proved that the T cell-specific molecules T4 (CD4) and T8 (CD8) functioned as accessory molecules in antigen recognition by defining MHC class II or class I restriction respectively. As well, the expression of the T3 (CD3) molecules, found to be common to all T cells, were shown further to be obligatory for functional antigen-specific T cell signaling. The monoclonal IL-2-dependent T cells were also instrumental in the isolation and purification of the IL-2 molecule to homogeneity, the first interleukin molecule to be identified and characterized. These advances then led to the generation of pure radiolabeled IL-2 molecules that were used to identify the first interleukin cellular receptors, and as well the generation of the first MoAbs reactive with both IL-2 and IL-2 receptors. All of these advances led subsequently to the isolation of the first cDNA clones recognizing one of the two chains comprising the T cell antigen recognition elements (β-chain), as well cDNA clones encoding IL-2. Accordingly, armed with all of these unique cellular and molecular reagents, it was possible to determine that antigen triggering of the TCR complex initiates IL-2 production and IL-2 receptor expression, which in turn initiate the T cell clonal proliferative expansion, envisioned by Burnet in his formulation of the clonal selection theory. Thus, adaptive immunity receives antigen-specific activation signals from the environment and turns them into antigen non-specific endogenous action signals.

Overexpression of immunoglobulin (CD79a) binding protein1 (IGBP‐1) in small lung adenocarcinomas and its clinicopathological significance

Immunoglobulin binding protein 1 (IGBP-1) was initially identified as a signal transduction molecule coprecipitating with MB1 (Igα) of the B cell antigen receptor (BCR) complex and was later found to be broadly expressed. Immunoglobulin binding protein 1 has been characterized as an associated and regulatory component of the catalytic subunits of protein phosphatase 2A (PP2A), which is the most abundant phosphatase and plays important roles in cell growth and cell cycle control. The aim of this study was to investigate the expressional characteristics of IGBP-1 and PP2Ac during pulmonary adenocarcinogenesis. The positivity rate of IGBP-1 increased during the course of sequential progression from non-invasive carcinoma (8/30, 26.7%) to invasive adenocarcinoma (37/46, 80.4%) among cases that showed areas of lepidic growth. In contrast, all of the small adenocarcinomas showing a non-lepidic growth pattern were positive for IGBP-1 (20/20, 100%). All cancers that proved ultimately fatal were positive for IGBP-1, and log-rank analysis showed that IGBP-1 positivity was significantly correlated with a poor prognosis. In contrast, atypical adenomatous hyperplasias and lung adenocarcinomas were uniformly positive for PP2Ac. Protein phosphatase 2A was not associated with carcinoma progression. Thus we have demonstrated that IGBP-1 is expressed universally in advanced lung adenocarcinomas, and that its overexpression is significantly related to outcome.

Practical aspects of immunocytochemistry in canine lymphomas

The aim of this study was to evaluate the utility of immunocytochemistry in a standard veterinary practice and to determine the immunophenotype of tumor cells in cases of multicentric lymphoma in dogs by immunocytochemical analysis of fine-needle biopsy specimens. The study was performed on cytological samples collected from 54 dogs, in which multicentric lymphoma was recognised based on clinical data, cytology or cytology and histology, and follow-up information. Diagnosis of lymphoma was established according to the updated Kiel classification. Immunocytochemical assays were conducted using commercially available antibodies to the pan T-lymphocyte marker CD3 and B cell antigen receptor complex CD79 alpha. Among all animals examined B cell lymphoma was recognized in 42/54 (77.8%) of cases, while in the remaining 12/54 (22.2%) of dogs T cell lymphoma was recognized. In 11 animals with lymphoma recognized cytologically, in which an entire lymph node was obtained for histology, the results of routine cytology and immunocytochemistry fully corresponded with findings revealed by histology and immunohistochemistry. Immunocytochemistry can be successfully conducted in smears stored at room temperature for 24 hours without changes of staining results. It can be stated that application of standard cytopathological assessment in connection with immunocytochemistry of lymph nodes samples collected from dogs with lymphoma is a method of choice for establishing final diagnosis, and avoids the need for reexamination or collection of tissue samples for histopathology and immunohistochemistry during surgical procedures in ambiguous cases.

Analysis of novel phospho-ITAM specific antibodies in a S2 reconstitution system for TCR–CD3 signalling

The T cell antigen receptor (TCR–CD3) complex contains 12 different cytoplasmic tyrosines, each of which is part of an immunoreceptor tyrosine-based activation motif and thus occurs in similar sequence context. Since phosphorylation of individual tyrosines can be correlated with the quality of the T cell response, monitoring their phosphorylation is important. We thus generated novel antibodies against phospho-tyrosines of the TCR–CD3 complex and tested the specificity in a synthetic biology approach. We utilized the Drosophila S2 reconstitution system testing several kinases and stimulation conditions that lead to optimal phosphorylation of the TCR–CD3 subunit ζ. Expressing TCR–CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immunopurification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of ζ (phospho-ζY1) and antisera raised against the first and the second phospho-tyrosine of CD3ɛ (phospho-ɛY1 and phospho-ɛY2).

ERK and cell death: ERK location and T cell selection

The selection of functional T cells is mediated by interactions between the T cell antigen receptor and self-peptide major histocompatibility complex expressed on thymic epithelium. These interactions either lead to survival and development or death. The T cell antigen receptor is an unusual receptor able to signal multiple cell fates. The precise mechanism by which this is achieved has been an area of intense research effort over the years. One model proposes that the differential activation of mitogen-activated protein kinase pathways contributes to this decision. Here, the role of extracellular signal-regulated kinase in promoting or preventing apoptosis during thymic selection is discussed.

TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation

The organization and dynamics of receptors and other molecules in the plasma membrane are not well understood. Here we analyzed the spatio-temporal dynamics of T cell antigen receptor (TCR) complexes and linker for activation of T cells (Lat), a key adaptor molecule in the TCR signaling pathway, in T cell membranes using high-speed photoactivated localization microscopy, dual-color fluorescence cross-correlation spectroscopy and transmission electron microscopy. In quiescent T cells, both molecules existed in separate membrane domains (protein islands), and these domains concatenated after T cell activation. These concatemers were identical to signaling microclusters, a prominent hallmark of T cell activation. This separation versus physical juxtapositioning of receptor domains and domains containing downstream signaling molecules in quiescent versus activated T cells may be a general feature of plasma membrane-associated signal transduction.

Resting and activated states of the B cell antigen receptor

Each B cell contains up to 100,000 B cell antigen receptor (BCR) complexes on its surface, which only become fully active on exposure of the B cell to its cognate antigen. Most studies on BCR function aims at a better understanding how the BCR becomes active and transmits its signal to the many signaling pathways inside the cell. Another, albeit related question, is how the B cells ensure that the many BCR complexes stay (in most cases) in a silent inert conformation. This problem of activation control the BCR shares with many of its signaling elements, be it kinases, phosphatases or adaptors, which all have to be regulated tightly in their localization and activity. For the later element autoinhibition plays an essential role in this regulation. In a biochemical study we previously have found that on resting B cells the BCR resides in oligomeric and monomeric forms. We now have conducted experiments that suggest that the BCR also can resume an autoinhibitory conformation and that BCR oligomerization plays an important role in this process. Specifically oligomerization of the BCR in conjunction with the kinase-phosphatase equilibrium at the BCR contributes to the tight control of BCR activation. Our studies resulted in a new model of the resting BCR and its activation. We also study how the cytosolic protein tyrosine kinase Syk is regulated and activated by the BCR. Syk is also regulated by auto-inhibition and only becomes fully active when recruited to the BCR. We now have identified Syk mutant that are altered in auto-inhibition and tested their activity in the S2 Schneider cell reconstitution system as well as in reconstituted Syk deficient B cell lines. Our data show that a tight Syk regulation is essential for normal B-lymphocytes development.