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  1. Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

    PubMed Central

    van Oers, N S; Tao, W; Watts, J D; Johnson, P; Aebersold, R; Teh, H S

    1993-01-01

    The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta. Images PMID:7689151

  2. TCR Signaling in T Cell Memory.

    PubMed

    Daniels, Mark A; Teixeiro, Emma

    2015-01-01

    T cell memory plays a critical role in our protection against pathogens and tumors. The antigen and its interaction with the T cell receptor (TCR) is one of the initiating elements that shape T cell memory together with inflammation and costimulation. Over the last decade, several transcription factors and signaling pathways that support memory programing have been identified. However, how TCR signals regulate them is still poorly understood. Recent studies have shown that the biochemical rules that govern T cell memory, strikingly, change depending on the TCR signal strength. Furthermore, TCR signal strength regulates the input of cytokine signaling, including pro-inflammatory cytokines. These highlight how tailoring antigenic signals can improve immune therapeutics. In this review, we focus on how TCR signaling regulates T cell memory and how the quantity and quality of TCR-peptide-MHC interactions impact the multiple fates a T cell can adopt in the memory pool. PMID:26697013

  3. Innate signals overcome acquired TCR signaling pathway regulation and govern the fate of human CD161(hi) CD8α⁺ semi-invariant T cells.

    PubMed

    Turtle, Cameron J; Delrow, Jeff; Joslyn, Rochelle C; Swanson, Hillary M; Basom, Ryan; Tabellini, Laura; Delaney, Colleen; Heimfeld, Shelly; Hansen, John A; Riddell, Stanley R

    2011-09-01

    Type 17 programmed CD161(hi)CD8α(+) T cells contribute to mucosal immunity to bacteria and yeast. In early life, microbial colonization induces proliferation of CD161(hi) cells that is dependent on their expression of a semi-invariant Vα7.2(+) TCR. Although prevalent in adults, CD161(hi)CD8α(+) cells exhibit weak proliferative and cytokine responses to TCR ligation. The mechanisms responsible for the dichotomous response of neonatal and adult CD161(hi) cells, and the signals that enable their effector function, have not been established. We describe acquired regulation of TCR signaling in adult memory CD161(hi)CD8α(+) T cells that is absent in cord CD161(hi) cells and adult CD161(lo) cells. Regulated TCR signaling in CD161(hi) cells was due to profound alterations in TCR signaling pathway gene expression and could be overcome by costimulation through CD28 or innate cytokine receptors, which dictated the fate of their progeny. Costimulation with IL-1β during TCR ligation markedly increased proinflammatory IL-17 production, while IL-12-induced Tc1-like function and restored the response to TCR ligation without costimulation. CD161(hi) cells from umbilical cord blood and granulocyte colony stimulating factor-mobilized leukaphereses differed in frequency and function, suggesting future evaluation of the contribution of CD161(hi) cells in hematopoietic stem cell grafts to transplant outcomes is warranted.

  4. THEMIS: a critical TCR signal regulator for ligand discrimination.

    PubMed

    Gascoigne, Nicholas R J; Acuto, Oreste

    2015-04-01

    Genetic approaches identified THEMIS as a critical element driving positive selection of CD4(+)CD8(+) thymocytes towards maturation. THEMIS is expressed only in the T-cell lineage, and is recruited to the proximity of signaling T-cell antigen receptors (TCR) by association with the membrane scaffold LAT. However, its molecular role remained an enigma until recently. Conventionally positively-selected T-cells are lacking in THEMIS-deficient mice, leading to the initial hypothesis that THEMIS positively regulates TCR signaling. Recent data show that THEMIS deficiency increases rather than decreases TCR signaling, leading to augmented apoptosis. The finding that THEMIS is constitutively bound to the tyrosine phosphatases SHP1 or SHP2, provides a mechanism for THEMIS action. When recruited onto LAT, THEMIS-SHP promotes immediate dephosphorylation of TCR-proximal signaling components. This negative feedback is central in setting sharp signaling thresholds and helps explain the exquisite ligand discrimination by the TCR, particularly during thymocyte selection.

  5. Spatiotemporal regulation of T cell co-stimulation by TCR-CD28 microclusters through PKCθ translocation

    PubMed Central

    Yokosuka, Tadashi; Kobayashi, Wakana; Sakata-Sogawa, Kumiko; Hashimoto-Tane, Akiko; Dustin, Michel L.; Tokunaga, Makio; Saito, Takashi

    2010-01-01

    Summary T cell activation is mediated by microclusters (MCs) containing TCRs, kinases, and adaptors. Although TCR-MCs translocate to form a central supramolecular activation cluster (c-SMAC) of immunological synapse between T cells and antigen-presenting cells (APCs), the role of MC translocation in T cell signaling remains unclear. Here, we found that the accumulation of MCs in c-SMAC was important for T cell co-stimulation. Using planar bilayer system, co-stimulatory receptor CD28 was initially recruited coordinately with TCR to MCs and its signals was mediated through the assembly with PKCθ. Their co-localization and assembly is correlated withco-stimulatory function. The accumulation of MCs at c-SMAC was accompanied by segregation of CD28 from TCRs and both CD28 and PKCθ translocated to a spatially unique sub-zone of c-SMAC. Thus, co-stimulation is mediated by generating a novel co-stimulatory compartment in c-SMAC via the dynamic regulation of MC translocation. PMID:18848472

  6. Control of T cell antigen reactivity via programmed TCR downregulation.

    PubMed

    Gallegos, Alena M; Xiong, Huizhong; Leiner, Ingrid M; Sušac, Bože; Glickman, Michael S; Pamer, Eric G; van Heijst, Jeroen W J

    2016-04-01

    The T cell antigen receptor (TCR) is unique in that its affinity for ligand is unknown before encounter and can vary by orders of magnitude. How the immune system regulates individual T cells that display very different reactivity to antigen remains unclear. Here we found that activated CD4(+) T cells, at the peak of clonal expansion, persistently downregulated their TCR expression in proportion to the strength of the initial antigen recognition. This programmed response increased the threshold for cytokine production and recall proliferation in a clone-specific manner and ultimately excluded clones with the highest antigen reactivity. Thus, programmed downregulation of TCR expression represents a negative feedback mechanism for constraining T cell effector function with a suitable time delay to thereby allow pathogen control while avoiding excess inflammatory damage. PMID:26901151

  7. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  8. The protein tyrosine phosphatase PTPN4/PTP-MEG1, an enzyme capable of dephosphorylating the TCR ITAMs and regulating NF-κB, is dispensable for T cell development and/or T cell effector functions

    PubMed Central

    Young, Jennifer A.; Becker, Amy M.; Medeiros, Jennifer J.; Shapiro, Virginia S.; Wang, Andrew; Farrar, J. David; Quill, Timothy A.; van Huijsduijnen, Rob Hooft; van Oers, Nicolai S.C.

    2008-01-01

    T cell receptor signaling processes are controlled by the integrated actions of families of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Several distinct cytosolic protein tyrosine phosphatases have been described that are able to negatively regulate TCR signaling pathways, including SHP-1, SHP-2, PTPH1, and PEP. Using PTPase substrate-trapping mutants and wild type enzymes, we determined that PTPN4/PTP-MEG1, a PTPH1-family member, could complex and dephosphorylate the ITAMs of the TCR ζ subunit. In addition, the substrate-trapping derivative augmented basal and TCR-induced activation of NF-κB in T cells. To characterize the contribution of this PTPase in T cells, we developed PTPN4-deficient mice. T cell development and TCR signaling events were comparable between wild type and PTPN4-deficient animals. The magnitude and duration of TCR-regulated ITAM phosphorylation, as well as overall protein phosphorylation, was unaltered in the absence of PTPN4. Finally, Th1- and Th2-derived cytokines and in vivo immune responses to Listeria monocytogeneswere equivalent between wild type and PTPN4-deficient mice. These findings suggest that additional PTPases are involved in controlling ITAM phosphorylations. PMID:18614237

  9. The Phosphotyrosine Phosphatase SHP-2 Participates in a Multimeric Signaling Complex and Regulates T Cell Receptor (TCR) coupling to the Ras/Mitogen-activated Protein Kinase (MAPK) Pathway in Jurkat T Cells

    PubMed Central

    Frearson, Julie A.; Alexander, Denis R.

    1998-01-01

    Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process

  10. Requirement of full TCR repertoire for regulatory T cells to maintain intestinal homeostasis

    PubMed Central

    Nishio, Junko; Baba, Minato; Atarashi, Koji; Tanoue, Takeshi; Negishi, Hideo; Yanai, Hideyuki; Habu, Sonoko; Hori, Shohei; Honda, Kenya; Taniguchi, Tadatsugu

    2015-01-01

    The regulation of intestinal homeostasis by the immune system involves the dynamic interplay between gut commensal microbiota and resident immune cells. It is well known that a large and diverse lymphocyte antigen receptor repertoire enables the immune system to recognize and respond to a wide range of invading pathogens. There is also an emerging appreciation for a critical role the T-cell receptor (TCR) repertoire serves in the maintenance of peripheral tolerance by regulatory T cells (Tregs). Nevertheless, how the diversity of the TCR repertoire in Tregs affects intestinal homeostasis remains unknown. To address this question, we studied mice whose T cells express a restricted TCR repertoire. We observed the development of spontaneous colitis, accompanied by the induction of T-helper type 17 cells in the colon that is driven by gut commensal microbiota. We provide further evidence that a restricted TCR repertoire causes a loss of tolerogenicity to microbiota, accompanied by a paucity of peripherally derived, Helios− Tregs and hyperactivation of migratory dendritic cells. These results thus reveal a new facet of the TCR repertoire in which Tregs require a diverse TCR repitoire for intestinal homeostasis, suggesting an additional driving force in the evolutional significance of the TCR repertoire. PMID:26420876

  11. Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance

    PubMed Central

    Guittard, Geoffrey C.; Franco, Zulmarie; Crompton, Joseph G.; Eil, Robert L.; Patel, Shashank J.; Ji, Yun; Van Panhuys, Nicholas; Klebanoff, Christopher A.; Sukumar, Madhusudhanan; Clever, David; Chichura, Anna; Roychoudhuri, Rahul; Varma, Rajat; Wang, Ena; Gattinoni, Luca; Marincola, Francesco M.; Balagopalan, Lakshmi; Samelson, Lawrence E.

    2015-01-01

    Improving the functional avidity of effector T cells is critical in overcoming inhibitory factors within the tumor microenvironment and eliciting tumor regression. We have found that Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8+ T cells and inhibits their functional avidity against tumors. Genetic deletion of Cish in CD8+ T cells enhances their expansion, functional avidity, and cytokine polyfunctionality, resulting in pronounced and durable regression of established tumors. Although Cish is commonly thought to block STAT5 activation, we found that the primary molecular basis of Cish suppression is through inhibition of TCR signaling. Cish physically interacts with the TCR intermediate PLC-γ1, targeting it for proteasomal degradation after TCR stimulation. These findings establish a novel targetable interaction that regulates the functional avidity of tumor-specific CD8+ T cells and can be manipulated to improve adoptive cancer immunotherapy. PMID:26527801

  12. Exposure of Human CD4 T Cells to IL-12 Results in Enhanced TCR-Induced Cytokine Production, Altered TCR Signaling, and Increased Oxidative Metabolism

    PubMed Central

    2016-01-01

    Human CD4 T cells are constantly exposed to IL-12 during infections and certain autoimmune disorders. The current paradigm is that IL-12 promotes the differentiation of naïve CD4 T cells into Th1 cells, but recent studies suggest IL-12 may play a more complex role in T cell biology. We examined if exposure to IL-12 alters human CD4 T cell responses to subsequent TCR stimulation. We found that IL-12 pretreatment increased TCR-induced IFN-γ, TNF-α, IL-13, IL-4 and IL-10 production. This suggests that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. We observed that IL-12 mediated its effects through both transcriptional and post-transcriptional mechanisms. IL-12 pretreatment increased the phosphorylation of AKT, p38 and LCK following TCR stimulation without altering other TCR signaling molecules, potentially mediating the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that are not transcriptionally regulated was partially driven by increased oxidative metabolism. Our data uncover a novel function of IL-12 in human CD4 T cells; specifically, it enhances the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism. PMID:27280403

  13. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

    PubMed Central

    Shorter, Shayla K.; Schnell, Frederick J.; McMaster, Sean R.; Pinelli, David F.; Andargachew, Rakieb; Evavold, Brian D.

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant. PMID:26915099

  14. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    PubMed

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.

  15. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    PubMed

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies. PMID:17855129

  16. T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component*

    PubMed Central

    Brockmeyer, Claudia; Paster, Wolfgang; Pepper, David; Tan, Choon P.; Trudgian, David C.; McGowan, Simon; Fu, Guo; Gascoigne, Nicholas R. J.; Acuto, Oreste; Salek, Mogjiborahman

    2011-01-01

    Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive “signaling waves” revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function. PMID:21189249

  17. Selective Regulation of TCR Signaling Pathways by the CD45 Protein Tyrosine Phosphatase During Thymocyte Development1

    PubMed Central

    Falahati, Rustom; Leitenberg, David

    2009-01-01

    In CD45 deficient animals there is a severe defect in thymocyte positive selection, resulting in an absence of mature T cells and the accumulation of thymocytes at the double positive stage of development. However, the signaling defect(s) responsible for the block in development of mature single positive T cells are not well characterized. Previous studies have found that early signal transduction events in CD45 deficient cell lines and thymocytes are markedly diminished following stimulation with anti-CD3. Nevertheless, there are also situations in which T cell activation and TCR signaling events can be induced in the absence of CD45. For example, CD45 independent TCR signaling can be recovered upon simultaneous antibody cross-linking of CD3 and CD4 compared to cross-linking of CD3 alone. These data suggest that CD45 may differentially regulate TCR signaling events depending on the nature of the signal and/or on the differentiation state of the cell. In the current study we have assessed the role of CD45 in regulating primary thymocyte activation following physiologic stimulation with peptide. Unlike CD3 mediated stimulation, peptide stimulation of CD45 deficient thymocytes induces diminished, but readily detectable TCR mediated signaling events such as phosphorylation of TCR-associated zeta, ZAP70, LAT, and Akt, and increased intracellular calcium concentration. In contrast, phosphorylation of ERK, which is essential for positive selection, is more severely affected in the absence of CD45. These data suggest that CD45 has a selective role in regulating different aspects of T cell activation. PMID:18941197

  18. TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

    PubMed

    Hwang, SuJin; Palin, Amy C; Li, LiQi; Song, Ki-Duk; Lee, Jan; Herz, Jasmin; Tubo, Noah; Chu, Hamlet; Pepper, Marion; Lesourne, Renaud; Zvezdova, Ekaterina; Pinkhasov, Julia; Jenkins, Marc K; McGavern, Dorian; Love, Paul E

    2015-01-01

    The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated 'knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR-ligand interactions, but is not essential for 'general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire. PMID:25959494

  19. TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

    PubMed

    Hwang, SuJin; Palin, Amy C; Li, LiQi; Song, Ki-Duk; Lee, Jan; Herz, Jasmin; Tubo, Noah; Chu, Hamlet; Pepper, Marion; Lesourne, Renaud; Zvezdova, Ekaterina; Pinkhasov, Julia; Jenkins, Marc K; McGavern, Dorian; Love, Paul E

    2015-05-11

    The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated 'knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR-ligand interactions, but is not essential for 'general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire.

  20. Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.

    PubMed

    Dombroski, Derek; Houghtling, Richard A; Labno, Christine M; Precht, Patricia; Takesono, Aya; Caplen, Natasha J; Billadeau, Daniel D; Wange, Ronald L; Burkhardt, Janis K; Schwartzberg, Pamela L

    2005-02-01

    The Tec family kinase Itk is an important regulator of Ca(2+) mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

  1. CD8α+ Dendritic cells prime TCR-peptide-reactive regulatory CD4+FOXP3− T cells

    PubMed Central

    Smith, Trevor R. F.; Maricic, Igor; Ria, Francesco; Schneider, Susan; Kumar, Vipin

    2011-01-01

    Summary CD4+ T cells with immune regulatory function can be either FOXP3+ or FOXP3−. We have previously shown that priming of naturally occurring TCR-peptide-reactive regulatory CD4+FOXP3− T cells (Treg) specifically controls Vβ8.2+CD4+ T cells mediating experimental autoimmune encephalomyelitis (EAE). However, the mechanism by which these Treg are primed to recognize their cognate antigenic determinant, which is derived from the TCRVβ8.2-chain, is not known. In this study we show that antigen presenting cells (APC) derived from splenocytes of naïve mice are able to stimulate cloned CD4+ Treg in the absence of exogenous antigen, and their stimulation capacity is augmented during EAE. Among the APC populations DC were the most efficient in stimulating the Treg. Stimulation of CD4+ Treg was dependent upon processing and presentation of TCR peptides from ingested Vβ8.2TCR+ CD4+ T cells. Additionally, dendritic cells pulsed with TCR peptide or apoptotic Vβ8.2+ T cells are able to prime Treg in vivo and mediate protection from disease in a CD8-dependent fashion. These data highlight a novel mechanism for the priming of CD4+ Treg by CD8α+ DC, and suggest a pathway that can be exploited to prime antigen-specific regulation of T cell-mediated inflammatory disease. PMID:20394075

  2. Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals.

    PubMed

    Zarin, Payam; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Zúñiga-Pflücker, Juan Carlos

    2014-04-15

    Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice. PMID:24706811

  3. Optimization of T-cell reactivity by exploiting TCR chain centricity for the purpose of safe and effective antitumor TCR gene therapy

    PubMed Central

    Ochi, Toshiki; Nakatsugawa, Munehide; Chamoto, Kenji; Tanaka, Shinya; Yamashita, Yuki; Guo, Tingxi; Fujiwara, Hiroshi; Yasukawa, Masaki; Butler, Marcus O.; Hirano, Naoto

    2015-01-01

    Adoptive transfer of T cells redirected by a high affinity antitumor T-cell receptor (TCR) is a promising treatment modality for cancer patients. Safety and efficacy depend on the selection of a TCR that induces minimal toxicity and elicits sufficient antitumor reactivity. Many, if not all, TCRs possess cross-reactivity to unrelated MHC molecules in addition to reactivity to target self-MHC/peptide complexes. Some TCRs display chain centricity, in which recognition of MHC/peptide complexes is dominated by one of the TCR hemi-chains. In this study, we comprehensively studied how TCR chain centricity impacts reactivity to target self-MHC/peptide complexes and alloreactivity using the TCR, clone TAK1, which is specific for human leukocyte antigen-A*24:02/Wilms tumor 1235–243 (A24/WT1235) and cross-reactive with B*57:01 (B57). The TAK1β, but not the TAK1α, hemi-chain possessed chain centricity. When paired with multiple clonotypic TCRα counter-chains encoding TRAV12-2, 20, 36, or 38-2, the de novo TAK1β-containing TCRs showed enhanced, weakened, or absent reactivity to A24/WT1235 and/or to B57. T cells reconstituted with these TCRα genes along with TAK1β possessed a very broad range (>3 log orders) of functional and structural avidities. These results suggest that TCR chain centricity can be exploited to enhance desired antitumor TCR reactivity and eliminate unwanted TCR cross-reactivity. TCR reactivity to target MHC/peptide complexes and cross-reactivity to unrelated MHC molecules are not inextricably linked and are separable at the TCR sequence level. However, it is still mandatory to carefully monitor for possible harmful toxicities caused by adoptive transfer of T cells redirected by thymically-unselected TCRs. PMID:25943533

  4. A mechanism for TCR sharing between T cell subsets and individuals revealed by pyrosequencing.

    PubMed

    Venturi, Vanessa; Quigley, Máire F; Greenaway, Hui Yee; Ng, Pauline C; Ende, Zachary S; McIntosh, Tina; Asher, Tedi E; Almeida, Jorge R; Levy, Samuel; Price, David A; Davenport, Miles P; Douek, Daniel C

    2011-04-01

    The human naive T cell repertoire is the repository of a vast array of TCRs. However, the factors that shape their hierarchical distribution and relationship with the memory repertoire remain poorly understood. In this study, we used polychromatic flow cytometry to isolate highly pure memory and naive CD8(+) T cells, stringently defined with multiple phenotypic markers, and used deep sequencing to characterize corresponding portions of their respective TCR repertoires from four individuals. The extent of interindividual TCR sharing and the overlap between the memory and naive compartments within individuals were determined by TCR clonotype frequencies, such that higher-frequency clonotypes were more commonly shared between compartments and individuals. TCR clonotype frequencies were, in turn, predicted by the efficiency of their production during V(D)J recombination. Thus, convergent recombination shapes the TCR repertoire of the memory and naive T cell pools, as well as their interrelationship within and between individuals.

  5. Rapid TCR-mediated SHP-1 S591 phosphorylation regulates SHP-1 cellular localization and phosphatase activity

    PubMed Central

    Liu, Yin; Kruhlak, Michael J.; Hao, Jian-Jiang; Shaw, Stephen

    2007-01-01

    Since the tyrosine phosphatase SHP-1 plays a major role in regulating T-cell signaling, we investigated regulation thereof by Ser/Thr phosphorylation. We found that TCR stimulation induced fast (≤1min) and transient phosphorylation of SHP-1 S591 in both Jurkat and human peripheral blood T-cells (PBT). Phosphorylation of S591 in T-cells could be mediated artificially by a constitutive active PKC-theta construct, but the dose dependence of inhibition by PKC inhibitors indicated that PKCs were not the relevant basophilic kinase in the physiologic response. S591 phosphorylation inhibited phosphatase function since a S591D mutant had lower activity than the S591A mutant. Additional evidence that S591 phosphorylation alters SHP-1 function was provided by studies of Jurkat cells stably expressing SHP-1 wildtype or mutants. In those cells, S591D mutation reduced the capacity of transfected SHP-1 to inhibit TCR-induced phosphorylation of PLC-γ1. Interestingly, SHP-1 Y536 phosphorylation (previously shown to augment phosphatase activity) was also induced in PBT by TCR signal but at a much later time compared to S591 (~30 min). S591 phosphorylation also altered cellular distribution of SHP-1 because: 1) SHP-1 in lipid rafts and a sheared membrane fraction was hypo-phosphorylated; 2) In stably transfected Jurkat cell lines, S591D mutant protein had reduced presence in both lipid raft and the sheared membrane fraction; 3) S591 phosphorylation prevented nuclear localization of a C-terminal GFP tagged SHP-1 construct. Our studies also shed light on an additional mechanism regulating SHP-1 nuclear localization, namely conformational autoinhibition. These findings highlight elegant regulation of SHP-1 by sequential phosporylation of serine then tyrosine. PMID:17575265

  6. Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

    PubMed Central

    Giannoni, Francesca; Hardee, Cinnamon L; Wherley, Jennifer; Gschweng, Eric; Senadheera, Shantha; Kaufman, Michael L; Chan, Rebecca; Bahner, Ingrid; Gersuk, Vivian; Wang, Xiaoyan; Gjertson, David; Baltimore, David; Witte, Owen N; Economou, James S; Ribas, Antoni; Kohn, Donald B

    2013-01-01

    Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34+ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8+ and CD4+ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8+ and CD4+ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34+ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma. PMID:23380815

  7. Allelic exclusion and peripheral reconstitution by TCR transgenic T cells arising from transduced human hematopoietic stem/progenitor cells.

    PubMed

    Giannoni, Francesca; Hardee, Cinnamon L; Wherley, Jennifer; Gschweng, Eric; Senadheera, Shantha; Kaufman, Michael L; Chan, Rebecca; Bahner, Ingrid; Gersuk, Vivian; Wang, Xiaoyan; Gjertson, David; Baltimore, David; Witte, Owen N; Economou, James S; Ribas, Antoni; Kohn, Donald B

    2013-05-01

    Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34(+) HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8(+) and CD4(+) T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4-5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8(+) and CD4(+) T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34(+) HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.

  8. Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4+ T Helper Cells

    PubMed Central

    Hynes, Thomas R.; Yost, Evan A.; Yost, Stacy M.; Hartle, Cassandra M.; Ott, Braden J.

    2015-01-01

    Background: The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. Methods: The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4+ T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. Results: ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4+ T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2’,5’-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. Conclusions: GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels. PMID

  9. Differential Requirements of TCR Signaling in Homeostatic Maintenance and Function of Dendritic Epidermal T Cells.

    PubMed

    Zhang, Baojun; Wu, Jianxuan; Jiao, Yiqun; Bock, Cheryl; Dai, Meifang; Chen, Benny; Chao, Nelson; Zhang, Weiguo; Zhuang, Yuan

    2015-11-01

    Dendritic epidermal T cells (DETCs) are generated exclusively in the fetal thymus and maintained in the skin epithelium throughout postnatal life of the mouse. DETCs have restricted antigenic specificity as a result of their exclusive usage of a canonical TCR. Although the importance of the TCR in DETC development has been well established, the exact role of TCR signaling in DETC homeostasis and function remains incompletely defined. In this study, we investigated TCR signaling in fully matured DETCs by lineage-restricted deletion of the Lat gene, an essential signaling molecule downstream of the TCR. We found that Lat deletion impaired TCR-dependent cytokine gene activation and the ability of DETCs to undergo proliferative expansion. However, linker for activation of T cells-deficient DETCs were able to maintain long-term population homeostasis, although with a reduced proliferation rate. Mice with Lat deletion in DETCs exhibited delayed wound healing accompanied by impaired clonal expansion within the wound area. Our study revealed differential requirements for TCR signaling in homeostatic maintenance of DETCs and in their effector function during wound healing. PMID:26408667

  10. Retroviral vector expression in TCR transgenic CD4⁺ T cells.

    PubMed

    Choi, Youn Soo; Crotty, Shane

    2015-01-01

    The regulation of gene expression is key to understand the function of genes of interest. To explore the biological functions of genes, transgenic knock-in or knockout technologies have served as invaluable tools. While recent advances in molecular biology have introduced new techniques (i.e., CRISPR mediated gene editing) (Cong et al., Science 339(6121):819-823, 2013; Wang et al., Cell 153(4):910-918, 2013) for the generation of transgenic mice in a relatively short period of time, it can still take a long time to test biological hypotheses from scratch to design how to generate knock-in or knockout mice. Here, we describe methods to manipulate gene expression in T cell receptor (TCR) transgenic CD4 T cells, which allow us to investigate gene functions in the study of differentiation pathways of follicular helper T (Tfh) cells.

  11. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire

    PubMed Central

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-01-01

    Abstract The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals. Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR. Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides. Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination. PMID:26962778

  12. Micro-adhesion rings surrounding TCR microclusters are essential for T cell activation.

    PubMed

    Hashimoto-Tane, Akiko; Sakuma, Machie; Ike, Hiroshi; Yokosuka, Tadashi; Kimura, Yayoi; Ohara, Osamu; Saito, Takashi

    2016-07-25

    The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.

  13. Fully functional HLA B27-restricted CD4+ as well as CD8+ T cell responses in TCR transgenic mice.

    PubMed

    Roddis, Matthew; Carter, Robert W; Sun, Mei-Yi; Weissensteiner, Thomas; McMichael, Andrew J; Bowness, Paul; Bodmer, Helen C

    2004-01-01

    The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restricted CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo. PMID:14688321

  14. Micropatterning of TCR and LFA-1 ligands reveals complementary effects on cytoskeleton mechanics in T-cells

    PubMed Central

    Tabdanov, Erdem; Gondarenko, Sasha; Kumari, Sudha; Liapis, Anastasia; Dustin, Michael L.; Sheetz, Michael P.; Kam, Lance C.; Iskratsch, Thomas

    2015-01-01

    Summary The formation of the immunological synapse between a T-cell and the antigen-presenting cell (APC) is critically dependent on actin dynamics, downstream of T-cell receptor (TCR) and integrin (LFA-1) signalling. There is also accumulating evidence that mechanical forces, generated by actin polymerization and/or myosin contractility regulate T-cell signalling. Because both receptor pathways are intertwined, their contributions towards the cytoskeletal organization remain elusive. Here, we identify the specific roles of TCR and LFA-1 by using a combination of micropatterning to spatially separate signalling systems and nanopillar arrays for high-precision analysis of cellular forces. We identify that Arp2/3 acts downstream of TCRs to nucleate dense actin foci but propagation of the network requires LFA-1 and the formin FHOD1. LFA-1 adhesion enhances actomyosin forces, which in turn modulate actin assembly downstream of the TCR. Together our data shows a mechanically cooperative system through which ligands presented by an APC modulate T-cell activation. PMID:26156536

  15. The influence of age on T cell generation and TCR diversity.

    PubMed

    Naylor, Keith; Li, Guangjin; Vallejo, Abbe N; Lee, Won-Woo; Koetz, Kerstin; Bryl, Ewa; Witkowski, Jacek; Fulbright, James; Weyand, Cornelia M; Goronzy, Jörg J

    2005-06-01

    The ability to mount protective immune responses depends on the diversity of T cells. T cell diversity may be compromised by the declining thymic output of new T cells. The aging process imposes a threat to diversity, because thymic function deteriorates. In this study we have examined the relationship between thymic production, homeostatic T cell proliferation and TCR beta-chain diversity in young (approximately 25 years), middle-aged ( approximately 60 years), and elderly adults (approximately 75 years). TCR excision circles (TREC) as a marker of thymic output exponentially decreased by >95% between 25 and 60 years of age. The frequency of Ki67(+) cycling CD4 T cells remained steady, and surprisingly, the diversity of the naive CD4 T cell repertoire was maintained at approximately 2 x 10(7) different TCR beta-chains. After the age of 70 years, TRECs only slightly declined, but homeostatic proliferation doubled. The diversity of the T cell pool drastically contracted to 200,000 TCR beta-chains. Also, the phenotypic distinction between naive and memory CD4 T cells became fuzzy. The collapse in CD4 T cell diversity during the seventh and eighth decades indicates substantial T cell loss and implies that therapeutic measures to improve vaccine responses will have to include strategies for T cell replenishment.

  16. Mixed lymphocyte cultures can predict TCR Vbeta repertoires of T cells infiltrating kidney transplants during acute rejection episodes.

    PubMed

    Paraoan, Marius T; Bakran, Ali; Hammad, Abdul; Sells, Robert A; Christmas, Stephen E

    2005-12-27

    Alloreactive T cell populations can show skewing of T-cell antigen receptor (TCR) Vbeta gene usage. The aims of the experiments were to compare in vivo and in vitro T cell alloresponses against donor alloantigens for TCR Vbeta gene usage. T-cell cultures from renal biopsies taken during acute rejection and pretransplant mixed lymphocyte cultures (MLC) were established from five renal transplant patients. TCR Vbeta gene usage, assessed with Vbeta family specific antibodies, showed that up to five different Vbeta families were significantly expanded. In four of five cases, there was close concordance between Vbeta families expanded from the biopsy and in MLC. T-cell clones from one renal biopsy were specific for the mismatched donor alloantigen and showed similar TCR Vbeta gene usage to the original T-cell line. The results show very similar patterns of TCR Vbeta gene usage in alloreactive T cells generated ex vivo or in vitro.

  17. Piceatannol inhibits effector T cell functions by suppressing TcR signaling.

    PubMed

    Kim, Do-Hyun; Lee, Yong-Gab; Park, Hong-Jai; Lee, Jung-Ah; Kim, Hyun Jung; Hwang, Jae-Kwan; Choi, Je-Min

    2015-04-01

    Piceatannol, a metabolite of resveratrol found in red wine and grapes, displays a wide spectrum of biological activity. Although the anti-oxidant, anti-inflammatory, and anti-tumorigenesis activity of piceatannol has been extensively studied, its role in the adaptive immune response has received less attention. Here we investigated the role of piceatannol, a well-known Syk inhibitor, in T cell activation, proliferation, and differentiation using isolated murine splenic T cells from C57BL/6 mice. Piceatannol treatment inhibited surface expression of CD4 and CD8 T cell activation markers CD25 and CD69, reduced production of cytokines IFNγ, IL-2, and IL-17, and suppressed proliferation of activated T cells. Moreover, piceatannol treatment significantly inhibited differentiation of CD4(+)CD25(-)CD62L(+) naïve CD4 T cells into Th1, Th2, and Th17 cells, presumably due to inhibition of TcR signaling through p-Erk, p-Akt, and p-p38. Piceatannol appears to be a useful nutritional or pharmacological biomolecule that regulates effector T cell functions such as cytokine production, differentiation, and proliferation. PMID:25676533

  18. Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress.

    PubMed

    Malcolm, Tim I M; Villarese, Patrick; Fairbairn, Camilla J; Lamant, Laurence; Trinquand, Amélie; Hook, C Elizabeth; Burke, G A Amos; Brugières, Laurence; Hughes, Katherine; Payet, Dominique; Merkel, Olaf; Schiefer, Ana-Iris; Ashankyty, Ibraheem; Mian, Shahid; Wasik, Mariusz; Turner, Martin; Kenner, Lukas; Asnafi, Vahid; Macintyre, Elizabeth; Turner, Suzanne D

    2016-01-01

    Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM-ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy. PMID:26753883

  19. Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress

    PubMed Central

    Malcolm, Tim I. M.; Villarese, Patrick; Fairbairn, Camilla J.; Lamant, Laurence; Trinquand, Amélie; Hook, C. Elizabeth; Burke, G. A. Amos; Brugières, Laurence; Hughes, Katherine; Payet, Dominique; Merkel, Olaf; Schiefer, Ana-Iris; Ashankyty, Ibraheem; Mian, Shahid; Wasik, Mariusz; Turner, Martin; Kenner, Lukas; Asnafi, Vahid; Macintyre, Elizabeth; Turner, Suzanne D.

    2016-01-01

    Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM–ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy. PMID:26753883

  20. Jarid2 is induced by TCR signalling and controls iNKT cell maturation.

    PubMed

    Pereira, Renata M; Martinez, Gustavo J; Engel, Isaac; Cruz-Guilloty, Fernando; Barboza, Bianca A; Tsagaratou, Ageliki; Lio, Chan-Wang J; Berg, Leslie J; Lee, Youngsook; Kronenberg, Mitchell; Bandukwala, Hozefa S; Rao, Anjana

    2014-08-08

    Jarid2 is a reported component of three lysine methyltransferase complexes, polycomb repressive complex 2 (PRC2) that methylates histone 3 lysine 27 (H3K27), and GLP-G9a and SETDB1 complexes that methylate H3K9. Here we show that Jarid2 is upregulated upon TCR stimulation and during positive selection in the thymus. Mice lacking Jarid2 in T cells display an increase in the frequency of IL-4-producing promyelocytic leukemia zinc finger (PLZF)(hi) immature invariant natural killer T (iNKT) cells and innate-like CD8(+) cells; Itk-deficient mice, which have a similar increase of innate-like CD8(+) cells, show blunted upregulation of Jarid2 during positive selection. Jarid2 binds to the Zbtb16 locus, which encodes PLZF, and thymocytes lacking Jarid2 show increased PLZF and decreased H3K9me3 levels. Jarid2-deficient iNKT cells perturb Th17 differentiation, leading to reduced Th17-driven autoimmune pathology. Our results establish Jarid2 as a novel player in iNKT cell maturation that regulates PLZF expression by modulating H3K9 methylation.

  1. TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8(+) T cells.

    PubMed

    Takada, Kensuke; Van Laethem, Francois; Xing, Yan; Akane, Kazuyuki; Suzuki, Haruhiko; Murata, Shigeo; Tanaka, Keiji; Jameson, Stephen C; Singer, Alfred; Takahama, Yousuke

    2015-10-01

    In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8(+) T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I-associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8(+) T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8(+) T cells in the periphery.

  2. T cell epitope redundancy: cross-conservation of the TCR face between pathogens and self and its implications for vaccines and autoimmunity.

    PubMed

    Moise, Leonard; Beseme, Sarah; Tassone, Ryan; Liu, Rui; Kibria, Farzana; Terry, Frances; Martin, William; De Groot, Anne S

    2016-05-01

    T cells are extensively trained on 'self' in the thymus and then move to the periphery, where they seek out and destroy infections and regulate immune response to self-antigens. T cell receptors (TCRs) on T cells' surface recognize T cell epitopes, short linear strings of amino acids presented by antigen-presenting cells. Some of these epitopes activate T effectors, while others activate regulatory T cells. It was recently discovered that T cell epitopes that are highly conserved on their TCR face with human genome sequences are often associated with T cells that regulate immune response. These TCR-cross-conserved or 'redundant epitopes' are more common in proteins found in pathogens that have co-evolved with humans than in other non-commensal pathogens. Epitope redundancy might be the link between pathogens and autoimmune disease. This article reviews recently published data and addresses epitope redundancy, the "elephant in the room" for vaccine developers and T cell immunologists.

  3. Too much of a good thing? Tim-3 and TCR signaling in T cell exhaustion.

    PubMed

    Ferris, Robert L; Lu, Binfeng; Kane, Lawrence P

    2014-08-15

    T cell exhaustion is thought to be a natural mechanism for limiting immune pathology, although it may be desirable to circumvent this mechanism to help eliminate viral reservoirs or tumors. Although there are no definitive markers, a fingerprint for exhausted T cells has been described that includes the transmembrane proteins PD-1, LAG3, and Tim-3. However, apart from the recruitment of tyrosine phosphatases to PD-1, little is known about the biochemical mechanisms by which these proteins contribute to the development or maintenance of exhaustion. Tim-3 contains no known motifs for the recruitment of inhibitory phosphatases, but it may actually increase signaling downstream of TCR/CD3, at least under acute conditions. Other studies showed that T cell exhaustion results from chronic stimulation that extends the effector phase of T cell activation, at the expense of T cell memory. We suggest that Tim-3 may contribute to T cell exhaustion by enhancing TCR-signaling pathways.

  4. Id1 expression promotes T regulatory cell differentiation by facilitating TCR costimulation.

    PubMed

    Liu, Chen; Wang, Hong-Cheng; Yu, Sen; Jin, Rong; Tang, Hui; Liu, Yuan-Feng; Ge, Qing; Sun, Xiao-Hong; Zhang, Yu

    2014-07-15

    T regulatory (Treg) cells play crucial roles in the regulation of cellular immunity. The development of Treg cells depends on signals from TCRs and IL-2Rs and is influenced by a variety of transcription factors. The basic helix-loop-helix proteins are known to influence TCR signaling thresholds. Whether this property impacts Treg differentiation is not understood. In this study, we interrogated the role of basic helix-loop-helix proteins in the production of Treg cells using the CD4 promoter-driven Id1 transgene. We found that Treg cells continued to accumulate as Id1 transgenic mice aged, resulting in a significant increase in Treg cell counts in the thymus as well as in the periphery compared with wild-type controls. Data from mixed bone marrow assays suggest that Id1 acts intrinsically on developing Treg cells. We made a connection between Id1 expression and CD28 costimulatory signaling because Id1 transgene expression facilitated the formation of Treg precursors in CD28(-/-) mice and the in vitro differentiation of Treg cells on thymic dendritic cells despite the blockade of costimulation by anti-CD80/CD86. Id1 expression also allowed in vitro Treg differentiation without anti-CD28 costimulation, which was at least in part due to enhanced production of IL-2. Notably, with full strength of costimulatory signals, however, Id1 expression caused modest but significant suppression of Treg induction. Finally, we demonstrate that Id1 transgenic mice were less susceptible to the induction of experimental autoimmune encephalomyelitis, thus illustrating the impact of Id1-mediated augmentation of Treg cell levels on cellular immunity.

  5. The activation threshold of CD4+ T cells is defined by TCR/peptide-MHC class II interactions in the thymic medulla.

    PubMed

    Stephen, Tom Li; Tikhonova, Anastasia; Riberdy, Janice M; Laufer, Terri M

    2009-11-01

    Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-associated signaling pathways of single-positive thymocytes are attenuated to respond appropriately to the peptide-MHC molecules that will be encountered in the periphery. In this study, we explore the mechanisms that regulate the tuning of CD4(+) single-positive T cells to MHC class II encountered in the thymic medulla. Experiments with murine BM chimeras demonstrate that tuning can be mediated by MHC class II expressed by either thymic medullary epithelial cells or thymic dendritic cells. Tuning does not require the engagement of CD4 by MHC class II on stromal cells. Rather, it is mediated by interactions between MHC class II and the TCR. To understand the molecular changes that distinguish immature hyperactive T cells from tuned mature CD4(+) T cells, we compared their responses to TCR stimulation. The altered response of mature CD4 single-positive thymocytes is characterized by the inhibition of ERK activation by low-affinity self-ligands and increased expression of the inhibitory tyrosine phosphatase SHP-1. Thus, persistent TCR engagement by peptide-MHC class II on thymic medullary stroma inhibits reactivity to self-Ags and prevents autoreactivity in the mature repertoire.

  6. CD28 ligation in the absence of TCR stimulation up-regulates IL-17A and pro-inflammatory cytokines in relapsing-remitting multiple sclerosis T lymphocytes.

    PubMed

    Camperio, Cristina; Muscolini, Michela; Volpe, Elisabetta; Di Mitri, Diletta; Mechelli, Rosella; Buscarinu, Maria C; Ruggieri, Serena; Piccolella, Enza; Salvetti, Marco; Gasperini, Claudio; Battistini, Luca; Tuosto, Loretta

    2014-01-01

    CD28 is a crucial costimulatory receptor necessary full T cell activation. The role of CD28 in multiple sclerosis (MS) has been evaluated as the source of costimulatory signals integrating those delivered by TCR. However, CD28 is also able to act as a unique signaling receptor and to deliver TCR-independent autonomous signals, which regulate the expression and production of pro-inflammatory cytokines and chemokines. By comparing the cytokine/chemokine profiles of CD4(+) T cells from relapsing-remitting multiple sclerosis (RRMS) patients and healthy donors (HD), we found that CD28 engagement without TCR strongly up-regulates IL-8 and IL-6 expression in RRMS compared to HD. More interestingly, in RRMS but not in HD, CD28 stimulation selectively induces the expression of IL-17A by cooperating with IL-6-mediated signals. By using specific inhibitory drugs, we also identify the phosphatidylinositol 3 kinase (PI3K) as the critical regulator of CD28 proinflammatory functions in MS.

  7. Affinity and dose of TCR engagement yield proportional enhancer and gene activity in CD4+ T cells

    PubMed Central

    Allison, Karmel A; Sajti, Eniko; Collier, Jana G; Gosselin, David; Troutman, Ty Dale; Stone, Erica L; Hedrick, Stephen M; Glass, Christopher K

    2016-01-01

    Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of mouse CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis plays an important role in translating TCR signal strength into proportional activation of enhancers and genes essential for T cell function. DOI: http://dx.doi.org/10.7554/eLife.10134.001 PMID:27376549

  8. T cells survey the stability of the self: a testable hypothesis on the homeostatic role of TCR-MHC interactions.

    PubMed

    Bakács, Tibor; Mehrishi, Jitendra N; Szabados, Tamás; Varga, László; Szabó, Miklós; Tusnády, Gábor

    2007-01-01

    In the lifetime of an individual, every single gene will have undergone mutation on about 10(10) separate occasions. Nevertheless, cancer occurs mainly with advancing age. Here, we hypothesize that the evolutionary pressure driving the creation of the T cell receptor (TCR) repertoire was primarily the homeostatic surveillance of the genome. The subtly variable T cells may in fact constitute an evolutionary link between the invariable innate and hypervariable B cell systems. The new model is based on the homeostatic role of T cells, suggesting that molecular complementarity between the positively selected TCR and the self peptide-presenting major histocompatibility complex molecules establishes and regulates homeostasis, strictly limiting variations of its components. Notwithstanding, the 'homeostatic role of T cells' model offers a more realistic explanation as to how a naïve clonal immune system can cope with the much faster replicating pathogens, despite a limited repertoire that is capable of facing only a small fraction of the vast antigenic universe at a time.

  9. Localization of T cell receptor (TCR)-gamma delta + T cells into human colorectal cancer: flow cytometric analysis of TCR-gamma delta expression in tumour-infiltrating lymphocytes.

    PubMed Central

    Watanabe, N; Hizuta, A; Tanaka, N; Orita, K

    1995-01-01

    We analysed TCR-gamma delta expression in tumour-infiltrating lymphocytes (TIL) obtained from 13 patients with colorectal cancer and simultaneously isolated the T lymphocytes from normal intestinal tissue (IL) to compare the frequencies of TCR-gamma delta expression in TIL, IL, and peripheral blood lymphocytes (PBL) in the same patient. Flow cytometric analysis showed that the frequency of TCR-gamma delta expression in TIL (2.75 +/- 1.84%) was significantly lower than that in IL (15.28 +/- 9.45%, P < 0.01). However, a larger quantity of TIL was separated than IL per unit weight of specimen, so the total number of gamma delta T cells obtained per unit weight was not different between tumour tissue and normal intestine. In addition, phenotypic analysis revealed that about half of the TCR-gamma delta + TIL were CD8+ (CD4+, 3.0 +/- 3.1%; CD8+, 54.7 +/- 19.9%, mean +/- s.d. of five patients), and a very similar result was obtained in TCR-gamma delta + IL (CD4+, 2.7 +/- 2.4%; CD8+, 53.1 +/- 17.4%). In contrast, most TCR-gamma delta + PBL were double-negative (CD4+, 3.2 +/- 3.0%; CD8+, 20.6 +/- 7.4%). These results indicated that TCR-gamma delta + CD8+ T cells selectively and consistently localized in colorectal tumour tissue, similarly to normal intestinal epithelium. PMID:7554384

  10. Complete TCR-α gene locus control region activity in T cells derived in vitro from embryonic stem cells.

    PubMed

    Lahiji, Armin; Kucerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D

    2013-07-01

    Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.

  11. TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.

    PubMed

    Kirsch, Ilan R; Watanabe, Rei; O'Malley, John T; Williamson, David W; Scott, Laura-Louise; Elco, Christopher P; Teague, Jessica E; Gehad, Ahmed; Lowry, Elizabeth L; LeBoeuf, Nicole R; Krueger, James G; Robins, Harlan S; Kupper, Thomas S; Clark, Rachael A

    2015-10-01

    Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor γ (TCRγ) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting that a niche of finite size may exist for benign T cells in skin. Last, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases, and provided insights into the cell of origin and location of malignant CTCL cells in skin. PMID:26446955

  12. Immunoregulation of encephalitogenic MBP-NAc1-11-reactive T cells by CD4+ TCR-specific T cells involves IL-4, IL-10 and IFN-gamma.

    PubMed

    Adlard, K; Tsaknardis, L; Beam, A; Bebo, B F; Vandenbark, A A; Offner, H

    1999-01-01

    The generation of TCR transgenic (Tg) mice expressing a BV8S2 (Vbeta8 subfamily 2) chain specific for the encephalitogenic NAc1-11 region of MBP provides a unique system for evaluating the mechanisms involved in anti-TCR immunoregulation of EAE. In a previous study, we showed that vaccination with BV8S2 protein induced specific T cells that inhibited proliferation responses and encephalitogenic activity of MBP-reactive T cells in vitro, and resulted in a skewed production of Th2 cytokines by the MBP-reactive T cells. These data suggested that regulation of the encephalitogenic T cells was mediated by inhibitory cytokines rather than through a deletional mechanism. In the current study, we have employed the BV8S2 Tg mouse model to address the issue of which cytokines produced by anti-TCR-reactive T cells can regulate the function of encephalitogenic Th1 cells. Utilizing neutralizing anti-cytokine antibodies to reverse inhibitory effects of supernatants from BV8S2-specific T cells, we found that IL-4, IL-10, and to a lesser extent, IFN-gamma and TGF-beta, were the major regulatory cytokines responsible for inhibiting encephalitogenic activity, proliferation, and IFN-gamma secretion of MBP-NAc1-11-reactive Th1 cells. These results indicate that cytokine regulation is the major mechanism through which TCR specific CD4+ T cells regulate encephalitogenic and potentially other bystander Th1 cells.

  13. TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL

    PubMed Central

    O'Malley, John T.; Williamson, David W.; Scott, Laura-Louise; Elco, Christopher P.; Teague, Jessica E.; Gehad, Ahmed; Lowry, Elizabeth L.; LeBoeuf, Nicole R.; Krueger, James G.; Robins, Harlan S.; Kupper, Thomas S.; Clark, Rachael A.

    2016-01-01

    Early diagnosis of CTCL is difficult and takes on average six years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL but the TCRγ PCR analysis in current clinical use detect clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46/46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting a niche of finite size may exist for benign T cells in skin. Lastly, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases and provided insights into the cell of origin and location of malignant CTCL cells in skin. PMID:26446955

  14. Single TCR-Vβ2 evaluation discloses the circulating T cell clone in Sezary syndrome: one family fits all!

    PubMed

    Scala, Enrico; Abeni, Damiano; Pomponi, Debora; Russo, Nicoletta; Russo, Giandomenico; Narducci, Maria Grazia

    2015-08-01

    Sézary Syndrome (SS/L-CTCL) is a rare but aggressive variant of cutaneous T cell lymphoma (CTCL), characterized by erythroderma, lymphadenopathy, and the presence of a circulating memory CD4(+) T cell malignant clone with a skin homing behavior, lacking CD26 and CD49d and over-expressing CD60. The availability of a panel of monoclonal antibodies recognizing distinct TCR-Vβ families, allows to typify the clone by flow cytometry in about 70 % of cases. The TCR-Vβ repertoire of 533 individuals, comprising 308 patients affected by CTCL, 50 healthy donors, and subjects affected by various non-neoplastic dermatological affections was evaluated by flow cytometry. Statistical analyses were performed using the SPSS statistical software package for Microsoft Windows (SPSS, version 21, Chicago, IL). TCR-Vβ2 levels below 5.4 % or above 39.5 %, within total CD4(+) T cells, showed the best balance between sensitivity (98.1 %) and specificity (96 %) to identify the presence of a clone in the peripheral blood of patients affected by SS. Based on this observation, a "two-step" procedure in the detection of the malignant T cell clone in CTCLs is herein suggested. TCR-Vβ2 assessment in all cases (first step). In the case of TCR-Vβ2 levels above 39.5 %, the presence of a clonal expansion of this family is suggested, deserving further confirmation by means of T cell gene rearrangement evaluation. In patients having a TCR-Vβ2 reactivity below 5.4 % (second step), the entire TCR-Vβ repertoire should be evaluated to typify the expanded clone. In conclusion, the single TCR-Vβ2 expression check, instead of the entire repertoire assessment, represents an easy and cost-effective method for the recognition of CTCL aggressive leukemic variant.

  15. Inhibition of FOXP3/NFAT Interaction Enhances T Cell Function after TCR Stimulation.

    PubMed

    Lozano, Teresa; Villanueva, Lorea; Durántez, Maika; Gorraiz, Marta; Ruiz, Marta; Belsúe, Virginia; Riezu-Boj, José I; Hervás-Stubbs, Sandra; Oyarzábal, Julen; Bandukwala, Hozefa; Lourenço, Ana R; Coffer, Paul J; Sarobe, Pablo; Prieto, Jesús; Casares, Noelia; Lasarte, Juan J

    2015-10-01

    Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4(+) T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-γ, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-β. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.

  16. Expression of TCR-Vβ peptides by murine bone marrow cells does not identify T-cell progenitors

    PubMed Central

    Abbey, Janice L; Karsunky, Holger; Serwold, Thomas; Papathanasiou, Peter; Weissman, Irving L; O’Neill, Helen C

    2015-01-01

    Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2+Cβ− are found in several lymphoid sites, and among the lineage-negative (Lin−) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin−Vβ8.2+Cβ− BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin−Vβ8.2+Cβ− BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development. PMID:25754612

  17. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

  18. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    PubMed

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells. PMID:20164855

  19. The CD3 gamma epsilon/delta epsilon signaling module provides normal T cell functions in the absence of the TCR zeta immunoreceptor tyrosine-based activation motifs.

    PubMed

    Pitcher, Lisa A; Mathis, Meredith A; Young, Jennifer A; DeFord, Laura M; Purtic, Bozidar; Wulfing, Christoph; van Oers, Nicolai S C

    2005-12-01

    T cell receptor (TCR) signal transduction is mediated by the immunoreceptor tyrosine-based activation motifs (ITAM). The ten ITAM in the TCR complex are distributed in two distinct signaling modules termed TCR zetazeta and CD3 gammaepsilon/deltaepsilon. To delineate the specific role of the zeta ITAM in T cell development and TCR signal transmission, we compared the properties of T cells from different TCR zeta-transgenic lines wherein tyrosine-to-phenylalanine substitutions had been introduced in the zeta subunit. These lines lack selected phosphorylated forms of TCR zeta including just p23, both p21 and p23, or all phospho-zeta derivatives. We report herein that the efficiency of positive selection in HY TCR-transgenic female mice was directly related to the number of zeta ITAM in the TCR. In contrast, TCR-mediated signal transmission and T cell proliferative responses following agonist peptide stimulation were similar and independent of the zeta ITAM. Only the duration of MAPK activation was affected by multiple zeta ITAM substitutions. These results strongly suggest that the ITAM in the CD3 gammaepsilon/deltaepsilon module can provide normal TCR signal transmission, with zeta ITAM providing a secondary function facilitating MAPK activation and positive selection.

  20. T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

    PubMed Central

    Gold, R; Giegerich, G; Hartung, H P; Toyka, K V

    1995-01-01

    Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE. Images Fig. 4 Fig. 5 PMID:7597040

  1. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

    PubMed Central

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

  2. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

    PubMed

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.

  3. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

    PubMed

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

  4. T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells

    PubMed Central

    Conley, James M.; Gallagher, Michael P.; Berg, Leslie J.

    2016-01-01

    Signaling downstream of the T cell receptor (TCR) is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength. T cells rapidly alter their gene expression upon activation. Multiple signaling pathways downstream of the TCR activate transcription factors, which are critical for this process. The dynamics between proximal TCR signaling, transcription factor activation and CD8 T cell function are discussed here. We propose that inducible T cell kinase (ITK) acts as a rheostat for gene expression. This unique regulation of TCR signaling by ITK provides a possible signaling mechanism for the promotion of a diverse T cell repertoire in response to pathogen. PMID:26973653

  5. Generating HPV specific T helper cells for the treatment of HPV induced malignancies using TCR gene transfer

    PubMed Central

    2011-01-01

    Background Infection with high risk Human Papilloma Virus (HPV) is associated with cancer of the cervix, vagina, penis, vulva, anus and some cases of head and neck carcinomas. The HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore potential targets for T cell mediated adoptive immunotherapy. Effective immunotherapy is dependent on the presence of both CD4+ and CD8+ T cells. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. An alternative to generate HPV specific CD4+ and CD8+ T cells is TCR gene transfer. Methods HPV specific CD4+ T cells were generated using either a MHC class I or MHC class II restricted TCR (from clones A9 and 24.101 respectively) directed against HPV16 antigens. Functional analysis was performed by interferon-γ secretion, proliferation and cytokine production assays. Results Introduction of HPV16 specific TCRs into blood derived CD4+ recipient T cells resulted in recognition of the relevant HPV16 epitope as determined by IFN-γ secretion. Importantly, we also show recognition of the endogenously processed and HLA-DP1 presented HPV16E6 epitope by 24.101 TCR transgenic CD4+ T cells and recognition of the HLA-A2 presented HPV16E7 epitope by A9 TCR transgenic CD4+ T cells. Conclusion Our data indicate that TCR transfer is feasible as an alternative strategy to generate human HPV16 specific CD4+ T helper cells for the treatment of patients suffering from cervical cancer and other HPV16 induced malignancies. PMID:21892941

  6. Lck Mediates Signal Transmission from CD59 to the TCR/CD3 Pathway in Jurkat T Cells

    PubMed Central

    Lipp, Anna M.; Juhasz, Kata; Paar, Christian; Ogris, Christoph; Eckerstorfer, Paul; Thuenauer, Roland; Hesse, Jan; Nimmervoll, Benedikt; Stockinger, Hannes; Schütz, Gerhard J.; Bodenhofer, Ulrich

    2014-01-01

    The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca2+ imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca2+ response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca2+ signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses. PMID:24454946

  7. Correlation of contact sensitizer potency with T cell frequency and TCR repertoire diversity.

    PubMed

    Esser, Philipp R; Kimber, Ian; Martin, Stefan F

    2014-01-01

    Allergic contact dermatitis is a T cell-mediated skin disease. Many hundreds of organic chemicals and some metal ions are contact sensitizers. They induce an innate inflammatory immune response in the skin that results in the priming of contact sensitizer-specific T cells by dendritic cells in the draining lymph nodes. The factors that determine the strength of this T cell response and thereby define the potency of a contact sensitizer are largely unknown. This chapter highlights different variables such as precursor frequency of antigen-specific T cells, possible bystander activation, and T cell receptor diversity or avidity of the TCR/peptide-MHC interactions, which might impact the quality and strength of T cell responses to contact sensitizers. In addition, different methods available to determine both the frequency of antigen-specific T cells and T cell receptor repertoires are discussed. Identification of the factors determining potency may allow for the development of suitable in vitro assays for potency assessment of contact sensitizers.

  8. Low-affinity TCR engagement drives IL-2-dependent post-thymic maintenance of naive CD4+ T cells in aged humans

    PubMed Central

    van der Geest, Kornelis S M; Abdulahad, Wayel H; Teteloshvili, Nato; Tete, Sarah M; Peters, Jorieke H; Horst, Gerda; Lorencetti, Pedro G; Bos, Nicolaas A; Lambeck, Annechien; Roozendaal, Caroline; Kroesen, Bart-Jan; Koenen, Hans J P M; Joosten, Irma; Brouwer, Elisabeth; Boots, Annemieke M H

    2015-01-01

    Insight into the maintenance of naive T cells is essential to understand defective immune responses in the context of aging and other immune compromised states. In humans, naive CD4+ T cells, in contrast to CD8+ T cells, are remarkably well retained with aging. Here, we show that low-affinity TCR engagement is the main driving force behind the emergence and accumulation of naive-like CD4+ T cells with enhanced sensitivity to IL-2 in aged humans. In vitro, we show that these CD45RA+CD25dimCD4+ T cells can develop from conventional naive CD25−CD4+ T cells upon CD3 cross-linking alone, in the absence of costimulation, rather than via stimulation by the homeostatic cytokines IL-2, IL-7, or IL-15. In vivo, TCR engagement likely occurs in secondary lymphoid organs as these cells were detected in lymph nodes and spleen where they showed signs of recent activation. CD45RA+CD25dimCD4+ T cells expressed a broad TCRVβ repertoire and could readily differentiate into functional T helper cells. Strikingly, no expansion of CD45RA+CD25dimCD8+ T cells was detected with aging, thereby implying that maintenance of naive CD4+ T cells is uniquely regulated. Our data provide novel insight into the homeostasis of naive T cells and may guide the development of therapies to preserve or restore immunity in the elderly. PMID:26010129

  9. Pre-TCR ligand binding impacts thymocyte development before αβTCR expression

    PubMed Central

    Mallis, Robert J.; Bai, Ke; Arthanari, Haribabu; Hussey, Rebecca E.; Handley, Maris; Li, Zhenhai; Chingozha, Loice; Duke-Cohan, Jonathan S.; Lu, Hang; Wang, Jia-Huai; Zhu, Cheng; Wagner, Gerhard; Reinherz, Ellis L.

    2015-01-01

    Adaptive cellular immunity requires accurate self- vs. nonself-discrimination to protect against infections and tumorous transformations while at the same time excluding autoimmunity. This vital capability is programmed in the thymus through selection of αβT-cell receptors (αβTCRs) recognizing peptides bound to MHC molecules (pMHC). Here, we show that the pre-TCR (preTCR), a pTα-β heterodimer appearing before αβTCR expression, directs a previously unappreciated initial phase of repertoire selection. Contrasting with the ligand-independent model of preTCR function, we reveal through NMR and bioforce-probe analyses that the β-subunit binds pMHC using Vβ complementarity-determining regions as well as an exposed hydrophobic Vβ patch characteristic of the preTCR. Force-regulated single bonds akin to those of αβTCRs but with more promiscuous ligand specificity trigger calcium flux. Thus, thymic development involves sequential β- and then, αβ-repertoire tuning, whereby preTCR interactions with self pMHC modulate early thymocyte expansion, with implications for β-selection, immunodominant peptide recognition, and germ line-encoded MHC interaction. PMID:26056289

  10. Pre-TCR ligand binding impacts thymocyte development before αβTCR expression.

    PubMed

    Mallis, Robert J; Bai, Ke; Arthanari, Haribabu; Hussey, Rebecca E; Handley, Maris; Li, Zhenhai; Chingozha, Loice; Duke-Cohan, Jonathan S; Lu, Hang; Wang, Jia-Huai; Zhu, Cheng; Wagner, Gerhard; Reinherz, Ellis L

    2015-07-01

    Adaptive cellular immunity requires accurate self- vs. nonself-discrimination to protect against infections and tumorous transformations while at the same time excluding autoimmunity. This vital capability is programmed in the thymus through selection of αβT-cell receptors (αβTCRs) recognizing peptides bound to MHC molecules (pMHC). Here, we show that the pre-TCR (preTCR), a pTα-β heterodimer appearing before αβTCR expression, directs a previously unappreciated initial phase of repertoire selection. Contrasting with the ligand-independent model of preTCR function, we reveal through NMR and bioforce-probe analyses that the β-subunit binds pMHC using Vβ complementarity-determining regions as well as an exposed hydrophobic Vβ patch characteristic of the preTCR. Force-regulated single bonds akin to those of αβTCRs but with more promiscuous ligand specificity trigger calcium flux. Thus, thymic development involves sequential β- and then, αβ-repertoire tuning, whereby preTCR interactions with self pMHC modulate early thymocyte expansion, with implications for β-selection, immunodominant peptide recognition, and germ line-encoded MHC interaction. PMID:26056289

  11. Mushroom acidic glycosphingolipid induction of cytokine secretion from murine T cells and proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in vitro

    SciTech Connect

    Nozaki, Hirofumi; Itonori, Saki; Sugita, Mutsumi; Nakamura, Kimihide; Ohba, Kiyoshi; Suzuki, Akemi; Kushi, Yasunori

    2008-08-29

    Interferon (IFN)-{gamma} and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- {gamma} and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of {alpha}-galactosylceramide ({alpha}-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by {alpha}-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 {alpha}/{beta} TCR-double positive cells in splenocytes. Administration of a mixture of {alpha}-GalCer and AGLs affected the stimulation of {alpha}-GalCer and generally induced a subtle Th1 bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.

  12. Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

    PubMed Central

    Munson, Daniel J.; Egelston, Colt A.; Chiotti, Kami E.; Parra, Zuly E.; Bruno, Tullia C.; Moore, Brandon L.; Nakano, Taizo A.; Simons, Diana L.; Jimenez, Grecia; Yim, John H.; Rozanov, Dmitri V.; Falta, Michael T.; Fontenot, Andrew P.; Reynolds, Paul R.; Leach, Sonia M.; Borges, Virginia F.; Kappler, John W.; Spellman, Paul T.; Slansky, Jill E.

    2016-01-01

    Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha–beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients’ tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer. PMID:27307436

  13. Naive CD8+ T-cell precursors display structured TCR repertoires and composite antigen-driven selection dynamics

    PubMed Central

    Neller, Michelle A; Ladell, Kristin; McLaren, James E; Matthews, Katherine K; Gostick, Emma; Pentier, Johanne M; Dolton, Garry; Schauenburg, Andrea JA; Koning, Dan; Fontaine Costa, Ana Isabel CA; Watkins, Thomas S; Venturi, Vanessa; Smith, Corey; Khanna, Rajiv; Miners, Kelly; Clement, Mathew; Wooldridge, Linda; Cole, David K; van Baarle, Debbie; Sewell, Andrew K; Burrows, Scott R; Price, David A; Miles, John J

    2015-01-01

    Basic parameters of the naive antigen (Ag)-specific T-cell repertoire in humans remain poorly defined. Systematic characterization of this ‘ground state' immunity in comparison with memory will allow a better understanding of clonal selection during immune challenge. Here, we used high-definition cell isolation from umbilical cord blood samples to establish the baseline frequency, phenotype and T-cell antigen receptor (TCR) repertoire of CD8+ T-cell precursor populations specific for a range of viral and self-derived Ags. Across the board, these precursor populations were phenotypically naive and occurred with hierarchical frequencies clustered by Ag specificity. The corresponding patterns of TCR architecture were highly ordered and displayed partial overlap with adult memory, indicating biased structuring of the T-cell repertoire during Ag-driven selection. Collectively, these results provide new insights into the complex nature and dynamics of the naive T-cell compartment. PMID:25801351

  14. Regulation of TCR signalling by tyrosine phosphatases: from immune homeostasis to autoimmunity

    PubMed Central

    Stanford, Stephanie M; Rapini, Novella; Bottini, Nunzio

    2012-01-01

    More than half of the known protein tyrosine phosphatases (PTPs) in the human genome are expressed in T cells, and significant progress has been made in elucidating the biology of these enzymes in T-cell development and function. Here we provide a systematic review of the current understanding of the roles of PTPs in T-cell activation, providing insight into their mechanisms of action and regulation in T-cell receptor signalling, the phenotypes of their genetically modified mice, and their possible involvement in T-cell-mediated autoimmune disease. Our projection is that the interest in PTPs as mediators of T-cell homeostasis will continue to rise with further functional analysis of these proteins, and PTPs will be increasingly considered as targets of immunomodulatory therapies. PMID:22862552

  15. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    PubMed Central

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  16. Chimeric antigen receptor (CAR) and T cell receptor (TCR) Modified T cells Enter Main Street and Wall Street

    PubMed Central

    Barrett, David M; Grupp, Stephan A; June, Carl H

    2015-01-01

    The field of adoptive cell transfer (ACT) is currently comprised of CAR and TCR engineered T cells and has emerged from principles of basic immunology to paradigm-shifting clinical immunotherapy. ACT of T cells engineered to express artificial receptors that target cells of choice is an exciting new approach for cancer, and holds equal promise for chronic infection and autoimmunity. Using principles of synthetic biology, advances in immunology and genetic engineering have made it possible to generate human T-cells that display desired specificities and enhanced functionalities. Clinical trials in patients with advanced B cell leukemias and lymphomas treated with CD19-specific CAR T cells have induced durable remissions in adults and children. The prospects for the widespread availability of engineered T cells have changed dramatically given the recent entry of the pharmaceutical industry to this arena. Here, we discuss some of the challenges and opportunities that face the field of ACT. PMID:26188068

  17. Specific roles of each TCR hemichain in generating functional chain-centric TCR

    PubMed Central

    Nakatsugawa, Munehide; Yamashita, Yuki; Ochi, Toshiki; Tanaka, Shinya; Chamoto, Kenji; Guo, Tingxi; Butler, Marcus O.; Hirano, Naoto

    2015-01-01

    T cell receptor (TCR)α and β chains cooperatively recognize peptide-MHC (pMHC) complexes. It has been shown that a ‘chain-centric’ TCR hemichain can, by itself, dictate MHC-restricted antigen specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α chain-centric HLA-A*02:01(A2)/MART127–35 TCRα, clone SIG35α, into A2-matched and unmatched post-thymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127–35. While the generated A2/MART127–35-specific T cells used various TRBV genes, TRBV27 predominated with >102 highly diverse and unique clonotypic CDR3β sequences. T cells individually reconstituted with various A2/MART127–35 TRBV27 TCRβ genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127–35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-antigens. Our results indicate that, although a chain-centric TCR hemichain determines antigen specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising antigen specificity. TCR chain centricity can be exploited to generate a thymically unselected antigen-specific T cell repertoire, which can be used to isolate high avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery due to tolerance. PMID:25710913

  18. Membrane-Mediated Regulation of the Intrinsically Disordered CD3ϵ Cytoplasmic Tail of the TCR

    PubMed Central

    López, Cesar A.; Sethi, Anurag; Goldstein, Byron; Wilson, Bridget S.; Gnanakaran, S.

    2015-01-01

    The regulation of T-cell-mediated immune responses depends on the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on T-cell receptors. Although many details of the signaling cascades are well understood, the initial mechanism and regulation of ITAM phosphorylation remains unknown. We used molecular dynamics simulations to study the influence of different compositions of lipid bilayers on the membrane association of the CD3ϵ cytoplasmic tails of the T-cell receptors. Our results show that binding of CD3ϵ to membranes is modulated by both the presence of negatively charged lipids and the lipid order of the membrane. Free-energy calculations reveal that the protein-membrane interaction is favored by the presence of nearby basic residues and the ITAM tyrosines. Phosphorylation minimizes membrane association, rendering the ITAM motif more accessible to binding partners. In systems mimicking biological membranes, the CD3ϵ chain localization is modulated by different facilitator lipids (e.g., gangliosides or phosphoinositols), revealing a plausible regulatory effect on activation through the regulation of lipid composition in cell membranes. PMID:25992726

  19. Generation of TCR-engineered T cells and their use to control the performance of T cell assays.

    PubMed

    Bidmon, Nicole; Attig, Sebastian; Rae, Richard; Schröder, Helene; Omokoko, Tana A; Simon, Petra; Kuhn, Andreas N; Kreiter, Sebastian; Sahin, Ugur; Gouttefangeas, Cécile; van der Burg, Sjoerd H; Britten, Cedrik M

    2015-06-15

    The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients' immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simple, stable, robust, and scalable technology to generate reference samples that contain defined numbers of functional Ag-specific T cells. First, we show that RNA-engineered lymphocytes, equipped with selected TCRs, can repetitively deliver functional readouts of a controlled size across multiple assay platforms. We further describe a concept for the application of TCR-engineered reference samples to keep assay performance within or across institutions under tight control. Finally, we provide evidence that these novel control reagents can sensitively detect assay variation resulting from typical sources of error, such as low cell quality, loss of reagent stability, suboptimal hardware settings, or inaccurate gating. PMID:25957167

  20. A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen*

    PubMed Central

    Bianchi, Valentina; Bulek, Anna; Fuller, Anna; Lloyd, Angharad; Attaf, Meriem; Rizkallah, Pierre J.; Dolton, Garry; Sewell, Andrew K.; Cole, David K.

    2016-01-01

    Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination. PMID:26917722

  1. Immune selection of tumor cells in TCR β-chain transgenic mice.

    PubMed

    Silaeva, Yulia Yu; Grinenko, Tatyana S; Vagida, Murad S; Kalinina, Anastasia A; Khromykh, Ludmila M; Kazansky, Dmitry B

    2014-10-01

    The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single β-chain transgenic B10.D2(R101) (K(d)I(d)D(b)) mice to allogeneic EL4 (H-2(b)) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2K(b) molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8(+) T-lymphocytes with endogenous β-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic β-chains became CD8(+)CD44(+)CD62L(-) effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40-60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2K(b) molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses.

  2. Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning.

    PubMed

    Paensuwan, Pussadee; Hartl, Frederike A; Yousefi, O Sascha; Ngoenkam, Jatuporn; Wipa, Piyamaporn; Beck-Garcia, Esmeralda; Dopfer, Elaine P; Khamsri, Boonruang; Sanguansermsri, Donruedee; Minguet, Susana; Schamel, Wolfgang W; Pongcharoen, Sutatip

    2016-01-01

    Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning. PMID:26590318

  3. Endogenous galectin-1 enforces class I–restricted TCR functional fate decisions in thymocytes

    PubMed Central

    Liu, Scot D.; Whiting, Chan C.; Tomassian, Tamar; Pang, Mabel; Bissel, Stephanie J.; Baum, Linda G.; Mossine, Valeri V.; Poirier, Françoise; Huflejt, Margaret E.

    2008-01-01

    During thymocyte development, the T-cell receptor (TCR) can discriminate major histocompatibility complex (MHC)/peptide ligands over a narrow range of affinities and translate subtle differences into functional fate decisions. How small differences in TCR input are translated into absolute differences in functional output is unclear. We examined the effects of galectin-1 ablation in the context of class-I–restricted thymocyte development. Galectin-1 expression opposed TCR partial agonist-driven positive selection, but promoted TCR agonist-driven negative selection of conventional CD8+ T cells. Galectin-1 expression also promoted TCR agonist-driven CD8αα intestinal intraepithelial lymphocytes (IEL) development. Recombinant galectin-1 enhanced TCR binding to agonist/MHC complexes and promoted a negative-selection-signaling signature, reflected in intensified rapid and transient extracellular signal-regulated kinase (ERK) activation. In contrast, galectin-1 expression antagonized ERK activity in thymocytes undergoing positive selection. We propose that galectin-1 aids in discriminating TCR-directed fate decisions by promoting TCR binding to agonist/MHC complexes and enforcing agonist-driven signals, while opposing partial-agonist signals. In this way, galectin-1 widens the distinction between TCR-directed functional fate cues. PMID:18323414

  4. [Development of Tax-redirected T-cell immunotherapy using TCR gene transduction in patients with ATL].

    PubMed

    Tanaka, Yukie; Kanda, Yoshinobu

    2015-07-01

    ATL is an aggressive T-cell malignancy caused by HTLV-1 virus infection. Tax, which is the most important regulatory protein of HTLV-1, is associated with aggressive proliferation of host cells and is also a major target antigen for CD8⁺ cytotoxic T-cells (CTLs). Recently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has proven effective for ATL, and donor-derived Tax-specific CTL might contribute to graft-versus-ATL effects in some recipients who maintained complete remission after allo-HSCT. We, for the first time, analyzed the Tax-specific T-cell receptor (TCR) repertoire, phenotypes and functions of Tax-specific CTLs at single-cell levels in HLA-A24⁺ ATL patients who underwent allo-HSCT. We found that 1) a particular amino acid sequence motif (PDR) in the CDR3 region of TCR-β was conserved in different patients and also within the same patient before and after allo-HSCT, and 2) the PDR⁺ Tax-specific CTL clone selectively expanded in ATL long-term survivors as less-differentiated effector memory CTLs. Actually, the PDR⁺ CTL showed not only strong binding activity for the Tax-tetramer but also strong killing activity against patients' HTLV-1-infected T-cells without any reaction against normal cells. We are presently evaluating the killing activities of PDR⁺ TCR-transduced T-cells against Tax in immunodeficient mice, with the aim of developing a new immunotherapy for ATL.

  5. Specific roles of each TCR hemichain in generating functional chain-centric TCR.

    PubMed

    Nakatsugawa, Munehide; Yamashita, Yuki; Ochi, Toshiki; Tanaka, Shinya; Chamoto, Kenji; Guo, Tingxi; Butler, Marcus O; Hirano, Naoto

    2015-04-01

    TCRα- and β-chains cooperatively recognize peptide-MHC complexes. It has been shown that a "chain-centric" TCR hemichain can, by itself, dictate MHC-restricted Ag specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric, hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α-chain-centric HLA-A*02:01(A2)/MART127-35 TCRα, clone SIG35α, into A2-matched and unmatched postthymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127-35. Although the generated A2/MART127-35-specific T cells used various TRBV genes, TRBV27 predominated with >10(2) highly diverse and unique clonotypic CDR3β sequences. T cells individually reconstituted with various A2/MART127-35 TRBV27 TCRβ genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127-35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-Ags. Our results indicate that, although a chain-centric TCR hemichain determines Ag specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising Ag specificity. TCR chain centricity can be exploited to generate a thymically unselected Ag-specific T cell repertoire, which can be used to isolate high-avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery because of tolerance. PMID:25710913

  6. The Protein Phosphatase 2A Regulatory Subunit B56γ Mediates Suppression of T Cell Receptor (TCR)-induced Nuclear Factor-κB (NF-κB) Activity*

    PubMed Central

    Breuer, Rebecca; Becker, Michael S.; Brechmann, Markus; Mock, Thomas; Arnold, Rüdiger; Krammer, Peter H.

    2014-01-01

    NF-κB is an important transcription factor in the immune system, and aberrant NF-κB activity contributes to malignant diseases and autoimmunity. In T cells, NF-κB is activated upon TCR stimulation, and signal transduction to NF-κB activation is triggered by a cascade of phosphorylation events. However, fine-tuning and termination of TCR signaling are only partially understood. Phosphatases oppose the role of kinases by removing phosphate moieties. The catalytic activity of the protein phosphatase PP2A has been implicated in the regulation of NF-κB. PP2A acts in trimeric complexes in which the catalytic subunit is promiscuous and the regulatory subunit confers substrate specificity. To understand and eventually target NF-κB-specific PP2A functions it is essential to define the regulatory PP2A subunit involved. So far, the regulatory PP2A subunit that mediates NF-κB suppression in T cells remained undefined. By performing a siRNA screen in Jurkat T cells harboring a NF-κB-responsive luciferase reporter, we identified the PP2A regulatory subunit B56γ as negative regulator of NF-κB in TCR signaling. B56γ was strongly up-regulated upon primary human T cell activation, and B56γ silencing induced increased IκB kinase (IKK) and IκBα phosphorylation upon TCR stimulation. B56γ silencing enhanced NF-κB activity, resulting in increased NF-κB target gene expression including the T cell cytokine IL-2. In addition, T cell proliferation was increased upon B56γ silencing. These data help to understand the physiology of PP2A function in T cells and the pathophysiology of diseases involving PP2A and NF-κB. PMID:24719332

  7. Human MHC Class I-restricted high avidity CD4(+) T cells generated by co-transfer of TCR and CD8 mediate efficient tumor rejection in vivo.

    PubMed

    Xue, Shao-An; Gao, Liquan; Ahmadi, Maryam; Ghorashian, Sara; Barros, Rafael D; Pospori, Constandina; Holler, Angelika; Wright, Graham; Thomas, Sharyn; Topp, Max; Morris, Emma C; Stauss, Hans J

    2013-01-01

    In this study, we generated human MHC Class I-restricted CD4(+) T cells specific for Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two herpesviridae associated with lymphoma, nasopharyngeal carcinoma and medulloblastoma, respectively. Retroviral transfer of virus-specific, HLA-A2-restricted TCR-coding genes generated CD4(+) T cells that recognized HLA-A2/peptide multimers and produced cytokines when stimulated with MHC Class II-deficient cells presenting the relevant viral peptides in the context of HLA-A2. Peptide titration revealed that CD4(+) T cells had a 10-fold lower avidity than CD8(+) T cells expressing the same TCR. The impaired avidity of CD4(+) T cells was corrected by simultaneously transferring TCR- and CD8-coding genes. The CD8 co-receptor did not alter the cytokine signature of CD4(+) T cells, which remained distinct from that of CD8(+) T cells. Using the xenogeneic NOD/SCID mouse model, we demonstrated that human CD4(+) T cells expressing a specific TCR and CD8 can confer efficient protection against the growth of tumors expressing the EBV or CMV antigens recognized by the TCR. In summary, we describe a robust approach for generating therapeutic CD4(+) T cells capable of providing MHC Class I-restricted immunity against MHC Class II-negative tumors in vivo.

  8. The feature of distribution and clonality of TCR γ/δ subfamilies T cells in patients with B-cell non-Hodgkin lymphoma.

    PubMed

    Wang, Liang; Xu, Meng; Wang, Chunyan; Zhu, Lihua; Hu, Junyan; Chen, Shaohua; Wu, Xiuli; Li, Bo; Li, Yangqiu

    2014-01-01

    Restricted T-cell receptor (TCR) Vα/Vβ repertoire expression and clonal expansion of αβ T cells especially for putative tumor-associated antigens were observed in patients with hematological malignancies. To further characterize the γδ T-cell immune status in B-cell non-Hodgkin lymphoma (B-NHL), we investigated the distribution and clonality of TCR Vγ/Vδ repertoire in peripheral blood (PB), bone marrow (BM), and lymph node (LN) from patients with B-NHL. Four newly diagnosed B-NHL cases, including three with diffuse large B-cell lymphoma (DLBCL) and one with small lymphocytic lymphoma (SLL), were enrolled. The restrictive expression of TCR Vγ/Vδ subfamilies with different distribution patterns could be detected in PB, BM, or LN from all of four patients, and partial subfamily T cells showed clonal proliferation. At least one clonally expanded Vδ subfamily member was found in PB from each patient. However, the expression pattern and clonality of TCR Vγ/Vδ changed in different immune organs and showed individual feature in different patients. The clonally expanded Vδ5, Vδ6, and Vδ8 were detected only in PB but neither in BM nor LN while clonally expanded Vδ2 and Vδ3 could be detected in both PB and BM/LN. In conclusion, the results provide a preliminary profile of distribution and clonality of TCR γ/δ subfamilies T cells in PB, BM, and LN from B-NHL; similar clonally expanded Vδ subfamily T cells in PB and BM may be related to the same B-cell lymphoma-associated antigens, while the different reactive clonally expanded Vγ/Vδ T cells may be due to local immune response. PMID:24963496

  9. SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

    PubMed

    Friend, Samantha F; Peterson, Lisa K; Kedl, Ross M; Dragone, Leonard L

    2013-03-01

    How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

  10. Generation of the First TCR Transgenic Mouse with CD4(+) T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide.

    PubMed

    Jansen, Manon A A; van Herwijnen, Martijn J C; van Kooten, Peter J S; Hoek, Aad; van der Zee, Ruurd; van Eden, Willem; Broere, Femke

    2016-01-01

    Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such -antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen--specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4(+) T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4(+) T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4(+)CD25(+) Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis. PMID:27014269

  11. Generation of the First TCR Transgenic Mouse with CD4+ T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide

    PubMed Central

    Jansen, Manon A. A.; van Herwijnen, Martijn J. C.; van Kooten, Peter J. S.; Hoek, Aad; van der Zee, Ruurd; van Eden, Willem; Broere, Femke

    2016-01-01

    Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such ­antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen-­specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4+ T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4+ T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4+CD25+ Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis. PMID:27014269

  12. The thymic cortical epithelium determines the TCR repertoire of IL-17-producing γδT cells

    PubMed Central

    Nitta, Takeshi; Muro, Ryunosuke; Shimizu, Yukiko; Nitta, Sachiko; Oda, Hiroyo; Ohte, Yuki; Goto, Motohito; Yanobu-Takanashi, Rieko; Narita, Tomoya; Takayanagi, Hiroshi; Yasuda, Hisataka; Okamura, Tadashi; Murata, Shigeo; Suzuki, Harumi

    2015-01-01

    The thymus provides a specialized microenvironment in which distinct subsets of thymic epithelial cells (TECs) support T-cell development. Here, we describe the significance of cortical TECs (cTECs) in T-cell development, using a newly established mouse model of cTEC deficiency. The deficiency of mature cTECs caused a massive loss of thymic cellularity and impaired the development of αβT cells and invariant natural killer T cells. Unexpectedly, the differentiation of certain γδT-cell subpopulations—interleukin-17-producing Vγ4 and Vγ6 cells—was strongly dysregulated, resulting in the perturbation of γδT-mediated inflammatory responses in peripheral tissues. These findings show that cTECs contribute to the shaping of the TCR repertoire, not only of “conventional” αβT cells but also of inflammatory “innate” γδT cells. PMID:25770130

  13. Visualizing TCR-induced POLKADOTS formation and NF-κB activation in the D10 T-cell clone and mouse primary effector T cells.

    PubMed

    Paul, Suman; Schaefer, Brian C

    2015-01-01

    T cells are an immune cell lineage that play a central role in protection against pathogen infection. Antigen, in the form of pathogen-derived peptides, stimulates the T-cell receptor (TCR), leading to activation of the transcription factor, nuclear factor kappa B (NF-κB). The subsequent NF-κB-dependent gene expression program drives expansion and effector differentiation of antigen-specific T cells, leading to the adaptive anti-pathogen immune response. The cell surface TCR transmits activating signals to cytosolic NF-κB by a complex signaling cascade, in which the adapter protein Bcl10 plays a key role. We have previously demonstrated that TCR engagement leads to the formation of cytosolic Bcl10 clusters, called POLKADOTS, that provide a platform for the assembly of the terminal signaling complex that ultimately mediates NF-κB activation. In this chapter, we describe the methods utilized to visualize the formation of TCR-induced POLKADOTS and to study the temporal association between POLKADOTS formation and nuclear translocation of the NF-κB subunit, RelA/p65.

  14. MHC-I restricted Melanoma Antigen Specific TCR Engineered Human CD4+ T Cells Exhibit Multifunctional Effector and Helper Responses, In Vitro

    PubMed Central

    Ray, Swagatam; Chhabra, Arvind; Chakraborty, Nitya G.; Hegde, Upendra; Dorsky, David I.; Chodon, Thinle; von Euw, Erika; Comin-Anduix, Begonya; Koya, Richard C.; Ribas, Antoni; Economou, James S.; Rosenberg, Steven A.; Mukherji, Bijay

    2010-01-01

    MHC class 1-restricted human melanoma epitope MART-127–35 specific TCR engineered CD4+CD25− T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25− T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-γ, TNF-α, IL-2, MIP1ß), lyse target cells, and “help” the expansion of the MART-127–35 specific CD8+ T cells when stimulated by the MART-127–35 peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-127–35 specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR-transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies. PMID:20547105

  15. Pro-inflammatory self-reactive T cells are found within murine TCR-αβ(+) CD4(-) CD8(-) PD-1(+) cells.

    PubMed

    Rodríguez-Rodríguez, Noé; Apostolidis, Sokratis A; Fitzgerald, Lauren; Meehan, Bronwyn S; Corbett, Alexandra J; Martín-Villa, José Manuel; McCluskey, James; Tsokos, George C; Crispín, José C

    2016-06-01

    TCR-αβ(+) double negative (DN) T cells (CD3(+) TCR-αβ(+) CD4(-) CD8(-) NK1.1(-) CD49b(-) ) represent a minor heterogeneous population in healthy humans and mice. These cells have been ascribed pro-inflammatory and regulatory capacities and are known to expand during the course of several autoimmune diseases. Importantly, previous studies have shown that self-reactive CD8(+) T cells become DN after activation by self-antigens, suggesting that self-reactive T cells may exist within the DN T-cell population. Here, we demonstrate that programmed cell death 1 (PD-1) expression in unmanipulated mice identifies a subset of DN T cells with expression of activation-associated markers and a phenotype that strongly suggests they are derived from self-reactive CD8(+) cells. We also found that, within DN T cells, the PD-1(+) subset generates the majority of pro-inflammatory cytokines. Finally, using a TCR-activation reporter mouse (Nur77-GFP), we confirmed that in the steady-state PD-1(+) DN T cells engage endogenous antigens in healthy mice. In conclusion, we provide evidence that indicates that the PD-1(+) fraction of DN T cells represents self-reactive cells.

  16. Human regulatory T cells control TCR signaling and susceptibility to suppression in CD4+ T cells.

    PubMed

    Chellappa, Stalin; Lieske, Nora V; Hagness, Morten; Line, Pål D; Taskén, Kjetil; Aandahl, Einar M

    2016-07-01

    Human CD4(+)CD25(hi)FOXP3(+) regulatory T cells maintain immunologic tolerance and prevent autoimmune and inflammatory immune responses. Regulatory T cells undergo a similar activation cycle as conventional CD4(+) T cells upon antigen stimulation. Here, we demonstrate that T cell receptors and costimulation are required to activate the regulatory T cell suppressive function. Regulatory T cells suppressed the T cell receptor signaling in effector T cells in a time-dependent manner that corresponded with inhibition of cytokine production and proliferation. Modulation of the activation level and thereby the suppressive capacity of regulatory T cells imposed distinct T cell receptor signaling signatures and hyporesponsiveness in suppressed and proliferating effector T cells and established a threshold for effector T cell proliferation. The immune suppression of effector T cells was completely reversible upon removal of regulatory T cells. However, the strength of prior immune suppression by regulatory T cells and corresponding T cell receptor signaling in effector T cells determined the susceptibility to suppression upon later reexposure to regulatory T cells. These findings demonstrate how the strength of the regulatory T cell suppressive function determines intracellular signaling, immune responsiveness, and the later susceptibility of effector T cells to immune suppression and contribute to unveiling the complex interactions between regulatory T cells and effector T cells. PMID:26715685

  17. The combination of a chemokine, cytokine and TCR-based T cell stimulus for effective gene therapy of cancer.

    PubMed

    Paul, Stephane; Regulier, Etienne; Poitevin, Yves; Hormann, Horst; Acres, R Bruce

    2002-12-01

    Cytotoxic T cells can recognize and kill tumor cells that present peptides derived from tumor-associated antigens (TAA) on their surface when associated with major histocompatibility complex (MHC) class I molecules. However, immune responses to tumor-associated antigens are often suppressed by a tumor-induced state of immune anergy. Previous work has attempted to overcome tumor-induced T cell anergy by the direct injection of vectors carrying genes encoding one of a variety of cytokines. Polyclonal stimulation of T cells, preferably via the TCR complex, results in a cascade of cytokines associated with T cell activation and thus may be better able to overcome T cell anergy. We have previously reported the use of the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, antibodies specific for the CD3epsilon chain (KT3). When injected into growing tumors, these constructs induce the activation of immune effector cells and result in rejection of the tumor. A variety of recombinant adenovirus (Ad) vectors expressing immunostimulatory and/or immunoattractant molecules have now been produced. With this collection of viruses, we have carried out in vivo analyses of combinations of vectors in tumor therapy experiments. For example, we have tested, in murine tumor models, the combination of MVA-KT3 with Ad expressing recently identified cytokines [for example interleukin-12 (IL-12), IL-18] as well as chemokines (e.g. RANTES, MIP1beta). One combination, MVA-KT3/Ad-IL-12/Ad-MIP1beta causes rejection of 100% of growing RENCA tumors. Much attention has been focused on cancer gene therapy using gene transfer of single agents. These data show that antigenic stimulation via the MHCI/TCR-CD3+cytokine+chemokine combination may provide a new and promising approach to cancer gene therapy which is more likely to bypass tumor immunosuppression mechanisms. PMID:12439610

  18. Quantitative proteomic analysis of signalosome dynamics in primary T cells identifies the CD6 surface receptor as a Lat-independent TCR signaling hub

    PubMed Central

    Fiore, Fréderic; Liang, Yinming; Chen, Zhi; Sansoni, Amandine; Kanduri, Kartiek; Joly, Rachel; Malzac, Aurélie; Lähdesmäki, Harri; Lahesmaa, Riitta; Yamasaki, Sho; Saito, Takashi; Malissen, Marie; Aebersold, Ruedi; Gstaiger, Matthias; Malissen, Bernard

    2014-01-01

    T cell antigen receptor (TCR)-mediated T cell activation requires the interaction of dozens of proteins. We used quantitative mass spectrometry and activated primary CD4+ T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes forming around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high confidence time-resolved protein interactions we observed were novel. The CD6 surface receptor was found capable of initiating its own signaling pathway by recruiting SLP-76 and Vav1, irrespective of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub contributing to TCR signal diversification. PMID:24584089

  19. T Cell Receptor (TCR) Interacting Molecule (TRIM), A Novel Disulfide-linked Dimer Associated with the TCR–CD3–ζ Complex, Recruits Intracellular Signaling Proteins to the Plasma Membrane

    PubMed Central

    Bruyns, Eddy; Marie-Cardine, Anne; Kirchgessner, Henning; Sagolla, Karin; Shevchenko, Andrej; Mann, Matthias; Autschbach, Frank; Bensussan, Armand; Meuer, Stefan; Schraven, Burkhart

    1998-01-01

    The molecular mechanisms regulating recruitment of intracellular signaling proteins like growth factor receptor–bound protein 2 (Grb2), phospholipase Cγ1, or phosphatidylinositol 3-kinase (PI3-kinase) to the plasma membrane after stimulation of the T cell receptor (TCR)– CD3–ζ complex are not very well understood. We describe here purification, tandem mass spectrometry sequencing, molecular cloning, and biochemical characterization of a novel transmembrane adaptor protein which associates and comodulates with the TCR–CD3–ζ complex in human T lymphocytes and T cell lines. This protein was termed T cell receptor interacting molecule (TRIM). TRIM is a disulfide-linked homodimer which is comprised of a short extracellular domain of 8 amino acids, a 19–amino acid transmembrane region, and a 159–amino acid cytoplasmic tail. In its intracellular domain, TRIM contains several tyrosine-based signaling motifs that could be involved in SH2 domain–mediated protein–protein interactions. Indeed, after T cell activation, TRIM becomes rapidly phosphorylated on tyrosine residues and then associates with the 85-kD regulatory subunit of PI3-kinase via an YxxM motif. Thus, TRIM represents a TCR-associated transmembrane adaptor protein which is likely involved in targeting of intracellular signaling proteins to the plasma membrane after triggering of the TCR. PMID:9687533

  20. Autologous stem cell transplantation aids autoimmune patients by functional renewal and TCR diversification of regulatory T cells.

    PubMed

    Delemarre, Eveline M; van den Broek, Theo; Mijnheer, Gerdien; Meerding, Jenny; Wehrens, Ellen J; Olek, Sven; Boes, Marianne; van Herwijnen, Martijn J C; Broere, Femke; van Royen, Annet; Wulffraat, Nico M; Prakken, Berent J; Spierings, Eric; van Wijk, Femke

    2016-01-01

    Autologous hematopoietic stem cell transplantation (HSCT) is increasingly considered for patients with severe autoimmune diseases whose prognosis is poor with standard treatments. Regulatory T cells (Tregs) are thought to be important for disease remission after HSCT. However, eliciting the role of donor and host Tregs in autologous HSCT is not possible in humans due to the autologous nature of the intervention. Therefore, we investigated their role during immune reconstitution and re-establishment of immune tolerance and their therapeutic potential following congenic bone marrow transplantation (BMT) in a proteoglycan-induced arthritis (PGIA) mouse model. In addition, we determined Treg T-cell receptor (TCR) CDR3 diversity before and after HSCT in patients with juvenile idiopathic arthritis and juvenile dermatomyositis. In the PGIA BMT model, after an initial predominance of host Tregs, graft-derived Tregs started dominating and displayed a more stable phenotype with better suppressive capacity. Patient samples revealed a striking lack of diversity of the Treg repertoire before HSCT. This ameliorated after HSCT, confirming reset of the Treg compartment following HSCT. In the mouse model, a therapeutic approach was initiated by infusing extra Foxp3(GFP+) Tregs during BMT. Infusion of Foxp3(GFP+) Tregs did not elicit additional clinical improvement but conversely delayed reconstitution of the graft-derived T-cell compartment. These data indicate that HSCT-mediated amelioration of autoimmune disease involves renewal of the Treg pool. In addition, infusion of extra Tregs during BMT results in a delayed reconstitution of T-cell compartments. Therefore, Treg therapy may hamper development of long-term tolerance and should be approached with caution in the clinical autologous setting. PMID:26480932

  1. In vivo 6-thioguanine-resistant T cells from melanoma patients have public TCR and share TCR beta amino acid sequences with melanoma-reactive T cells

    PubMed Central

    Zuleger, Cindy L.; Macklin, Michael D.; Bostwick, Bret L.; Pei, Qinglin; Newton, Michael A.; Albertini, Mark R.

    2011-01-01

    In vivo hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient T cells (MT) from melanoma patients are enriched for T cells with in vivo clonal amplifications that traffic between blood and tumor tissues. Melanoma is thus a model cancer to test the hypothesis that in vivo MT from cancer patients can be used as immunological probes for immunogenic tumor antigens. MT were obtained by 6-thioguanine (TG) selection of lymphocytes from peripheral blood and tumor tissues, and wild-type T cells (WT) were obtained analogously without TG selection. cDNA sequences of the T cell receptor beta chains (TRB) were used as unambiguous biomarkers of in vivo clonality and as indicators of T cell specificity. Public TRB were identified in MT from the blood and tumor of different melanoma patients. Such public TRB were not found in normal control MT or WT. As an indicator of T cell specificity for melanoma, the >2600 MT and WT TRB, including the public TRB from melanoma patients, were compared to a literature-derived empirical database of >1270 TRB from melanoma-reactive T cells. Various degrees of similarity, ranging from 100% conservation to 3-amino acid motifs (3-mer), were found between both melanoma patient MT and WT TRBs and the empirical database. The frequency of 3-mer and 4-mer TRB matching to the empirical database was significantly higher in MT compared with WT in the tumor (p=0.0285 and p=0.006, respectively). In summary, in vivo MT from melanoma patients contain public TRB as well as T cells with specificity for characterized melanoma antigens. We conclude that in vivo MT merit study as novel probes for uncharacterized immunogenic antigens in melanoma and other malignancies. PMID:21182840

  2. In vivo 6-thioguanine-resistant T cells from melanoma patients have public TCR and share TCR beta amino acid sequences with melanoma-reactive T cells.

    PubMed

    Zuleger, Cindy L; Macklin, Michael D; Bostwick, Bret L; Pei, Qinglin; Newton, Michael A; Albertini, Mark R

    2011-02-28

    In vivo hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient T cells (MT) from melanoma patients are enriched for T cells with in vivo clonal amplifications that traffic between blood and tumor tissues. Melanoma is thus a model cancer to test the hypothesis that in vivo MT from cancer patients can be used as immunological probes for immunogenic tumor antigens. MT were obtained by 6-thioguanine (TG) selection of lymphocytes from peripheral blood and tumor tissues, and wild-type T cells (WT) were obtained analogously without TG selection. cDNA sequences of the T cell receptor beta chains (TRB) were used as unambiguous biomarkers of in vivo clonality and as indicators of T cell specificity. Public TRB were identified in MT from the blood and tumor of different melanoma patients. Such public TRB were not found in normal control MT or WT. As an indicator of T cell specificity for melanoma, the >2600 MT and WT TRB, including the public TRB from melanoma patients, were compared to a literature-derived empirical database of >1270 TRB from melanoma-reactive T cells. Various degrees of similarity, ranging from 100% conservation to 3-amino acid motifs (3-mer), were found between both melanoma patient MT and WT TRBs and the empirical database. The frequency of 3-mer and 4-mer TRB matching to the empirical database was significantly higher in MT compared with WT in the tumor (p=0.0285 and p=0.006, respectively). In summary, in vivo MT from melanoma patients contain public TRB as well as T cells with specificity for characterized melanoma antigens. We conclude that in vivo MT merit study as novel probes for uncharacterized immunogenic antigens in melanoma and other malignancies. PMID:21182840

  3. Hepatitis C virus-cross-reactive TCR gene-modified T cells: a model for immunotherapy against diseases with genomic instability.

    PubMed

    Spear, Timothy T; Riley, Timothy P; Lyons, Gretchen E; Callender, Glenda G; Roszkowski, Jeffrey J; Wang, Yuan; Simms, Patricia E; Scurti, Gina M; Foley, Kendra C; Murray, David C; Hellman, Lance M; McMahan, Rachel H; Iwashima, Makio; Garrett-Mayer, Elizabeth; Rosen, Hugo R; Baker, Brian M; Nishimura, Michael I

    2016-09-01

    A major obstacle hindering the development of effective immunity against viral infections, their associated disease, and certain cancers is their inherent genomic instability. Accumulation of mutations can alter processing and presentation of antigens recognized by antibodies and T cells that can lead to immune escape variants. Use of an agent that can intrinsically combat rapidly mutating viral or cancer-associated antigens would be quite advantageous in developing effective immunity against such disease. We propose that T cells harboring cross-reactive TCRs could serve as a therapeutic agent in these instances. With the use of hepatitis C virus, known for its genomic instability as a model for mutated antigen recognition, we demonstrate cross-reactivity against immunogenic and mutagenic nonstructural protein 3:1406-1415 and nonstructural protein 3:1073-1081 epitopes in PBL-derived, TCR-gene-modified T cells. These single TCR-engineered T cells can CD8-independently recognize naturally occurring and epidemiologically relevant mutant variants. TCR-peptide MHC modeling data allow us to rationalize how TCR structural properties accommodate recognition of certain mutated epitopes and how these substitutions impact the requirement of CD8 affinity enhancement for recognition. A better understanding of such TCRs' promiscuous behavior may allow for exploitation of these properties to develop novel, adoptive T cell-based therapies for viral infections and cancers exhibiting similar genomic instability. PMID:26921345

  4. Hepatitis C virus-cross-reactive TCR gene-modified T cells: a model for immunotherapy against diseases with genomic instability.

    PubMed

    Spear, Timothy T; Riley, Timothy P; Lyons, Gretchen E; Callender, Glenda G; Roszkowski, Jeffrey J; Wang, Yuan; Simms, Patricia E; Scurti, Gina M; Foley, Kendra C; Murray, David C; Hellman, Lance M; McMahan, Rachel H; Iwashima, Makio; Garrett-Mayer, Elizabeth; Rosen, Hugo R; Baker, Brian M; Nishimura, Michael I

    2016-09-01

    A major obstacle hindering the development of effective immunity against viral infections, their associated disease, and certain cancers is their inherent genomic instability. Accumulation of mutations can alter processing and presentation of antigens recognized by antibodies and T cells that can lead to immune escape variants. Use of an agent that can intrinsically combat rapidly mutating viral or cancer-associated antigens would be quite advantageous in developing effective immunity against such disease. We propose that T cells harboring cross-reactive TCRs could serve as a therapeutic agent in these instances. With the use of hepatitis C virus, known for its genomic instability as a model for mutated antigen recognition, we demonstrate cross-reactivity against immunogenic and mutagenic nonstructural protein 3:1406-1415 and nonstructural protein 3:1073-1081 epitopes in PBL-derived, TCR-gene-modified T cells. These single TCR-engineered T cells can CD8-independently recognize naturally occurring and epidemiologically relevant mutant variants. TCR-peptide MHC modeling data allow us to rationalize how TCR structural properties accommodate recognition of certain mutated epitopes and how these substitutions impact the requirement of CD8 affinity enhancement for recognition. A better understanding of such TCRs' promiscuous behavior may allow for exploitation of these properties to develop novel, adoptive T cell-based therapies for viral infections and cancers exhibiting similar genomic instability.

  5. New Insights into How Trafficking Regulates T Cell Receptor Signaling

    PubMed Central

    Lou, Jieqiong; Rossy, Jérémie; Deng, Qiji; Pageon, Sophie V.; Gaus, Katharina

    2016-01-01

    There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions. PMID:27508206

  6. Regulation of T cell-dendritic cell interactions by IL-7 governs T-cell activation and homeostasis

    PubMed Central

    Saini, Manoj; Pearson, Claire

    2009-01-01

    Interleukin-7 (IL-7) plays a central role in the homeostasis of the T-cell compartment by regulating T-cell survival and proliferation. Whether IL-7 can influence T-cell receptor (TCR) signaling in T cells remains controversial. Here, using IL-7–deficient hosts and TCR-transgenic T cells that conditionally express IL-7R, we examined antigen-specific T-cell responses in vitro and in vivo to viral infection and lymphopenia to determine whether IL-7 signaling influences TCR-triggered cell division events. In vitro, we could find no evidence that IL-7 signaling could costimulate T-cell activation over a broad range of conditions, suggesting that IL-7 does not directly tune TCR signaling. In vivo, however, we found an acute requirement for IL-7 signaling for efficiently triggering T-cell responses to influenza A virus challenge. Furthermore, we found that IL-7 was required for the enhanced homeostatic TCR signaling that drives lymphopenia-induced proliferation by a mechanism involving efficient contacts of T cells with dendritic cells. Consistent with this, saturating antigen-presenting capacity in vivo overcame the triggering defect in response to cognate peptide. Thus, we demonstrate a novel role for IL-7 in regulating T cell–dendritic cell interactions that is essential for both T-cell homeostasis and activation in vivo. PMID:19357399

  7. The Sts Proteins Target Tyrosine Phosphorylated, Ubiquitinated Proteins within TCR Signaling Pathways

    SciTech Connect

    Carpino, N.; Chen, Y; Nassar, N; Oh, H

    2009-01-01

    The T cell receptor (TCR) detects the presence of infectious pathogens and activates numerous intracellular signaling pathways. Protein tyrosine phosphorylation and ubiquitination serve as key regulatory mechanisms downstream of the TCR. Negative regulation of TCR signaling pathways is important in controlling the immune response, and the Suppressor of TCR Signaling proteins (Sts-1 and Sts-2) have been shown to function as critical negative regulators of TCR signaling. Although their mechanism of action has yet to be fully uncovered, it is known that the Sts proteins possess intrinsic phosphatase activity. Here, we demonstrate that Sts-1 and Sts-2 are instrumental in down-modulating proteins that are dually modified by both protein tyrosine phosphorylation and ubiquitination. Specifically, both naive and activated T cells derived from genetically engineered mice that lack the Sts proteins display strikingly elevated levels of tyrosine phosphorylated, ubiquitinated proteins following TCR stimulation. The accumulation of the dually modified proteins is transient, and in activated T cells but not naive T cells is significantly enhanced by co-receptor engagement. Our observations hint at a novel regulatory mechanism downstream of the T cell receptor.

  8. Phenolic-glycolipid-1 and lipoarabinomannan preferentially modulate TCR- and CD28-triggered proximal biochemical events, leading to T-cell unresponsiveness in mycobacterial diseases

    PubMed Central

    2012-01-01

    Background Advanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling. Results We observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted. Conclusion Altogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum. PMID:22985026

  9. Structure of the TCR expressed on a gastritogenic T cell clone, II-6, and frequent appearance of similar clonotypes in mice bearing autoimmune gastritis.

    PubMed

    Katakai, T; Agata, Y; Shimizu, A; Ohshima, C; Nishio, A; Inaba, M; Kasakura, S; Mori, K J; Masuda, T

    1997-12-01

    A parietal cell-specific Th1 clone, II-6, which was established from a BALB/c mouse bearing post-thymectomy autoimmune gastritis (AIG), recognizes a peptide of the alpha subunit (alpha891-905) of H+/K+-ATPase and induces gastritis in nu/nu BALB/c mice by adoptive cell transfer. In the present study, the primary structure of the TCR of II-6 was determined as Valpha10-Jalpha c5a-Calpha and Vbeta14-Jbeta2.3-Cbeta2 by cDNA cloning. Using PCR with specific primers, we defined the use of this II-6 TCR in nu/nu mice with transferred II-6 cells and in mice that spontaneously developed AIG by thymectomy on day 3 after birth (d3-Tx). II-6 TCR mRNAs were detected in the gastric mucosa of all of the nu/nu mice, suggesting that II-6 cells indeed home to the gastric mucosa and thereby were directly involved in the destruction of target parietal cells. TCR beta chain mRNAs encoding CDR3 region sequences almost identical with that of II-6 were also found in the gastric mucosa in 43% (six of 14 mice tested) of the d3-Tx AIG mice at 4-12 weeks old by nested RT-PCR. Such a frequent appearance of similar clonotypes in independent individuals suggests that T cells bearing II-6-like TCR including the II-6 itself might be directly involved in, although not essential for, the pathogenesis of AIG in 3d-Tx mice.

  10. Naive CD8⁺ T-cell precursors display structured TCR repertoires and composite antigen-driven selection dynamics.

    PubMed

    Neller, Michelle A; Ladell, Kristin; McLaren, James E; Matthews, Katherine K; Gostick, Emma; Pentier, Johanne M; Dolton, Garry; Schauenburg, Andrea J A; Koning, Dan; Fontaine Costa, Ana Isabel C A; Watkins, Thomas S; Venturi, Vanessa; Smith, Corey; Khanna, Rajiv; Miners, Kelly; Clement, Mathew; Wooldridge, Linda; Cole, David K; van Baarle, Debbie; Sewell, Andrew K; Burrows, Scott R; Price, David A; Miles, John J

    2015-08-01

    Basic parameters of the naive antigen (Ag)-specific T-cell repertoire in humans remain poorly defined. Systematic characterization of this 'ground state' immunity in comparison with memory will allow a better understanding of clonal selection during immune challenge. Here, we used high-definition cell isolation from umbilical cord blood samples to establish the baseline frequency, phenotype and T-cell antigen receptor (TCR) repertoire of CD8(+) T-cell precursor populations specific for a range of viral and self-derived Ags. Across the board, these precursor populations were phenotypically naive and occurred with hierarchical frequencies clustered by Ag specificity. The corresponding patterns of TCR architecture were highly ordered and displayed partial overlap with adult memory, indicating biased structuring of the T-cell repertoire during Ag-driven selection. Collectively, these results provide new insights into the complex nature and dynamics of the naive T-cell compartment.

  11. TCR variable gene involvement in chromosome inversion between 14q11 and 14q24 in adult T-cell leukemia.

    PubMed

    Haider, Shawkat; Hayakawa, Kousuke; Itoyama, Takahiro; Sadamori, Naoki; Kurosawa, Nobuyuki; Isobe, Masaharu

    2006-01-01

    Chromosomal translocations in T-cell malignancies frequently involve the T-cell receptor (TCR)alpha/delta locus at chromosome 14q11. Although 14q11 abnormalities are found in about 10% of adult T-cell leukemia (ATL) cases, until now there has been no direct evidence showing involvement of the TCR locus in ATL-a malignancy closely associated with HTLV-1 infection. The breakpoints of T-cell malignancies most commonly occur within the Jalpha or Jdelta region of the TCR locus. In ATL, however, despite extensive searching no breakpoint has yet been found in that region. Using fluorescence in situ hybridization with a panel of cosmid and bacterial artificial chromosome probes derived from chromosome 14, including the variable region of the TCRalpha locus, comprehensive analysis of an ATL patient carrying inv(14)(q11q32) revealed that the TCR locus was indeed involved in this inversion. Molecular cloning of the breakpoint revealed the juxtaposition of TCR Valpha to the 14q24 region as a result of two consecutive inversions: inv(14)(q11q32) and inv(14)(q11q24). We also found a gene near the breakpoint at the 14q24 region that is downregulated in this ATL patient and is assigned in the database as a pseudogene of ADAM21 (a disintegrin and metalloproteinase domain 21). Our expression analysis, however, showed that this pseudogene was actually expressed and was capable of encoding a protein similar to ADAM21; thus we have named this gene ADAM21-like (ADAM21-L).

  12. A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis.

    PubMed

    Carpenter, Stephen M; Nunes-Alves, Cláudio; Booty, Matthew G; Way, Sing Sing; Behar, Samuel M

    2016-01-01

    T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

  13. A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

    PubMed Central

    Carpenter, Stephen M.; Nunes-Alves, Cláudio; Booty, Matthew G.; Way, Sing Sing; Behar, Samuel M.

    2016-01-01

    T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection. PMID:26745507

  14. [Homology modeling and eukaryotic expression of a modified αβ TCR harboring the immunoglobulin-like domain of γδ TCR].

    PubMed

    Tao, Changli; Shao, Hongwei; Shen, Han; Huang, Shulin

    2016-08-01

    Objective To design, construct and express a chimeric αβ TCR harboring the immunoglobulin-like (Ig) domain of γδ TCR in Jurkat T cells. Methods The fusion sites of TCR δIg were determined by bioinformatics analysis. Then the protein structures of TCR α δIg and TCR β δIg were predicted by homology modeling. Furthermore, the structures of TCR α δIg and TCR β δIg were compared with the wild type (wt) TCR α and TCR β respectively by combinatorial extension (CE). After that, the TCR α δIg and TCR β δIg were fused to fluorescent protein ECFP and EYFP respectively via the overlap PCR, and then the fusion genes (TCR α δIg-ECFP and TCR β δIg-EYFP) were cloned into pIRES2-EGFP vector and respectively located at the upstream and downstream of an internal ribosome entry site (IRES). The recombinant prokaryotic expression vector pIRES-TCR βδIg-EYFP/TCR αδIg-ECFP was transferred into Jurkat T cells. Finally, the expression of TCR δIg in Jurkat T cells was monitored by confocal laser scanning microscopy (CLSM). Results The variable region structure of the TCR δIg did not change and the antigen recognition active regions remained stable compared to the wtTCR. The recombinant expression plasmid was successfully constructed as confirmed by PCR identification and sequencing analysis. CLSM showed that TCR δIg was expressed and located at the plasma membrane of Jurkat T cells. Conclusion The design of TCR δIg was reasonable and the TCR δIg could be expressed on Jurkat T cell surface. PMID:27412930

  15. Improved expression and reactivity of transduced tumor-specific TCRs in human lymphocytes by specific silencing of endogenous TCR.

    PubMed

    Okamoto, Sachiko; Mineno, Junichi; Ikeda, Hiroaki; Fujiwara, Hiroshi; Yasukawa, Masaki; Shiku, Hiroshi; Kato, Ikunoshin

    2009-12-01

    Adoptive T-cell therapy using lymphocytes genetically engineered to express tumor antigen-specific TCRs is an attractive strategy for treating patients with malignancies. However, there are potential drawbacks to this strategy: mispairing of the introduced TCR alpha/beta chains with the endogenous TCR subunits and competition of CD3 molecules between the introduced and endogenous TCRs can impair cell surface expression of the transduced TCR, resulting in insufficient function and potential generation of autoreactive T cells. In addition, the risk of tumor development following the infusion of cells with aberrant vector insertion sites increases with the vector copy number in the transduced cells. In this study, we developed retroviral vectors encoding both small interfering RNA constructs that specifically down-regulate endogenous TCR and a codon-optimized, small interfering RNA-resistant TCR specific for the human tumor antigens MAGE-A4 or WT1. At low copy numbers of the integrated vector, the transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR and reduced expression of endogenous TCRs. In consequence, the vector-transduced lymphocytes showed enhanced cytotoxic activity against antigen-expressing tumor cells. Therefore, our novel TCR gene therapy may open a new gate for effective immunotherapy in cancer patients. PMID:19903853

  16. Therapeutic targeting of naturally presented myeloperoxidase-derived HLA peptide ligands on myeloid leukemia cells by TCR-transgenic T cells.

    PubMed

    Klar, R; Schober, S; Rami, M; Mall, S; Merl, J; Hauck, S M; Ueffing, M; Admon, A; Slotta-Huspenina, J; Schwaiger, M; Stevanović, S; Oostendorp, R A J; Busch, D H; Peschel, C; Krackhardt, A M

    2014-12-01

    T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.

  17. NSOM/QD-Based Direct Visualization of CD3-Induced and CD28-Enhanced Nanospatial Coclustering of TCR and Coreceptor in Nanodomains in T Cell Activation

    PubMed Central

    Lu, Xiaoxu; Wang, Richard C.; Gong, Guangming; Yan, Lin; Huang, Dan; Chen, Zheng W.

    2009-01-01

    Direct molecular imaging of nano-spatial relationship between T cell receptor (TCR)/CD3 and CD4 or CD8 co-receptor before and after activation of a primary T cell has not been reported. We have recently innovated application of near-field scanning optical microscopy (NSOM) and immune-labeling quantum dots (QD) to image Ag-specific TCR response during in vivo clonal expansion, and now up-graded the NSOM/QD-based nanotechnology through dipole-polarization and dual-color imaging. Using this imaging system scanning cell-membrane molecules at a best-optical lateral resolution, we demonstrated that CD3, CD4 or CD8 molecules were distinctly distributed as single QD-bound molecules or nano-clusters equivalent to 2–4 QD fluorescence-intensity/size on cell-membrane of un-stimulated primary T cells, and ∼6–10% of CD3 were co-clustering with CD4 or CD8 as 70–110 nm nano-clusters without forming nano-domains. The ligation of TCR/CD3 on CD4 or CD8 T cells led to CD3 nanoscale co-clustering or interaction with CD4 or CD8 co-receptors forming 200–500 nm nano-domains or >500 nm micro-domains. Such nano-spatial co-clustering of CD3 and CD4 or CD3 and CD8 appeared to be an intrinsic event of TCR/CD3 ligation, not purely limited to MHC engagement, and be driven by Lck phosphorylation. Importantly, CD28 co-stimulation remarkably enhanced TCR/CD3 nanoscale co-clustering or interaction with CD4 co-receptor within nano- or micro-domains on the membrane. In contrast, CD28 co-stimulation did not enhance CD8 clustering or CD3–CD8 co-clustering in nano-domains although it increased molecular number and density of CD3 clustering in the enlarged nano-domains. These nanoscale findings provide new insights into TCR/CD3 interaction with CD4 or CD8 co-receptor in T-cell activation. PMID:19536289

  18. The ζ isoform of diacylglycerol kinase plays a predominant role in regulatory T cell development and TCR-mediated ras signaling.

    PubMed

    Joshi, Rohan P; Schmidt, Amanda M; Das, Jayajit; Pytel, Dariusz; Riese, Matthew J; Lester, Melissa; Diehl, J Alan; Behrens, Edward M; Kambayashi, Taku; Koretzky, Gary A

    2013-11-26

    Diacylglycerol (DAG) is a critical second messenger that mediates T cell receptor (TCR)-stimulated signaling. The abundance of DAG is reduced by the diacylglycerol kinases (DGKs), which catalyze the conversion of DAG to phosphatidic acid (PA) and thus inhibit DAG-mediated signaling. In T cells, the predominant DGK isoforms are DGKα and DGKζ, and deletion of the genes encoding either isoform enhances DAG-mediated signaling. We found that DGKζ, but not DGKα, suppressed the development of natural regulatory T (T(reg)) cells and predominantly mediated Ras and Akt signaling downstream of the TCR. The differential functions of DGKα and DGKζ were not attributable to differences in protein abundance in T cells or in their localization to the contact sites between T cells and antigen-presenting cells. RasGRP1, a key DAG-mediated activator of Ras signaling, associated to a greater extent with DGKζ than with DGKα; however, in silico modeling of TCR-stimulated Ras activation suggested that a difference in RasGRP1 binding affinity was not sufficient to cause differences in the functions of each DGK isoform. Rather, the model suggested that a greater catalytic rate for DGKζ than for DGKα might lead to DGKζ exhibiting increased suppression of Ras-mediated signals compared to DGKα. Consistent with this notion, experimental studies demonstrated that DGKζ was more effective than DGKα at catalyzing the metabolism of DAG to PA after TCR stimulation. The enhanced effective enzymatic production of PA by DGKζ is therefore one possible mechanism underlying the dominant functions of DGKζ in modulating T(reg) cell development.

  19. The ζ Isoform of Diacylglycerol Kinase Plays a Predominant Role in Regulatory T Cell Development and TCR-Mediated Ras Signaling

    PubMed Central

    Joshi, Rohan P.; Schmidt, Amanda M.; Das, Jayajit; Pytel, Dariusz; Riese, Matthew J.; Lester, Melissa; Diehl, J. Alan; Behrens, Edward M.; Kambayashi, Taku; Koretzky, Gary A.

    2014-01-01

    Diacylglycerol (DAG) is a critical second messenger that mediates T cell receptor (TCR)–stimulated signaling. The abundance of DAG is reduced by the diacylglycerol kinases (DGKs), which catalyze the conversion of DAG to phosphatidic acid (PA) and thus inhibit DAG-mediated signaling. In T cells, the predominant DGK isoforms are DGKα and DGKζ, and deletion of the genes encoding either isoform enhances DAG-mediated signaling. We found that DGKζ, but not DGKα, suppressed the development of natural regulatory T (Treg) cells and predominantly mediated Ras and Akt signaling downstream of the TCR. The differential functions of DGKα and DGKζ were not attributable to differences in protein abundance in T cells or in their localization to the contact sites between T cells and antigen-presenting cells. RasGRP1, a key DAG-mediated activator of Ras signaling, associated to a greater extent with DGKζ than with DGKα; however, in silico modeling of TCR-stimulated Ras activation suggested that a difference in RasGRP1 binding affinity was not sufficient to cause differences in the functions of each DGK isoform. Rather, the model suggested that a greater catalytic rate for DGKζ than for DGKα might lead to DGKζ exhibiting increased suppression of Ras-mediated signals compared to DGKα. Consistent with this notion, experimental studies demonstrated that DGKζ was more effective than DGKα at catalyzing the metabolism of DAG to PA after TCR stimulation. The enhanced effective enzymatic production of PA by DGKζ is therefore one possible mechanism underlying the dominant functions of DGKζ in modulating Treg cell development. PMID:24280043

  20. Otud7b facilitates T cell activation and inflammatory responses by regulating Zap70 ubiquitination.

    PubMed

    Hu, Hongbo; Wang, Hui; Xiao, Yichuan; Jin, Jin; Chang, Jae-Hoon; Zou, Qiang; Xie, Xiaoping; Cheng, Xuhong; Sun, Shao-Cong

    2016-03-01

    Signal transduction from the T cell receptor (TCR) is crucial for T cell-mediated immune responses and, when deregulated, also contributes to the development of autoimmunity. How TCR signaling is regulated is incompletely understood. In this study, we demonstrate a ubiquitin-dependent mechanism in which the deubiquitinase Otud7b has a crucial role in facilitating TCR signaling. Upon TCR ligation, Otud7b is rapidly recruited to the tyrosine kinase Zap70, a central mediator of TCR-proximal signaling. Otud7b deficiency attenuates the activation of Zap70 and its downstream pathways and impairs T cell activation and differentiation, rendering mice refractory to T cell-mediated autoimmune and inflammatory responses. Otud7b facilitated Zap70 activation by deubiquitinating Zap70, thus preventing the association of Zap70 with the negative-regulatory phosphatases Sts1 and Sts2. These findings establish Otud7b as a positive regulator of TCR-proximal signaling and T cell activation, highlighting the importance of deubiquitination in regulating Zap70 function. PMID:26903241

  1. NKG2D performs two functions in invariant NKT cells: Direct TCR-independent activation of NK-like cytolysis, and co-stimulation of activation by CD1d

    PubMed Central

    Kuylenstierna, Carlotta; Björkström, Niklas K.; Andersson, Sofia K.; Sahlström, Peter; Bosnjak, Lidija; Paquin-Proulx, Dominic; Malmberg, Karl-Johan; Ljunggren, Hans-Gustaf; Moll, Markus; Sandberg, Johan K.

    2012-01-01

    Invariant NKT cells are important in the activation and regulation of immune responses. They can also function as CD1d-restricted killer cells. However, the role of activating innate NK cell receptors expressed on NKT cells in triggering cytolytic function is poorly characterized. Here, we initially confirmed that the cellular stress-ligand receptor NKG2D is expressed on CD4− NKT cells, whereas most CD4+ NKT cells lack this receptor. Interestingly, NKG2D+ NKT cells frequently expressed perforin, and both NKG2D and perforin localized at the site of contact with NKG2D ligand-expressing target cells. CD4− NKT cells degranulated in response to NKG2D engagement in a redirected activation assay independently of stimulation via their invariant TCR. NKT cells killed P815 cells coated with anti-NKG2D mAb and CD1d-negative K562 tumor target cells in an NKG2D-dependent manner. Furthermore, NKG2D engagement co-stimulated TCR-mediated NKT cell activation in response to endogenous CD1d-presented ligands or suboptimal levels of anti-CD3 triggering. These data indicate that the CD4− subset of human NKT cells can mediate direct lysis of target cells via NKG2D engagement independently of CD1d, and that NKG2D also functions as a co-stimulatory receptor in these cells. NKG2D thus plays both a direct and a co-stimulatory role in the activation of NKT cells. PMID:21590763

  2. Restricted TCR-alpha CDR3 diversity disadvantages natural regulatory T cell development in the B6.2.16 beta-chain transgenic mouse.

    PubMed

    Singh, Yogesh; Ferreira, Cristina; Chan, Andrew C Y; Dyson, Julian; Garden, Oliver A

    2010-09-15

    To date, analysis of mice expressing TCR-beta transgenes derived from CD4(+) T cell clones has demonstrated equivalent or higher TCR diversity in naturally occurring regulatory CD4(+) T cells (Tregs) versus conventional CD4(+) T cells (Tcons). However, TCR-alpha-chain diversity in these mice may be influenced by the inherent bias toward the CD4(+) lineage in the selected repertoires. We wished to determine whether the choice of TCR-beta-chain influences the relative diversity of the Treg and Tcon repertoires, examining as a model the B6.2.16beta-transgenic mouse, in which the fixed beta-chain is derived from a CD8(+) T cell clone. B6.2.16beta Treg thymocytes showed significantly lower TRAV17 (AV9) CDR3 sequence diversity than both syngeneic Tcon thymocytes, and Treg and Tcon thymocytes from wild-type C57BL/6 (B6) mice. The ratio of single-positive CD4(+)/single-positive CD8(+) thymocytes in B6.2.16beta mice was similar to that in B6, yet both the proportional frequency and absolute number of CD4(+)Foxp3(+) cells was significantly lower in the thymi and peripheral lymph nodes of B6.2.16beta mice. Furthermore, B6 + B6.2.16beta-->B6 mixed bone marrow chimeras revealed that the transgenic beta-chain disadvantaged Treg development in a competitive environment. These data underline the importance of the beta-chain in assessments of Treg alpha-chain diversity and provide further support for the notion that interclonal competition for entry into the Treg lineage is a significant factor in determining the composition of this lineage.

  3. TCR stimulation without co-stimulatory signals induces expression of "tolerogenic" genes in memory CD4 T cells but does not compromise cell proliferation.

    PubMed

    Xie, Aini; Zheng, Xiong; Khattar, Mithun; Schroder, Paul; Stepkowski, Stanislaw; Xia, Jiahong; Chen, Wenhao

    2015-02-01

    Memory T cells resist co-stimulatory blockade and present a unique therapeutic challenge in transplantation and autoimmune diseases. Herein, we determined whether memory T cells express less "tolerogenic" genes than naïve T cells to reinforce a proliferative response under the deprivation of co-stimulatory signals. The expression of ∼40 tolerogenic genes in memory and naïve CD4(+) T cells was thus assessed during an in vitro TCR stimulation without co-stimulation. Briefly, upon TCR stimulation with an anti-CD3 mAb alone, memory CD4(+) T cells exhibited more proliferation than naïve CD4(+) T cells. To our surprise, at 24h upon anti-CD3 mAb stimulation, memory CD4(+) T cells expressed more than a 5-fold higher level of the transcription factor Egr2 and a 20-fold higher level of the transmembrane E3 ubiquitin ligase GRAIL than those in naïve T cells. Hence, the high-level expression of tolerogenic genes, Egr2 and GRAIL, in memory CD4(+) T cells does not prevent cell proliferation. Importantly, anti-CD3 mAb-stimulated memory CD4(+) T cells expressed high protein/gene levels of phosphorylated STAT5, Nedd4, Bcl-2, and Bcl-XL. Therefore, co-stimulation-independent proliferation of memory CD4(+) T cells may be due to elevated expression of molecules that support cell proliferation and survival, but not lack of tolerogenic molecules.

  4. Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappaB and NFAT.

    PubMed

    Zhan, Yifan; Gerondakis, Steve; Coghill, Elise; Bourges, Dorothee; Xu, Yuekang; Brady, Jamie L; Lew, Andrew M

    2008-10-15

    Although the transcription factor Foxp3 is implicated in regulating glucocorticoid-induced TNF receptor (GITR) expression in the T regulatory cell lineage, little is known about how GITR is transcriptionally regulated in conventional T cells. In this study, we provide evidence that TCR-mediated GITR expression depends on the ligand affinity and the maturity of conventional T cells. A genetic dissection of GITR transcriptional control revealed that of the three transcription factors downstream of the classical NF-kappaB pathway (RelA, cRel, and NF-kappaB1), RelA is a critical positive regulator of GITR expression, although cRel and NF-kappaB1 also play a positive regulatory role. Consistent with this finding, inhibiting NF-kappaB using Bay11-7082 reduces GITR up-regulation. In contrast, NFAT acts as a negative regulator of GITR expression. This was evidenced by our findings that agents suppressing NFAT activity (e.g., cyclosporin A and FK506) enhanced TCR-mediated GITR expression, whereas agents enhancing NFAT activity (e.g., lithium chloride) suppressed TCR-mediated GITR up-regulation. Critically, the induction of GITR was found to confer protection to conventional T cells from TCR-mediated apoptosis. We propose therefore that two major transcriptional factors activated downstream of the TCR, namely, NF-kappaB and NFAT, act reciprocally to balance TCR-mediated GITR expression in conventional T cells, an outcome that appears to influence cell survival.

  5. Modulation of MHC Binding by Lateral Association of TCR and Coreceptor

    PubMed Central

    Perica, Karlo; Bieler, Joan Glick; Edidin, Michael; Schneck, Jonathan

    2012-01-01

    The structure of a T cell receptor (TCR) and its affinity for cognate antigen are fixed, but T cells regulate binding sensitivity through changes in lateral membrane organization. TCR microclusters formed upon antigen engagement participate in downstream signaling. Microclusters are also found 3–4 days after activation, leading to enhanced antigen binding upon rechallenge. However, others have found an almost complete loss of antigen binding four days after T cell activation, when TCR clusters are present. To resolve these contradictory results, we compared binding of soluble MHC-Ig dimers by transgenic T cells stimulated with a high (100 μM) or low (100 fM) dose of cognate antigen. Cells activated by a high dose of peptide bound sixfold lower amounts of CD8-dependent ligand Kb-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand Ld-QL9 equally well. Consistent with the differences between binding of CD8-dependent and CD8-independent peptide/MHC, Förster resonance energy transfer (FRET) measurements of molecular proximity reported little nanoscale association of TCR with CD8 (16 FRET units) compared to their association on cells stimulated by low antigen dose (62 FRET units). Loss of binding induced by changes in lateral organization of TCR and CD8 may serve as a regulatory mechanism to avoid excessive inflammation and immunopathology in response to aggressive infection. PMID:23199917

  6. Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors.

    PubMed

    McCormack, Emmet; Adams, Katherine J; Hassan, Namir J; Kotian, Akhil; Lissin, Nikolai M; Sami, Malkit; Mujić, Maja; Osdal, Tereza; Gjertsen, Bjørn Tore; Baker, Deborah; Powlesland, Alex S; Aleksic, Milos; Vuidepot, Annelise; Morteau, Olivier; Sutton, Deborah H; June, Carl H; Kalos, Michael; Ashfield, Rebecca; Jakobsen, Bent K

    2013-04-01

    NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.

  7. Src-like Adaptor Protein (Slap) Is a Negative Regulator of T Cell Receptor Signaling

    PubMed Central

    Sosinowski, Tomasz; Pandey, Akhilesh; Dixit, Vishva M.; Weiss, Arthur

    2000-01-01

    Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)–, and interleukin 2–dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3ζ, ZAP-70, SH2 domain–containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling. PMID:10662792

  8. Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling.

    PubMed

    Sosinowski, T; Pandey, A; Dixit, V M; Weiss, A

    2000-02-01

    Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

  9. Anti-CD8 antibodies can trigger CD8+ T cell effector function in the absence of TCR engagement and improve peptide-MHCI tetramer staining.

    PubMed

    Clement, Mathew; Ladell, Kristin; Ekeruche-Makinde, Julia; Miles, John J; Edwards, Emily S J; Dolton, Garry; Williams, Tamsin; Schauenburg, Andrea J A; Cole, David K; Lauder, Sarah N; Gallimore, Awen M; Godkin, Andrew J; Burrows, Scott R; Price, David A; Sewell, Andrew K; Wooldridge, Linda

    2011-07-15

    CD8(+) T cells recognize immunogenic peptides presented at the cell surface bound to MHCI molecules. Ag recognition involves the binding of both TCR and CD8 coreceptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on Ag sensitivity. Anti-CD8 Abs have been used extensively to examine the role of CD8 in CD8(+) T cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 Abs per se are capable of inducing effector function. In this article, we report on the ability of seven monoclonal anti-human CD8 Abs to activate six human CD8(+) T cell clones with a total of five different specificities. Six of seven anti-human CD8 Abs tested did not activate CD8(+) T cells. In contrast, one anti-human CD8 Ab, OKT8, induced effector function in all CD8(+) T cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of Ag-specific CD8(+) T cells. The anti-mouse CD8 Abs, CT-CD8a and CT-CD8b, also activated CD8(+) T cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 Abs to trigger T cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of Ab-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CD8(+) T cell signaling.

  10. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT.

    PubMed

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I Jolanda M; Adema, Gosse J; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280-288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  11. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT

    PubMed Central

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I. Jolanda M.; Adema, Gosse J.; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280–288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  12. MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection

    PubMed Central

    Picarda, Elodie; Bézie, Séverine; Venturi, Vanessa; Echasserieau, Klara; Mérieau, Emmanuel; Delhumeau, Aurélie; Renaudin, Karine; Brouard, Sophie; Bernardeau, Karine; Anegon, Ignacio; Guillonneau, Carole

    2014-01-01

    In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II–derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function. PMID:24789907

  13. Inverted repeats in the promoter as an autoregulatory sequence for TcrX in Mycobacterium tuberculosis

    SciTech Connect

    Bhattacharya, Monolekha; Das, Amit Kumar

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer The regulatory sequences recognized by TcrX have been identified. Black-Right-Pointing-Pointer The regulatory region comprises of inverted repeats segregated by 30 bp region. Black-Right-Pointing-Pointer The mode of binding of TcrX with regulatory sequence is unique. Black-Right-Pointing-Pointer In silico TcrX-DNA docked model binds one of the inverted repeats. Black-Right-Pointing-Pointer Both phosphorylated and unphosphorylated TcrX binds regulatory sequence in vitro. -- Abstract: TcrY, a histidine kinase, and TcrX, a response regulator, constitute a two-component system in Mycobacterium tuberculosis. tcrX, which is expressed during iron scarcity, is instrumental in the survival of iron-dependent M. tuberculosis. However, the regulator of tcrX/Y has not been fully characterized. Crosslinking studies of TcrX reveal that it can form oligomers in vitro. Electrophoretic mobility shift assays (EMSAs) show that TcrX recognizes two regions in the promoter that are comprised of inverted repeats separated by {approx}30 bp. The dimeric in silico model of TcrX predicts binding to one of these inverted repeat regions. Site-directed mutagenesis and radioactive phosphorylation indicate that D54 of TcrX is phosphorylated by H256 of TcrY. However, phosphorylated and unphosphorylated TcrX bind the regulatory sequence with equal efficiency, which was shown with an EMSA using the D54A TcrX mutant.

  14. CD4+CD25− T cells transduced to express MHC class I-restricted epitope specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model

    PubMed Central

    Chhabra, Arvind; Yang, Lili; Wang, Pin; Comin-Anduix, Begoña; Das, Raja; Chakraborty, Nitya G.; Ray, Swagatam; Mehrotra, Shikhar; Yang, Haiguang; Hardee, Cinnamon L.; Hollis, Roger; Dorsky, David I.; Koya, Richard; Kohn, Donald B.; Ribas, Antoni; Economou, James S.; Baltimore, David; Mukherji, Bijay

    2009-01-01

    Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4+ T cells. Considering the difficulties in simultaneously engaging CD4+ and CD8+ T cells in tumor immunotherapy -- especially in an antigen specific manner -- “redirecting” CD4+ T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope specific T cell receptors (TCR) in CD4+ T cells has emerged as a strategic consideration. Such TCR engineered CD4+ T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have carried out a critical examination of functional characteristics of CD4+ T cells engineered to express the α and β chains of a high functional avidity TCR specific for the melanoma epitope, MART-127–35 (M1), as a prototypic human tumor antigen system. We found that unpolarized CD4+CD25− T cells engineered to express the M1 TCR selectively synthesize Th1 cytokines and exhibit a potent antigen-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8+ CTL. Such TCR engineered CD4+ T cells, therefore, might be useful in clinical immunotherapy. PMID:18606658

  15. Visualization of the human CD4{sup +} T-cell response in humanized HLA-DR4-expressing NOD/Shi-scid/γc{sup null} (NOG) mice by retrogenic expression of the human TCR gene

    SciTech Connect

    Takahashi, Takeshi Katano, Ikumi; Ito, Ryoji; Ito, Mamoru

    2015-01-02

    Highlights: • β-Lactoglobulin (BLG) specific TCR genes were introduced to human HSC by retrovirus. • Human HSC with BLG-specific TCR were transplanted into NOG-HLA-DR4 I-A{sup −/−} mice. • BLG-specific TCR induced positive selection of thymocytes. • BLG-specific TCR positive CD4{sup +} T cells mediated immune responses in humanized mice. - Abstract: The development of severe immunodeficient mouse strains containing various human genes, including cytokines or HLA, has enabled the reconstitution of functional human immune systems after transplantation of human hematopoietic stem cells (HSC). Accumulating evidence has suggested that HLA-restricted antigen-specific human T-cell responses can be generated in these humanized mice. To directly monitor immune responses of human CD4{sup +} T cells, we introduced β-lactoglobulin (BLG)-specific T cell receptor (TCR) genes derived from CD4{sup +} T-cell clones of cow-milk allergy patients into HSCs, and subsequently transplanted them into NOG-HLA-DR4 transgenic/I-Aβ deficient mice (NOG-DR4/I-A{sup o}). In the thymus, thymocytes with BLG-specific TCR preferentially differentiated into CD4{sup +}CD8{sup −} single-positive cells. Adoptive transfer of mature CD4{sup +} T cells expressing the TCR into recipient NOG-DR4/I-A{sup o} mice demonstrated that human CD4{sup +} T cells proliferated in response to antigenic stimulation and produced IFN-γ in vivo, suggesting that functional T-cell reactions (especially Th1-skewed responses) were induced in humanized mice.

  16. The cellular environment regulates in situ kinetics of T-cell receptor interaction with peptide major histocompatibility complex.

    PubMed

    Liu, Baoyu; Chen, Wei; Natarajan, Kannan; Li, Zhenhai; Margulies, David H; Zhu, Cheng

    2015-07-01

    T cells recognize antigens at the two-dimensional (2D) interface with antigen-presenting cells (APCs), which trigger T-cell effector functions. T-cell functional outcomes correlate with 2D kinetics of membrane-embedded T-cell receptors (TCRs) binding to surface-tethered peptide-major histocompatibility complex molecules (pMHCs). However, most studies have measured TCR-pMHC kinetics for recombinant TCRs in 3D by surface plasmon resonance, which differs drastically from 2D measurements. Here, we compared pMHC dissociation from native TCR on the T-cell surface to recombinant TCR immobilized on glass surface or in solution. Force on TCR-pMHC bonds regulated their lifetimes differently for native than recombinant TCRs. Perturbing the cellular environment suppressed 2D on-rates but had no effect on 2D off-rate regardless of whether force was applied. In contrast, for the TCR interacting with its monoclonal antibody, the 2D on-rate was insensitive to cellular perturbations and the force-dependent off-rates were indistinguishable for native and recombinant TCRs. These data present novel features of TCR-pMHC kinetics that are regulated by the cellular environment, underscoring the limitations of 3D kinetics in predicting T-cell functions and calling for further elucidation of the underlying molecular and cellular mechanisms that regulate 2D kinetics in physiological settings.

  17. OAS/PKR Pathways and α/β TCR+ T Cells are Required for Ad: IFN-γ Inhibition of HSV-1 in Cornea1

    PubMed Central

    Austin, Bobbie Ann; Halford, William P.; Williams, Bryan R. G.; Carr, Daniel J. J.

    2007-01-01

    An adenoviral vector containing the muIFN-γ transgene (Ad:IFN-γ) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad: IFN-γ potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-γ R. Moreover, Ad:IFN-γ was effective when delivered -72 and -24 h prior to infection as well as 24 h post infection. Associated with anti-viral opposition, TG from Ad: IFN-γ transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-γ was effective but attenuated in TG from α/β TCR deficient mice. In corneas, α/β TCR+ T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid anti-viral efficacy of Ad: IFN-γ was further investigated because type I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-γ inhibited viral reproduction in corneas and TG from IFN-α/β R deficient (CD118 −/−) mice, although viral titers were 2–3 fold higher in cornea and TG, compared to wild type. The absence of IFN-stimulated anti-viral proteins, 2’-5’ oligoadenylate synthetase/RNase L and ds RNA dependent protein kinase R, completely eliminated the anti-viral effectiveness of Ad:IFN-γ. Collectively, the results demonstrate: (1) nonexistence of type I IFN R does not abolish defense of Ad:IFN-γ against HSV-1; (2) anti-viral pathways, OAS/RNase L and PKR are mandatory; and (3) α/β TCR+ T cells are compulsory for Ad: IFN-γ effectiveness against HSV-1 in cornea but not in TG. PMID:17404299

  18. T-cell adoptive immunotherapy using tumor-infiltrating T cells and genetically engineered TCR-T cells.

    PubMed

    Ikeda, Hiroaki

    2016-07-01

    Immunotherapy has received the expectation that it should contribute to the therapy of cancer patients for >100 years. At long last, recent clinical trials of immunotherapy with immune checkpoint inhibitors and adoptive cell therapy with genetically engineered T cells have reported their remarkable efficacies. Nowadays, it is expected that T-cell adoptive immunotherapy can not only control tumor progression but even cure cancer in some patients. Conversely, severe adverse events associated with efficacy have frequently been reported in clinical trials, suggesting that the assessment and control of safety will be indispensable in the future development of the therapy. New approaches in T-cell adoptive immunotherapy such as reduction of adverse events, targeting of new antigens or utilization of allogeneic cells will open a new gate for less harmful and more effective immunological treatment of cancer patients. PMID:27127191

  19. Selective T-cell Ablation with Bismuth-213 Labeled Anti-TCR Alpha Beta as Nonmyeloablative Conditionaing for Allogeneic Canine Marrow Transplantion

    SciTech Connect

    Bethge, W. A.; Wilbur, D. Scott; Storb, R.; Hamlin, Donald K.; Santos, E. B.; Brechbiel, M. W.; Fisher, Darrell R.; Sandmaier, B. M.

    2003-06-15

    Two major immunological barriers, the host versus graft (HVG) and the graft versus host (GVH) reaction, must be overcome for successful allogeneic hematopoietic stem cell transplantation. T-cells are involved in these barriers in the major histocompatibility complex-identical settings. We hypothesized that selective ablation of T-cells using radioimmunotherapy, together with postgrafting immunosuppression, would ensure stable allogeneic engraftment. We developed a canine model of nonmyeloablative marrow transplantation in which host immune reactions are impaired by a single dose of 2 Gy total body irradiation (TBI), and where both GVH and residual HVG reactions are controlled by postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP). We substituted the alpha-emitter bismuth-213 linked to a monoclonal antibody against TCR(alpha,beta)using the metal-binding chelate CHX-A”-DTPA, for 2 Gy TBI. Biodistribution studies using a gamma-emitting indium-111-labeled anti-TCR mAb showed uptake primarily in blood, marrow, lymph nodes, spleen and liver. In a dosimetry study, 4 dogs were treated with 0.13-0.46 mg/kg TCR mAb labeled with 3.7-5.6 mCi/kg (137-207 MBq/kg) Bi-213. The treatment was administered in 6 injections on days -3 and -2 followed by transplantion of dog leukocyte antigen-identical marrow on day 0 and postgrafting immunosuppression with MMF and CSP. Therapy was well tolerated except for elevations of transaminases, which were transient in all but one dog. No other organ toxicities or signs of graft-versus-host-disease were noted. The dogs had prompt allogeneic hematopoietic engraftment and achieved stable mixed donor-host hematopoietic chimerism with donor contributions ranging from 5-55 % with >30 weeks follow up.

  20. TCR-MHC docking orientation: natural selection, or thymic selection?

    PubMed

    Collins, Edward J; Riddle, David S

    2008-01-01

    T cell receptors (TCR) dock on their peptide-major histocompatibility complex (pMHC) targets in a conserved orientation. Since amino acid sidechains are the foundation of specific protein-protein interactions, a simple explanation for the conserved docking orientation is that key amino acids encoded by the TCR and MHC genes have been selected and maintained through evolution in order to preserve TCR/pMHC binding. Expectations that follow from the hypothesis that TCR and MHC evolved to interact are discussed in light of the data that both support and refute them. Finally, an alternative and equally simple explanation for the driving force behind the conserved docking orientation is described.

  1. Analyzing the CDR3 Repertoire with respect to TCR-Beta Chain V-D-J and V-J Rearrangements in Peripheral T Cells using HTS.

    PubMed

    Ma, Long; Yang, Liwen; Bin Shi; He, Xiaoyan; Peng, Aihua; Li, Yuehong; Zhang, Teng; Sun, Suhong; Ma, Rui; Yao, Xinsheng

    2016-07-12

    V-D-J rearrangement of the TCR-beta chain follows the 12/23 rule and the beyond 12/23 restriction. Currently, the proportion and characteristics of TCR-beta chain V-J rearrangement is unclear. We used high-throughput sequencing to compare and analyze TCR-beta chain V-J rearrangement and V-D-J rearrangement in the CDR3 repertoires of T cells from the PBMCs of six volunteers and six BALB/c mice. The results showed that the percentage of V-J rearrangement of the volunteers was approximately 0.7%, whereas that of the mice was 2.2%. The clonality of mice V-J rearrangement was significantly reduced compared with the V-D-J rearrangement, whereas the clonality of human V-J rearrangement was slightly reduced compared with the V-D-J rearrangement. V-J rearrangement in CDR3 involved the significant usage of N, S, F and L, whereas V-D-J rearrangement in CDR3 involved the significant usage of R and G. The levels of V deletion and J deletion in V-J rearrangement were significantly reduced compared with V-D-J rearrangement. TRBD and TRBJ usage in V-J rearrangement differed from that of V-D-J rearrangement, including dominant usage of TRBV and TRBJ and their pairing. Taken together, these results provide new ideas and technology for studies of V-D-J rearrangement and V-J rearrangement in the CDR3 repertoire.

  2. Thymic commitment of regulatory T cells is a pathway of TCR-dependent selection that isolates repertoires undergoing positive or negative selection.

    PubMed

    Coutinho, A; Caramalho, I; Seixas, E; Demengeot, J

    2005-01-01

    autoimmune diseases (AID) result from Treg deficits, some of which might have a thymic origin, we also speculate on therapeutic strategies aiming at selectively stimulating their de novo production or peripheral function, within recent findings on Treg responses to inflammation (Caramalho et al. 2003; Lopes-Carvalho et al., submitted, Caramalho et al., submitted). In short, the MM96 argued that natural tolerance is dominant, established and maintained by the activity of Treg, which are selected upon high-affinity recognition of self-ligands on TECs, and committed intrathymically to a unique differentiative pathway geared to anti-inflammatory and antiproliferative effector functions. By postulating the intrathymic deletion of self-reactivities on hemopoietic stromal cells (THC), together with the inability of peripheral resident lymphocytes to engage in the regulatory pathway, the MM96 simultaneously explained the maintenance of responsiveness to non-self in a context of suppression mediating dominant self-tolerance. The major difficulty of the MM96 is related to the apparent tissue specificity of Treg repertoires generated intrathymically. This difficulty has now been principally solved by the work of Hanahan, Kyewski and others (Jolicoeur et al. 1994; Derbinski et al. 2001; Anderson et al. 2002; Gotter et al. 2004), demonstrating the selective expression of a variety of tissue-specific antigens by TECs, in topological patterns that are compatible with the MM96, but difficult to conciliate with recessive tolerance models (Kappler et al. 1987; Kisielow et al. 1988). While the developmentally regulated multireactivity of TCR repertoires (Gavin and Bevan 1995), as well as the peripheral recruitment of Treg among RTE (Modigliani et al. 1996a) might add to this process, it would seem that the establishment of tissue-specific tolerance essentially stems from the "promiscuous expression of tissue antigens" by TEC. The findings of AID resulting from natural mutations (reviewed in

  3. Thymic commitment of regulatory T cells is a pathway of TCR-dependent selection that isolates repertoires undergoing positive or negative selection.

    PubMed

    Coutinho, A; Caramalho, I; Seixas, E; Demengeot, J

    2005-01-01

    autoimmune diseases (AID) result from Treg deficits, some of which might have a thymic origin, we also speculate on therapeutic strategies aiming at selectively stimulating their de novo production or peripheral function, within recent findings on Treg responses to inflammation (Caramalho et al. 2003; Lopes-Carvalho et al., submitted, Caramalho et al., submitted). In short, the MM96 argued that natural tolerance is dominant, established and maintained by the activity of Treg, which are selected upon high-affinity recognition of self-ligands on TECs, and committed intrathymically to a unique differentiative pathway geared to anti-inflammatory and antiproliferative effector functions. By postulating the intrathymic deletion of self-reactivities on hemopoietic stromal cells (THC), together with the inability of peripheral resident lymphocytes to engage in the regulatory pathway, the MM96 simultaneously explained the maintenance of responsiveness to non-self in a context of suppression mediating dominant self-tolerance. The major difficulty of the MM96 is related to the apparent tissue specificity of Treg repertoires generated intrathymically. This difficulty has now been principally solved by the work of Hanahan, Kyewski and others (Jolicoeur et al. 1994; Derbinski et al. 2001; Anderson et al. 2002; Gotter et al. 2004), demonstrating the selective expression of a variety of tissue-specific antigens by TECs, in topological patterns that are compatible with the MM96, but difficult to conciliate with recessive tolerance models (Kappler et al. 1987; Kisielow et al. 1988). While the developmentally regulated multireactivity of TCR repertoires (Gavin and Bevan 1995), as well as the peripheral recruitment of Treg among RTE (Modigliani et al. 1996a) might add to this process, it would seem that the establishment of tissue-specific tolerance essentially stems from the "promiscuous expression of tissue antigens" by TEC. The findings of AID resulting from natural mutations (reviewed in

  4. Characterization of the epitope on murine T-cell receptor (TCR) alpha proteins recognized by H28-710 monoclonal antibody.

    PubMed

    Karaivanova, V; Suzuki, C; Howe, C; Kearse, K P

    1999-12-01

    Antigen recognition by alphabeta T lymphocytes is mediated via the multisubunit T-cell receptor (TCR) complex consisting of invariant CD3-gamma,delta,epsilon, and zeta chains associated with clonotypic TCRalpha,beta molecules. In the current report, we evaluated the molecular basis for recognition of murine TCRalpha proteins by H28-710 monoclonal antibody (MAb), specific for the constant region of murine TCRalpha chains. H28-710 is widely used in the study of the TCR complex as it is the only reagent currently available that recognizes all murine TCRalpha proteins, regardless of their clonotype. These data show that H28-710 is useful for the immunoprecipitation of TCRalpha proteins not associated with CD3 subunits, and that H28-710 effectively recognizes denatured TCRalpha proteins synthesized in several different cell types. Most importantly, these results demonstrate that H28 binding involves a serine/threonine-rich region between amino acids 150-177 on murine TCRalpha polypeptides.

  5. A Phosphatase Activity of Sts-1 Contributes to the Suppression of TCR Signaling

    SciTech Connect

    Mikhailik,A.; Ford, B.; Keller, J.; Chen, Y.; Nassar, N.; Carpino, N.

    2007-01-01

    Precise signaling by the T cell receptor (TCR) is crucial for a proper immune response. To ensure that T cells respond appropriately to antigenic stimuli, TCR signaling pathways are subject to multiple levels of regulation. Sts-1 negatively regulates signaling pathways downstream of the TCR by an unknown mechanism(s). Here, we demonstrate that Sts-1 is a phosphatase that can target the tyrosine kinase Zap-70 among other proteins. The X-ray structure of the Sts-1 C terminus reveals that it has homology to members of the phosphoglycerate mutase/acid phosphatase (PGM/AcP) family of enzymes, with residues known to be important for PGM/AcP catalytic activity conserved in nature and position in Sts-1. Point mutations that impair Sts-1 phosphatase activity in vitro also impair the ability of Sts-1 to regulate TCR signaling in T cells. These observations reveal a PGM/AcP-like enzyme activity involved in the control of antigen receptor signaling.

  6. Recognition and Regulation of T Cells by NK Cells

    PubMed Central

    Pallmer, Katharina; Oxenius, Annette

    2016-01-01

    Regulation of T cell responses by innate lymphoid cells (ILCs) is increasingly documented and studied. Direct or indirect crosstalk between ILCs and T cells early during and after T cell activation can affect their differentiation, polarization, and survival. Natural killer (NK) cells that belong to the ILC1 group were initially described for their function in recognizing and eliminating “altered self” and as source of early inflammatory cytokines, most notably type II interferon. Using signals conveyed by various germ-line encoded activating and inhibitory receptors, NK cells are geared to sense sudden cellular changes that can be caused by infection events, malignant transformation, or cellular stress responses. T cells, when activated by TCR engagement (signal 1), costimulation (signal 2), and cytokines (signal 3), commit to a number of cellular alterations, including entry into rapid cell cycling, metabolic changes, and acquisition of effector functions. These abrupt changes may alert NK cells, and T cells might thereby expose themselves as NK cell targets. Here, we review how activated T cells can be recognized and regulated by NK cells and what consequences such regulation bears for T cell immunity in the context of vaccination, infection, or autoimmunity. Conversely, we will discuss mechanisms by which activated T cells protect themselves against NK cell attack and outline the significance of this safeguard mechanism. PMID:27446081

  7. Id1 expression promotes peripheral CD4{sup +} T cell proliferation and survival upon TCR activation without co-stimulation

    SciTech Connect

    Liu, Chen; Jin, Rong; Wang, Hong-Cheng; Tang, Hui; Liu, Yuan-Feng; Qian, Xiao-Ping; Sun, Xiu-Yuan; Ge, Qing; Sun, Xiao-Hong; Zhang, Yu

    2013-06-21

    Highlights: •Id1 expression enables naïve T cell proliferation without anti-CD28 co-stimulation. •Id1 expression facilitates T cells survival when stimulated with anti-CD3. •Elevation of IL-2 production by Id1 contributes increased proliferation and survival. •Id1 potentiates NF-κB activation by anti-CD3 stimulation. -- Abstract: Although the role of E proteins in the thymocyte development is well documented, much less is known about their function in peripheral T cells. Here we demonstrated that CD4 promoter-driven transgenic expression of Id1, a naturally occurring dominant-negative inhibitor of E proteins, can substitute for the co-stimulatory signal delivered by CD28 to facilitate the proliferation and survival of naïve CD4{sup +} cells upon anti-CD3 stimulation. We next discovered that IL-2 production and NF-κB activity after anti-CD3 stimulation were significantly elevated in Id1-expressing cells, which may be, at least in part, responsible for the augmentation of their proliferation and survival. Taken together, results from this study suggest an important role of E and Id proteins in peripheral T cell activation. The ability of Id proteins to by-pass co-stimulatory signals to enable T cell activation has significant implications in regulating T cell immunity.

  8. Activating TCR Signaling to Thwart T-ALL.

    PubMed

    Lemonnier, François; Mak, Tak W

    2016-09-01

    Thymic negative selection is a process that aims to eliminate autoreactive T cells by inducing the apoptosis of thymocytes expressing a T-cell receptor (TCR) with high affinity for self-MHC. In this issue, Trinquand and colleagues demonstrate that TCR engagement or anti-CD3 stimulation of TCR-expressing T acute lymphoblastic leukemia cells results in their apoptosis. This cell death is reminiscent of thymic negative selection and has the potential for therapeutic exploitation. Cancer Discov; 6(9); 946-8. ©2016 AACR.See related article by Trinquand et al., p. 972. PMID:27587465

  9. Proinsulin Expression Shapes the TCR Repertoire but Fails to Control the Development of Low-Avidity Insulin-Reactive CD8+ T Cells.

    PubMed

    Pearson, James A; Thayer, Terri C; McLaren, James E; Ladell, Kristin; De Leenheer, Evy; Phillips, Amy; Davies, Joanne; Kakabadse, Dimitri; Miners, Kelly; Morgan, Peter; Wen, Li; Price, David A; Wong, F Susan

    2016-06-01

    NOD mice, a model strain for human type 1 diabetes, express proinsulin (PI) in the thymus. However, insulin-reactive T cells escape negative selection, and subsequent activation of the CD8(+) T-cell clonotype G9C8, which recognizes insulin B15-23 via an αβ T-cell receptor (TCR) incorporating TRAV8-1/TRAJ9 and TRBV19/TRBJ2-3 gene rearrangements, contributes to the development of diabetes. In this study, we used fixed TRAV8-1/TRAJ9 TCRα-chain transgenic mice to assess the impact of PI isoform expression on the insulin-reactive CD8(+) T-cell repertoire. The key findings were: 1) PI2 deficiency increases the frequency of insulin B15-23-reactive TRBV19(+)CD8(+) T cells and causes diabetes; 2) insulin B15-23-reactive TRBV19(+)CD8(+) T cells are more abundant in the pancreatic lymph nodes of mice lacking PI1 and/or PI2; 3) overexpression of PI2 decreases TRBV19 usage in the global CD8(+) T-cell compartment; 4) a biased repertoire of insulin-reactive CD8(+) T cells emerges in the periphery regardless of antigen exposure; and 5) low-avidity insulin-reactive CD8(+) T cells are less affected by antigen exposure in the thymus than in the periphery. These findings inform our understanding of the diabetogenic process and reveal new avenues for therapeutic exploitation in type 1 diabetes.

  10. Immune Tolerance Maintained by Cooperative Interactions between T Cells and Antigen Presenting Cells Shapes a Diverse TCR Repertoire.

    PubMed

    Best, Katharine; Chain, Benny; Watkins, Chris

    2015-01-01

    The T cell population in an individual needs to avoid harmful activation by self peptides while maintaining the ability to respond to an unknown set of foreign peptides. This property is acquired by a combination of thymic and extra-thymic mechanisms. We extend current models for the development of self/non-self discrimination to consider the acquisition of self-tolerance as an emergent system level property of the overall T cell receptor repertoire. We propose that tolerance is established at the level of the antigen presenting cell/T cell cluster, which facilitates and integrates cooperative interactions between T cells of different specificities. The threshold for self-reactivity is therefore imposed at a population level, and not at the level of the individual T cell/antigen encounter. Mathematically, the model can be formulated as a linear programing optimization problem that can be implemented as a multiplicative update algorithm, which shows a rapid convergence to a stable state. The model constrains self-reactivity within a predefined threshold, but maintains repertoire diversity and cross reactivity which are key characteristics of human T cell immunity. We show further that the size of individual clones in the model repertoire becomes heterogeneous, and that new clones can establish themselves even when the repertoire has stabilized. Our study combines the salient features of the "danger" model of self/non-self discrimination with the concepts of quorum sensing, and extends repertoire generation models to encompass the establishment of tolerance. Furthermore, the dynamic and continuous repertoire reshaping, which underlies tolerance in this model, suggests opportunities for therapeutic intervention to achieve long-term tolerance following transplantation. PMID:26300880

  11. Ubiquitylation as a Rheostat for TCR Signaling: From Targeted Approaches Toward Global Profiling

    PubMed Central

    O’Leary, Claire E.; Lewis, Emma L.; Oliver, Paula M.

    2015-01-01

    primary T cells. These methods provide an exciting opportunity for further defining how TCR signals are regulated and for identifying new targets for therapeutic modulation. PMID:26732666

  12. Regulation of T Cell Receptor Signaling by DENND1B in TH2 Cells and Allergic Disease.

    PubMed

    Yang, Chiao-Wen; Hojer, Caroline D; Zhou, Meijuan; Wu, Xiumin; Wuster, Arthur; Lee, Wyne P; Yaspan, Brian L; Chan, Andrew C

    2016-01-14

    The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.

  13. A soluble form of the human T cell differentiation antigen CD27 is released after triggering of the TCR/CD3 complex.

    PubMed

    Hintzen, R Q; de Jong, R; Hack, C E; Chamuleau, M; de Vries, E F; ten Berge, I J; Borst, J; van Lier, R A

    1991-07-01

    The human T cell Ag CD27 belongs to a recently defined family of cell surface receptors, including the nerve growth factor receptor, two distinct tumor necrosis factor receptors, and the B cell specific molecule CD40. On resting T cells, CD27 is a transmembrane homodimer with subunits of 50 to 55 kDa (p55). T cell activation via the TCR/CD3 complex causes a strong enhancement of p55 expression. Concomitantly, an alternative form of the CD27 molecule with a molecular mass of 28 to 32 kDa (p32) appears at the cell surface. With the use of ELISA, we here show that a soluble form of CD27 (sCD27) can be detected in the supernatant of T cells activated with anti-CD3 or combinations of anti-CD2 mAb. Moreover, sCD27 is found in both serum and urine from healthy donors. sCD27, purified from either culture supernatant or urine, has a molecular mass of 28 to 32 kDa and is, according to peptide mapping, structurally homologous to the p55 membrane form of CD27. Quantification of sCD27 levels may be used as a marker for T lymphocyte activation in vivo.

  14. Increased division of alpha beta TCR+ and gamma delta TCR+ intestinal intraepithelial lymphocytes after oral administration of cholera toxin.

    PubMed Central

    Penney, I; Kilshaw, P J; MacDonald, T T

    1996-01-01

    Cholera toxin (CT) or its subunits were given orally to mice and division of intestinal intraepithelial lymphocytes (IEL) in vivo measured by double immunofluorescence using 5-bromo-2'-deoxyuridine (BRdU) and membrane alpha beta T-cell receptors (TCR) or gamma delta TCR staining in frozen sections. Cholera toxin (10 micrograms) produced a two- to eightfold-increase in the uptake of BRdU in alpha beta TCR+ IEL in the duodenum and a two-to fivefold increase in gamma delta TCR IEL in the ileum. Increased uptake of BRdU was also seen after a dose of 100 micrograms of CT but this dose was also associated with the loss of alpha beta TCR+ IEL and gamma delta TCR+ IEL in the duodenum. CT-A and CT-B subunit produced increased BRdU incorporation by alpha beta TCR in the duodenum and by gamma delta TCR IEL in the ileum. Cholera toxin therefore appears to be mitogenic for IEL probably due to an indirect mechanism. Images Figure 1 PMID:8911140

  15. Vγ4 γδ T cell-derived IL-17A negatively regulates NKT cell function in Con A-induced fulminant hepatitis.

    PubMed

    Zhao, Na; Hao, Jianlei; Ni, Yuanyuan; Luo, Wei; Liang, Ruifang; Cao, Guangchao; Zhao, Yapu; Wang, Puyue; Zhao, Liqing; Tian, Zhigang; Flavell, Richard; Hong, Zhangyong; Han, Jihong; Yao, Zhi; Wu, Zhenzhou; Yin, Zhinan

    2011-11-15

    Con A-induced fulminant hepatitis is a well-known animal model for acute liver failure. However, the role of γδ T cells in this model is undefined. In this report, using TCR δ(-/-) mice, we demonstrated a protective role of γδ T cells in Con A-induced hepatitis model. TCR δ(-/-) mice showed significantly decreased levels of IL-17A and IL-17F in the Con A-treated liver tissue, and reconstitution of TCR δ(-/-) mice with wild-type (Wt), but not IL-17A(-/-), γδ T cells significantly reduced hepatitis, strongly suggesting a critical role of IL-17A in mediating the protective effect of γδ T cells. Interestingly, only Vγ4, but not Vγ1, γδ T cells exerted such a protective effect. Furthermore, depletion of NKT cells in TCR δ(-/-) mice completely abolished hepatitis, and NKT cells from Con A-challenged liver tissues of TCR δ(-/-) mice expressed significantly higher amounts of proinflammatory cytokine IFN-γ than those from Wt mice, indicating that γδ T cells protected hepatitis through targeting NKT cells. Finally, abnormal capacity of IFN-γ production by NKT cells of TCR δ(-/-) mice could only be downregulated by transferring Wt, but not IL-17(-/-), Vγ4 γδ T cells, confirming an essential role of Vγ4-derived IL-17A in regulating the function of NKT cells. In summary, our report thus demonstrated a novel function of Vγ4 γδ T cells in mediating a protective effect against Con A-induced fulminant hepatitis through negatively regulating function of NKT cells in an IL-17A-dependent manner, and transferring Vγ4 γδ T cells may provide a novel therapeutic approach for this devastating liver disease.

  16. Cytotoxic T cell recognition of allelic variants of HLA B35 bound to an Epstein-Barr virus epitope: influence of peptide conformation and TCR-peptide interaction.

    PubMed

    Khanna, R; Silins, S L; Weng, Z; Gatchell, D; Burrows, S R; Cooper, L

    1999-05-01

    Fine specificity analysis of HLA B35-restricted Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) clones revealed a unique heterogeneity whereby one group of these clones cross-recognized an EBV epitope (YPLHEQHGM) on virus-infected cells expressing either HLA B*3501 or HLA B*3503, while another group cross-recognized this epitope in association with either HLA B*3502 or HLA B*3503. Peptide binding and titration studies ruled out the possibility that these differences were due to variation in the efficiency of peptide presentation by the HLA B35 alleles. Sequence analysis of the TCR genetic elements showed that these clonotypes either expressed BV12/AV3 or BV14/ADV17S1 heterodimers. Interestingly, CTL analysis with monosubstituted alanine mutants of the YPLHEQHGM epitope indicated that the BV12/AV3+ clones preferentially recognized residues towards the C terminus of the peptide, while the BV14/ADV17S1+ clones interacted with residues towards N terminus of the peptide. Molecular modelling of the MHC-peptide complexes suggests that the differences in two floor positions (114 and 116) of the HLA B35 alleles dictate different conformations of the peptide residues L3 and/or H7 and directly contribute in the discerning allele-specific immune recognition by the CTL clonotypes. These results provide evidence for a critical role for the selective interaction of the TCR with specific residues within the peptide epitope in the fine specificity of CTL recognition of allelic variants of an HLA molecule.

  17. Metabolic regulation of T cell differentiation and function

    PubMed Central

    Park, Benjamin V.; Pan, Fan

    2016-01-01

    Upon encountering pathogens, T cells mount immune responses by proliferating, increasing cellular mass and differentiating. These cellular changes impose significant energetic challenges on T cells. It was believed that TCR and cytokine-mediated signaling are dominant dictators of T cell-mediated immune responses. Recently, it was recognized that T cells utilize metabolic transporters and metabolic sensors that allow them to rapidly respond to nutrient-limiting inflammatory environments. Metabolic sensors allow T cells to find a balance between energy consumption (anabolic metabolism) and production (catabolic metabolism) in order to mount effective immune responses. Also, metabolic regulators interact with cytokine-dependent transcriptional regulators, suggesting a more integrative and advanced model of T cell activation and differentiation. In this review, we will discuss recent discoveries regarding the roles of metabolic regulators in effector and memory T cell development and their interaction with canonical transcription factors. PMID:26277275

  18. Domains of the TCR beta-chain required for early thymocyte development

    PubMed Central

    1996-01-01

    The T cell receptor beta (TCR beta) chain controls the developmental transition from CD4-CD8- to CD4+8+thymocytes. We show that the extracellular constant region and the transmembrane region, but not the variable domain or cytoplasmic tail of the TCR beta chain are required for this differentiation step. TCR beta mutant chains lacking the cytoplasmic tail can be found at the cell surface both in functional TCR/CD3 complexes and in a GPI-anchored monomeric form indicating that the cytoplasmic tail of the TCR beta chain functions as an ER retention signal. The concordance between cell surface expression of the mutant chains as TCR/CD3 complexes and their capacity to mediate thymocyte differentiation supports the CD3 mediated feedback model in which preTCR/CD3 complexes control the developmental transition from CD4-CD8- to CD4+CD8+thymocytes. PMID:8920871

  19. T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2- restricted and melanocyte-lineage-specific CTL clone

    PubMed Central

    1993-01-01

    HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA- A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL

  20. Regulation of T-cell receptor signalling by membrane microdomains

    PubMed Central

    Razzaq, Tahir M; Ozegbe, Patricia; Jury, Elizabeth C; Sembi, Phupinder; Blackwell, Nathan M; Kabouridis, Panagiotis S

    2004-01-01

    There is now considerable evidence suggesting that the plasma membrane of mammalian cells is compartmentalized by functional lipid raft microdomains. These structures are assemblies of specialized lipids and proteins and have been implicated in diverse biological functions. Analysis of their protein content using proteomics and other methods revealed enrichment of signalling proteins, suggesting a role for these domains in intracellular signalling. In T lymphocytes, structure/function experiments and complementary pharmacological studies have shown that raft microdomains control the localization and function of proteins which are components of signalling pathways regulated by the T-cell antigen receptor (TCR). Based on these studies, a model for TCR phosphorylation in lipid rafts is presented. However, despite substantial progress in the field, critical questions remain. For example, it is unclear if membrane rafts represent a homogeneous population and if their structure is modified upon TCR stimulation. In the future, proteomics and the parallel development of complementary analytical methods will undoubtedly contribute in further delineating the role of lipid rafts in signal transduction mechanisms. PMID:15554919

  1. Proinsulin Expression Shapes the TCR Repertoire but Fails to Control the Development of Low-Avidity Insulin-Reactive CD8+ T Cells.

    PubMed

    Pearson, James A; Thayer, Terri C; McLaren, James E; Ladell, Kristin; De Leenheer, Evy; Phillips, Amy; Davies, Joanne; Kakabadse, Dimitri; Miners, Kelly; Morgan, Peter; Wen, Li; Price, David A; Wong, F Susan

    2016-06-01

    NOD mice, a model strain for human type 1 diabetes, express proinsulin (PI) in the thymus. However, insulin-reactive T cells escape negative selection, and subsequent activation of the CD8(+) T-cell clonotype G9C8, which recognizes insulin B15-23 via an αβ T-cell receptor (TCR) incorporating TRAV8-1/TRAJ9 and TRBV19/TRBJ2-3 gene rearrangements, contributes to the development of diabetes. In this study, we used fixed TRAV8-1/TRAJ9 TCRα-chain transgenic mice to assess the impact of PI isoform expression on the insulin-reactive CD8(+) T-cell repertoire. The key findings were: 1) PI2 deficiency increases the frequency of insulin B15-23-reactive TRBV19(+)CD8(+) T cells and causes diabetes; 2) insulin B15-23-reactive TRBV19(+)CD8(+) T cells are more abundant in the pancreatic lymph nodes of mice lacking PI1 and/or PI2; 3) overexpression of PI2 decreases TRBV19 usage in the global CD8(+) T-cell compartment; 4) a biased repertoire of insulin-reactive CD8(+) T cells emerges in the periphery regardless of antigen exposure; and 5) low-avidity insulin-reactive CD8(+) T cells are less affected by antigen exposure in the thymus than in the periphery. These findings inform our understanding of the diabetogenic process and reveal new avenues for therapeutic exploitation in type 1 diabetes. PMID:26953160

  2. TCR Signal Strength Alters T–DC Activation and Interaction Times and Directs the Outcome of Differentiation

    PubMed Central

    van Panhuys, Nicholas

    2016-01-01

    The ability of CD4+ T cells to differentiate into effector subsets underpins their ability to shape the immune response and mediate host protection. During T cell receptor-induced activation of CD4+ T cells, both the quality and quantity of specific activatory peptide/MHC ligands have been shown to control the polarization of naive CD4+ T cells in addition to co-stimulatory and cytokine-based signals. Recently, advances in two-­photon microscopy and tetramer-based cell tracking methods have allowed investigators to greatly extend the study of the role of TCR signaling in effector differentiation under in vivo conditions. In this review, we consider data from recent in vivo studies analyzing the role of TCR signal strength in controlling the outcome of CD4+ T cell differentiation and discuss the role of TCR in controlling the critical nature of CD4+ T cell interactions with dendritic cells during activation. We further propose a model whereby TCR signal strength controls the temporal aspects of T–DC interactions and the implications for this in mediating the downstream signaling events, which influence the transcriptional and epigenetic regulation of effector differentiation. PMID:26834747

  3. Attrition of TCR Vα7.2+ CD161++ MAIT Cells in HIV-Tuberculosis Co-Infection Is Associated with Elevated Levels of PD-1 Expression

    PubMed Central

    Saeidi, Alireza; Tien Tien, Vicky L.; Al-Batran, Rami; Al-Darraji, Haider A.; Tan, Hong Y.; Yong, Yean K.; Ponnampalavanar, Sasheela; Barathan, Muttiah; Rukumani, Devi V.; Ansari, Abdul W.; Velu, Vijayakumar; Kamarulzaman, Adeeba; Larsson, Marie; Shankar, Esaki M.

    2015-01-01

    Mucosal-associated invariant T (MAIT) cells are evolutionarily conserved antimicrobial MR1-restricted CD8+ T cells co-expressing the semi-invariant TCR Vα7.2, and are numerous in the blood and mucosal tissues of humans. MAIT cells appear to undergo exhaustion in chronic viral infections. However, their role in human immunodeficiency virus type 1 (HIV-1) mono-infection and HIV/tuberculosis (TB) co-infection have seldom been elaborately investigated. We conducted a cross-sectional study to investigate the frequencies and phenotypes of CD161++CD8+ T cells among anti-retroviral therapy (ART)/anti-TB therapy (ATT) treatment-naïve HIV/TB co-infected, ART/TB treated HIV/TB co-infected, ART naïve HIV-infected, ART-treated HIV-infected patients, and HIV negative healthy controls (HCs) by flow cytometry. Our data revealed that the frequency of MAIT cells was severely depleted in HIV mono- and HIV/TB co-infections. Further, PD-1 expression on MAIT cells was significantly increased in HIV mono- and HIV-TB co-infected patients. The frequency of MAIT cells did not show any significant increase despite the initiation of ART and/or ATT. Majority of the MAIT cells in HCs showed a significant increase in CCR6 expression as compared to HIV/TB co-infections. No marked difference was seen with expressions of chemokine co-receptor CCR5 and CD103 among the study groups. Decrease of CCR6 expression appears to explain why HIV-infected patients display weakened mucosal immune responses. PMID:25894562

  4. Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene.

    PubMed Central

    Ho, I C; Vorhees, P; Marin, N; Oakley, B K; Tsai, S F; Orkin, S H; Leiden, J M

    1991-01-01

    In addition to its role in the recognition of foreign antigens, the T cell receptor (TCR) alpha gene serves as a model system for studies of developmentally-regulated, lineage-specific gene expression in T cells. TCR alpha gene expression is restricted to cells of the TCR alpha/beta+ lineage, and is controlled by a T cell-specific transcriptional enhancer located 4.5 kb 3' to the C alpha gene segment. The TCR alpha enhancer contains four nuclear protein binding sites called T alpha 1-T alpha 4. In this report we describe the identification and characterization of a novel human cDNA, hGATA-3 that binds to the T alpha 3 element of the human TCR alpha enhancer. hGATA-3 contains a zinc finger domain that is highly related to the DNA-binding domain of the erythroid-specific transcription factor, GATA-1, and binds to a region of T alpha 3 that contains a consensus GATA binding site (AGATAG). Northern blot analyses of hematopoietic cell lines demonstrate that hGATA-3 is expressed exclusively in T cells. Overexpression of hGATA-3 in HeLa cells or human B cells specifically activated transcription from a co-transfected reporter plasmid containing two copies of the T alpha 3 binding site located upstream of the minimal SV40 promoter. Taken together these results demonstrate that hGATA-3 is a novel lineage-specific hematopoietic transcription factor that appears to play an important role in regulating the T cell-specific expression of the TCR alpha gene. Images PMID:1827068

  5. Synovial Regulatory T Cells Occupy a Discrete TCR Niche in Human Arthritis and Require Local Signals To Stabilize FOXP3 Protein Expression

    PubMed Central

    Giannakopoulou, Eirini; Lom, Hannah; Wedderburn, Lucy R.

    2015-01-01

    Although there is great interest in harnessing the immunosuppressive potential of FOXP3+ regulatory T cells (Tregs) for treating autoimmunity, a sizeable knowledge gap exists regarding Treg fate in human disease. In juvenile idiopathic arthritis (JIA) patients, we have previously reported that atypical CD25+FOXP3− Treg-like cells uniquely populate the inflamed site. Intriguingly, their proportions relative to CD25+FOXP3+ Tregs associate with arthritis course, suggesting a role in disease. The ontogeny of these FOXP3− Treg-like cells is, however, unknown. In this study, we interrogated clonal relationships between CD4+ T cell subsets in JIA, using high-throughput TCR repertoire analysis. We reveal that FOXP3+ Tregs possess highly exclusive TCRβ usage from conventional T cells, in blood, and also at the inflamed site, where they are clonally expanded. Intriguingly, the repertoires of FOXP3+ Tregs in synovial fluid are highly overlapping with CD25+FOXP3− Treg-like cells, indicating fluctuations in FOXP3 expression in the inflamed joint. Furthermore, cultured synovial Tregs rapidly downregulated FOXP3 protein (but not mRNA), and this process was prevented by addition of synovial fluid from JIA patients, through an IL-6–independent mechanism. Our findings suggest that most Tregs arise from a separate lineage from conventional T cells, and that this repertoire divergence is largely maintained under chronic inflammatory conditions. We propose that subsequent Treg expansions at the inflamed site creates an environment that leads to competition for limited resources within the synovium, resulting in the destabilization of FOXP3 expression in some Tregs. PMID:26561546

  6. Synovial Regulatory T Cells Occupy a Discrete TCR Niche in Human Arthritis and Require Local Signals To Stabilize FOXP3 Protein Expression.

    PubMed

    Bending, David; Giannakopoulou, Eirini; Lom, Hannah; Wedderburn, Lucy R

    2015-12-15

    Although there is great interest in harnessing the immunosuppressive potential of FOXP3(+) regulatory T cells (Tregs) for treating autoimmunity, a sizeable knowledge gap exists regarding Treg fate in human disease. In juvenile idiopathic arthritis (JIA) patients, we have previously reported that atypical CD25(+)FOXP3(-) Treg-like cells uniquely populate the inflamed site. Intriguingly, their proportions relative to CD25(+)FOXP3(+) Tregs associate with arthritis course, suggesting a role in disease. The ontogeny of these FOXP3(-) Treg-like cells is, however, unknown. In this study, we interrogated clonal relationships between CD4(+) T cell subsets in JIA, using high-throughput TCR repertoire analysis. We reveal that FOXP3(+) Tregs possess highly exclusive TCRβ usage from conventional T cells, in blood, and also at the inflamed site, where they are clonally expanded. Intriguingly, the repertoires of FOXP3(+) Tregs in synovial fluid are highly overlapping with CD25(+)FOXP3(-) Treg-like cells, indicating fluctuations in FOXP3 expression in the inflamed joint. Furthermore, cultured synovial Tregs rapidly downregulated FOXP3 protein (but not mRNA), and this process was prevented by addition of synovial fluid from JIA patients, through an IL-6-independent mechanism. Our findings suggest that most Tregs arise from a separate lineage from conventional T cells, and that this repertoire divergence is largely maintained under chronic inflammatory conditions. We propose that subsequent Treg expansions at the inflamed site creates an environment that leads to competition for limited resources within the synovium, resulting in the destabilization of FOXP3 expression in some Tregs.

  7. Regional variation in the proliferative rate and lifespan of alpha beta TCR+ and gamma delta TCR+ intraepithelial lymphocytes in the murine small intestine.

    PubMed Central

    Penney, L; Kilshaw, P J; MacDonald, T T

    1995-01-01

    Using double staining for T-cell receptor (TCR) and 5-bromo-2'-deoxyuridine (BRdU) we have examined the proliferation rates and lifespan of murine intraepithelial lymphocytes (IEL's) in vivo. After a 24-hr pulse of BRdU the number of labelled alpha beta TCR+ IEL was significantly higher in the ileum than the duodenum. In contrast, incorporation of BRdU into gamma delta TCR+ IEL was significantly higher in the duodenum than the ileum. This regional variation was also seen after a 4-hr pulse of BRdU indicating that the differences probably reflect local rates of proliferation in the epithelium. Over a 6-day labelling period, the accumulation of labelled alpha beta TCR+ and gamma delta TCR+ IEL was linear, which allowed IEL lifespan to be calculated. There was considerable variation between groups of mice but the 50% population renewal time for alpha beta TCR+ IEL was 12-36 days in the duodenum and 9-11 days in the ileum, and for gamma delta TCR+ IEL was 12-21 days in the duodenum and 26-100 days in the ileum. The incorporation of BRdU into V beta 8+ IEL showed the same regional variation as alpha beta TCR+ IEL and the V delta 4 population behaved like the total gamma delta TCR+ IEL population. In contrast V beta 11+, potentially self-reactive IEL, showed a regional pattern of labelling like gamma delta TCR+ IEL. Incorporation of BRdU into both alpha beta TCR+ and gamma delta TCR+ IEL in germ-free mice was very low and did not show marked regional variation. alpha beta TCR+ and gamma delta TCR+ IEL from both proximal and distal bowel were cytotoxic. Therefore alpha beta TCR+ and gamma delta TCR+ IEL show different rates of division in different sections of the gut, perhaps reflecting responses to different antigens. Both alpha beta TCR+ and gamma delta TCR+ IEL reside in the epithelium for weeks during which time the gut epithelial population will have been renewed many times. PMID:7490120

  8. Magnetic-Activated Cell Sorting of TCR-Engineered T Cells, Using tCD34 as a Gene Marker, but Not Peptide–MHC Multimers, Results in Significant Numbers of Functional CD4+ and CD8+ T Cells

    PubMed Central

    Govers, Coen; Berrevoets, Cor; Treffers-Westerlaken, Elike; Broertjes, Marieke

    2012-01-01

    Abstract T cell-sorting technologies with peptide–MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100280–288/HLA-A2-specific T cell receptor-αβ (TCRαβ) on magnetic-activated cell sorting (MACS) with various peptide–MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide–MHC multimers, we observed that Streptamer®, when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide–MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide–MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4+ T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4+ T cells. PMID:22871260

  9. Magnetic-activated cell sorting of TCR-engineered T cells, using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4+ and CD8+ T cells.

    PubMed

    Govers, Coen; Berrevoets, Cor; Treffers-Westerlaken, Elike; Broertjes, Marieke; Debets, Reno

    2012-06-01

    T cell-sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100(280-288)/HLA-A2-specific T cell receptor-αβ (TCRαβ) on magnetic-activated cell sorting (MACS) with various peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that Streptamer(®), when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide-MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide-MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4(+) T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4(+) T cells. PMID:22871260

  10. The Adapter Molecule Sin Regulates T-Cell-Receptor-Mediated Signal Transduction by Modulating Signaling Substrate Availability

    PubMed Central

    Xing, Luzhou; Donlin, Laura T.; Miller, Rebecca H.; Alexandropoulos, Konstantina

    2004-01-01

    Engagement of the T-cell receptor (TCR) results in the activation of a multitude of signaling events that regulate the function of T lymphocytes. These signaling events are in turn modulated by adapter molecules, which control the final functional output through the formation of multiprotein complexes. In this report, we identified the adapter molecule Sin as a new regulator of T-cell activation. We found that the expression of Sin in transgenic T lymphocytes and Jurkat T cells inhibited interleukin-2 expression and T-cell proliferation. This inhibitory effect was specific and was due to defective phospholipase C-γ (PLC-γ) phosphorylation and activation. In contrast to other adapters that become phosphorylated upon TCR stimulation, Sin was constitutively phosphorylated in resting cells by the Src kinase Fyn and bound to signaling intermediates, including PLC-γ. In stimulated cells, Sin was transiently dephosphorylated, which coincided with transient dissociation of Fyn and PLC-γ. Downregulation of Sin expression using Sin-specific short interfering RNA oligonucleotides inhibited transcriptional activation in response to TCR stimulation. Our results suggest that endogenous Sin influences T-lymphocyte signaling by sequestering signaling substrates and regulating their availability and/or activity in resting cells, while Sin is required for targeting these intermediates to the TCR for fast signal transmission during stimulation. PMID:15121874

  11. RhoH Regulates Subcellular Localization of ZAP-70 and Lck in T Cell Receptor Signaling

    PubMed Central

    Chae, Hee-Don; Siefring, Jamie E.; Hildeman, David A.; Gu, Yi; Williams, David A.

    2010-01-01

    RhoH is an hematopoietic-specific, GTPase-deficient Rho GTPase that plays a role in T development. We investigated the mechanisms of RhoH function in TCR signaling. We found that the association between Lck and CD3ζ was impaired in RhoH-deficient T cells, due to defective translocation of both Lck and ZAP-70 to the immunological synapse. RhoH with Lck and ZAP-70 localizes in the detergent-soluble membrane fraction where the complex is associated with CD3ζ phosphorylation. To determine if impaired translocation of ZAP-70 was a major determinant of defective T cell development, Rhoh-/- bone marrow cells were transduced with a chimeric myristoylation-tagged ZAP-70. Myr-ZAP-70 transduced cells partially reversed the in vivo defects of RhoH-associated thymic development and TCR signaling. Together, our results suggest that RhoH regulates TCR signaling via recruitment of ZAP-70 and Lck to CD3ζ in the immunological synapse. Thus, we define a new function for a RhoH GTPase as an adaptor molecule in TCR signaling pathway. PMID:21103055

  12. Fas apoptosis inhibitory molecule regulates T cell receptor-mediated apoptosis of thymocytes by modulating Akt activation and Nur77 expression.

    PubMed

    Huo, Jianxin; Xu, Shengli; Lam, Kong-Peng

    2010-04-16

    Fas apoptosis inhibitory molecule (FAIM) has been demonstrated to confer resistance to Fas-induced apoptosis of lymphocytes and hepatocytes in vitro and in vivo. Here, we show that FAIM is up-regulated in thymocytes upon T cell receptor (TCR) engagement and that faim(-/-) thymocytes are highly susceptible to TCR-mediated apoptosis with increased activation of caspase-8 and -9. Furthermore, injection of anti-CD3 antibodies leads to augmented depletion of CD4(+)CD8(+) T cells in the thymus of faim(-/-) mice compared with wild-type control, suggesting that FAIM plays a role in thymocyte apoptosis. Cross-linking of the TCR on faim(-/-) thymocytes leads to an elevated protein level of the orphan nuclear receptor Nur77, which plays a role in thymocyte apoptosis. Interestingly, in the absence of FAIM, there are reduced ubiquitination and degradation of the Nur77 protein. Faim(-/-) thymocytes also exhibit a defective TCR-induced activation of Akt whose activity we now show is required for Nur77 ubiquitination. Further analyses utilizing FAIM-deficient primary thymocytes and FAIM-overexpressing DO-11.10 T cells indicate that FAIM acts upstream of Akt during TCR signaling and influences the localization of Akt to lipid rafts, hence affecting its activation. Taken together, our study defined a TCR-induced FAIM/Akt/Nur77 signaling axis that is critical for modulating the apoptosis of developing thymocytes.

  13. The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

    PubMed

    Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L; Stein, Paul L; Wang, Chyung-Ru

    2014-12-01

    CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

  14. PTEN permits acute increases in D3-phosphoinositide levels following TCR stimulation but inhibits distal signaling events by reducing the basal activity of Akt.

    PubMed

    Seminario, Maria-Cristina; Precht, Patricia; Bunnell, Stephen C; Warren, Sarah E; Morris, Christa M; Taub, Dennis; Wange, Ronald L

    2004-11-01

    Phosphoinositide 3-kinase (PI3K) is important in TCR signaling. PI3K generates phosphatidylinositol 3, 4, 5-trisphosphate (PI-3,4,5-P3), which regulates membrane localization and/or activity of multiple signaling proteins. PTEN (phosphatase and tensin homologue deleted on chromosome 10) opposes PI3K, reversing this reaction. Maintaining the balance between these two enzymes is important for normal T cell function. Here we use the PTEN-null Jurkat T cell line to address the role of PTEN in modulating proximal and distal TCR-signaling events. PTEN expression at levels that restored low basal Akt phosphorylation (an indicator of PI-3,4,5-P3 levels), but which were not themselves cytotoxic, had minimal effect on TCR-stimulated activation of phospholipase Cgamma1 and Ca2+ flux, but reduced the duration of extracellular signal-regulated kinase (Erk) activation. Distal signaling events, including nuclear factor of activated T cells (NFAT) activation, CD69 expression and IL-2 production, were all inhibited by PTEN expression. Notably, PTEN did not block TCR-stimulated PI-3,4,5-P3 accumulation. The effect of PTEN on distal TCR signaling events was strongly correlated with the loss of the constitutive Akt activation and glycogen synthase kinase-3 (GSK3) inhibition that is typical of Jurkat cells, and could be reversed by expression of activated Akt or pharmacologic inhibition of GSK3. These results suggest that PTEN acts in T cells primarily to control basal PI-3,4,5-P3 levels, rather than opposing PI3K acutely during TCR stimulation.

  15. The role of membrane rafts in Lck transport, regulation and signalling in T-cells.

    PubMed

    Ventimiglia, Leandro N; Alonso, Miguel A

    2013-09-01

    Tyrosine phosphorylation is one of the key covalent modifications that occur in multicellular organisms. Since its discovery more than 30 years ago, tyrosine phosphorylation has come to be understood as a fundamentally important mechanism of signal transduction and regulation in all eukaryotic cells. The tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) plays a crucial role in the T-cell response by transducing early activation signals triggered by TCR (T-cell receptor) engagement. These signals result in the phosphorylation of immunoreceptor tyrosine-based activation motifs present within the cytosolic tails of the TCR-associated CD3 subunits that, once phosphorylated, serve as scaffolds for the assembly of a large supramolecular signalling complex responsible for T-cell activation. The existence of membrane nano- or micro-domains or rafts as specialized platforms for protein transport and cell signalling has been proposed. The present review discusses the signals that target Lck to membrane rafts and the importance of these specialized membranes in the transport of Lck to the plasma membrane, the regulation of Lck activity and the phosphorylation of the TCR.

  16. Charged MVB protein 5 is involved in T-cell receptor signaling.

    PubMed

    Wi, Sae Mi; Min, Yoon; Lee, Ki-Young

    2016-01-29

    Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)-mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5(KD)) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5(KD) Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5(KD) Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling.

  17. Functional evidence for TCR-intrinsic specificity for MHCII.

    PubMed

    Parrish, Heather L; Deshpande, Neha R; Vasic, Jelena; Kuhns, Michael S

    2016-03-15

    How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR-pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4(+) or CD8(+) T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR-MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or "shaved" peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR-MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII. PMID:26831112

  18. CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex.

    PubMed

    Koning, Dan; Costa, Ana I; Hoof, Ilka; Miles, John J; Nanlohy, Nening M; Ladell, Kristin; Matthews, Katherine K; Venturi, Vanessa; Schellens, Ingrid M M; Borghans, Jose A M; Kesmir, Can; Price, David A; van Baarle, Debbie

    2013-02-01

    CD8(+) T cells recognize infected or dysregulated cells via the clonotypically expressed αβ TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8(+) T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide-MHC I complex on the molecular frontline of CD8(+) T cell-mediated immune surveillance.

  19. Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1.

    PubMed

    Jia, Wei; He, Ming-Xiao; McLeod, Ian X; Guo, Jian; Ji, Dong; He, You-Wen

    2015-01-01

    The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8(+) T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.

  20. KIAA1530 protein is recruited by Cockayne syndrome complementation group protein A (CSA) to participate in transcription-coupled repair (TCR).

    PubMed

    Fei, Jia; Chen, Junjie

    2012-10-12

    Transcription-coupled repair (TCR) is the major pathway involved in the removal of UV-induced photolesions from the transcribed strand of active genes. Two Cockayne syndrome (CS) complementation group proteins, CSA and CSB, are important for TCR repair. The molecular mechanisms by which CS proteins regulate TCR remain elusive. Here, we report the characterization of KIAA1530, an evolutionarily conserved protein that participates in this pathway through its interaction with CSA and the TFIIH complex. We found that UV irradiation led to the recruitment of KIAA1530 onto chromatin in a CSA-dependent manner. Cells lacking KIAA1530 were highly sensitive to UV irradiation and displayed deficiency in TCR. In addition, KIAA1530 depletion abrogated stability of the CSB protein following UV irradiation. More excitingly, we found that a unique CSA mutant (W361C), which was previously identified in a patient with UV(s)S syndrome, showed defective KIAA1530 binding and resulted in a failure of recruiting KIAA1530 and stabilizing CSB after UV treatment. Together, our data not only reveal that KIAA1530 is an important player in TCR but also lead to a better understanding of the molecular mechanism underlying UV(s)S syndrome. PMID:22902626

  1. KIAA1530 Protein Is Recruited by Cockayne Syndrome Complementation Group Protein A (CSA) to Participate in Transcription-coupled Repair (TCR)

    PubMed Central

    Fei, Jia; Chen, Junjie

    2012-01-01

    Transcription-coupled repair (TCR) is the major pathway involved in the removal of UV-induced photolesions from the transcribed strand of active genes. Two Cockayne syndrome (CS) complementation group proteins, CSA and CSB, are important for TCR repair. The molecular mechanisms by which CS proteins regulate TCR remain elusive. Here, we report the characterization of KIAA1530, an evolutionarily conserved protein that participates in this pathway through its interaction with CSA and the TFIIH complex. We found that UV irradiation led to the recruitment of KIAA1530 onto chromatin in a CSA-dependent manner. Cells lacking KIAA1530 were highly sensitive to UV irradiation and displayed deficiency in TCR. In addition, KIAA1530 depletion abrogated stability of the CSB protein following UV irradiation. More excitingly, we found that a unique CSA mutant (W361C), which was previously identified in a patient with UVsS syndrome, showed defective KIAA1530 binding and resulted in a failure of recruiting KIAA1530 and stabilizing CSB after UV treatment. Together, our data not only reveal that KIAA1530 is an important player in TCR but also lead to a better understanding of the molecular mechanism underlying UVsS syndrome. PMID:22902626

  2. Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids

    PubMed Central

    2014-01-01

    Background Glucocorticoids affect peripheral immune responses, including modulation of T-cell activation, differentiation, and apoptosis. The quantity and quality of T-cell receptor (TCR)-triggered intracellular signals modulate T-cell function. Thus, glucocorticoids may affect T cells by interfering with the TCR signaling cascade. The purpose of the study was to search for glucocorticoid-modulated kinases downstream of the TCR. Methods Gene modulation in lymphoid cells either treated with glucocorticoids or from glucocorticoid-treated mice was studied using a RNase protection assay, real-time PCR, and western blotting. The sensitivity of genetically modified thymocytes to glucocorticoid-induced apoptosis was studied by performing hypotonic propidium iodide staining and flow cytometry. The Student’s t-test was employed for statistical evaluation. Results We found that transcription of Itk, a non-receptor tyrosine kinase of the Tec family, was up-regulated in a mouse T-cell hybridoma by the synthetic glucocorticoid dexamethasone. In contrast, dexamethasone down-regulated the expression of Txk, a Tec kinase that functions redundantly with Itk, and Lck, the Src kinase immediately downstream of the TCR. We investigated the expression of Itk, Txk, and Lck in thymocytes and mature lymphocytes following in vitro and in vivo dexamethasone treatment at different time points and doses. Kinase expression was differentially modulated and followed distinct kinetics. Itk was up-regulated in all cell types and conditions tested. Txk was strongly up-regulated in mature lymphocytes but only weakly up-regulated or non-modulated in thymocytes in vitro or in vivo, respectively. Conversely, Lck was down-regulated in thymocytes, but not modulated or up-regulated in mature lymphocytes in the different experimental conditions. This complex behaviour correlates with the presence of both positive and negative glucocorticoid responsive elements (GRE and nGRE, respectively) in the Itk, Txk

  3. Role of appendix in the development of inflammatory bowel disease in TCR-alpha mutant mice

    PubMed Central

    1996-01-01

    T cell receptor-alpha mutant mice (TCR-alpha-/-), created by gene targeting of the TCR-alpha gene in embryonic stem cells, spontaneously develop inflammatory bowel disease (IBD) resembling human ulcerative colitis. Since gut-associated lymphoid tissue is likely to play an important role in the development of chronic intestinal inflammation, we examined the changes in the appendix lymphoid follicle (ALF) and Peyer's patches (PP) in these mice. We found the structure of the ALF to be remarkably similar to that of the PP in the small intestine; in both instances, lymphoid follicles covered by surface epithelium (dome- formation) were found. The amount of proliferation in the lymphoid follicles of the appendix estimated by in vivo incorporation of 5-bromo- 2'deoxyuridine was more than two times that of PP in TCR-alpha-/- mice. ELISPOT assay showed an increase of IgA, IgG1, and IgG2a, but not IgM- secreting B cells in ALF of TCR-alpha-/- mice compared to TCR-alpha+/- control mice. Furthermore, TCR-alpha-/- mice revealed an increase of autoantibody-producing B cells against the cytoskeletal protein tropomyosin in ALF as compared to PP. When TCR-alpha-/- mice underwent appendectomy at a young age (3-5 wk), the number of mesenteric lymph nodes cells at 6-7 mo were markedly less than in the sham-operated TCR- alpha-/- mice. Furthermore, appendectomy at 1 mo of age suppressed the development of IBD, with only 3.3% of these mice developing IBD in the 6-7-mo period of observation. In contrast, approximately 80% of controls, including the sham-operated TCR-alpha-/- mice, developed IBD during this period. These results suggest that ALF, rather than PP, is the priming site of cells involved in the disease process and plays an important role in the development of IBD in TCR-alpha-/- mice. PMID:8760824

  4. Continuous antigenic stimulation of DO11.10 TCR transgenic mice in the presence or absence of IL-1β: possible implications for mechanisms of T cell depletion in HIV disease1

    PubMed Central

    Ladell, Kristin; Hazenberg, Mette D.; Fitch, Mark; Emson, Claire; McEvoy-Hein Asgarian, Bridget K.; Mold, Jeff E.; Miller, Corey; Busch, Robert; Price, David A.; Hellerstein, Marc K.; McCune, Joseph M.

    2015-01-01

    Untreated HIV disease is associated with chronic immune activation and CD4+ T cell depletion. A variety of mechanisms have been invoked to account for CD4+ T cell depletion in this context, but the quantitative contributions of these proposed mechanisms over time remains unclear. We turned to the DO11.10 TCR transgenic (tg) mouse model, where OVA is recognized in the context of H-2d, to explore the impact of chronic antigenic stimulation on CD4+ T cell dynamics. To model dichotomous states of persistent antigen exposure in the presence or absence of proinflammatory stimulation, we administered OVA peptide (OVAp) to these mice on a continuous basis with or without the prototypic proinflammatory cytokine, interleukin 1β (IL-1β). In both cases, circulating antigen-specific CD4+ T cells were depleted. However, in the absence of IL-1β, there was limited proliferation and effector/memory conversion of antigen-specific T cells, depletion of peripheral CD4+ T cells in hematolymphoid organs, and systemic induction of regulatory FoxP3+CD4+ T cells, as often observed in late-stage HIV disease. By contrast, when OVAp was administered in the presence of IL-1β, effector/memory phenotype T cells expanded and the typical symptoms of heightened immune activation were observed. Acknowledging the imperfect and incomplete relationship between antigen-stimulated DO11.10 TCR tg mice and HIV-infected humans, our data suggest that CD4+ T cell depletion in the setting of HIV disease may reflect, at least in part, chronic antigen exposure in the absence of proinflammatory signals and/or appropriate antigen-presenting cell functions. PMID:26416271

  5. T-cell receptors in ectothermic vertebrates.

    PubMed

    Charlemagne, J; Fellah, J S; De Guerra, A; Kerfourn, F; Partula, S

    1998-12-01

    The structure and expression of genes encoding molecules homologous to mammalian T-cell receptors (TCR) have been recently studied in ectothermic vertebrate species representative of chondrychthians, teleosts, and amphibians. The overall TCR chain structure is well conserved in phylogeny: TCR beta- and TCR alpha-like chains were detected in all the species analyzed; TCR gamma- and TCR delta-like chains were also present in a chondrychthian species. The diversity potential of the variable (V) and joining (J) segments is rather large and, as in mammals, conserved diversity (D) segments are associated to the TCR beta and TCR delta chains. An important level of junctional diversity occurred at the V-(D)-J junctions, with the potential addition of N- and P-nucleotides. Thus, the conservation of the structure and of the potential of diversity of TCR molecules have been under a permanent selective pressure during vertebrate evolution. The structure of MHC class I and class II molecules was also well conserved in jawed vertebrates. TCR and MHC molecules are strongly functionally linked and play a determinant role in the initiation and the regulation of the specific immune responses; thus, it is not surprising that their structures have been reciprocally frozen during evolution. PMID:9914905

  6. The MHC class II ligand lymphocyte activation gene-3 is co-distributed with CD8 and CD3-TCR molecules after their engagement by mAb or peptide-MHC class I complexes.

    PubMed

    Hannier, S; Triebel, F

    1999-11-01

    Previous studies indicated that signaling through lymphocyte activation gene-3 (LAG-3), a MHC class II ligand, induced by multivalent anti-receptor antibodies led to unresponsiveness to TCR stimulation. Here, lateral distribution of the LAG-3 molecules and its topological relationship (mutual proximity) to the TCR, CD8, CD4, and MHC class I and II molecules were studied in the plasma membrane of activated human T cells in co-capping experiments and conventional fluorescence microscopy. Following TCR engagement by either TCR-specific mAb or MHC-peptide complex recognition in T-B cell conjugates, LAG-3 was found to be specifically associated with the CD3-TCR complex. Similarly, following CD8 engagement LAG-3 and CD8 were co-distributed on the cell surface while only a low percentage of CD4-capped cells displayed LAG-3 co-caps. In addition, LAG-3 was found to be associated with MHC class II (i.e. DR, DP and DQ) and partially with MHC class I molecules. The supramolecular assemblies described here between LAG-3, CD3, CD8 and MHC class II molecules may result from an organization in raft microdomains, a phenomenon known to regulate early events of T cell activation.

  7. Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2.

    PubMed

    O'Hagan, Kyle L; Zhao, Jie; Pryshchep, Olga; Wang, Chyung-Ru; Phee, Hyewon

    2015-12-01

    NKT cells constitute a small population of T cells developed in the thymus that produce large amounts of cytokines and chemokines in response to lipid Ags. Signaling through the Vα14-Jα18 TCR instructs commitment to the NKT cell lineage, but the precise signaling mechanisms that instruct their lineage choice are unclear. In this article, we report that the cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), was essential for NKT cell development. Loss of Pak2 in T cells reduced stage III NKT cells in the thymus and periphery. Among different NKT cell subsets, Pak2 was necessary for the generation and function of NKT1 and NKT2 cells, but not NKT17 cells. Mechanistically, expression of Egr2 and promyelocytic leukemia zinc finger (PLZF), two key transcription factors for acquiring the NKT cell fate, were markedly diminished in the absence of Pak2. Diminished expression of Egr2 and PLZF were not caused by aberrant TCR signaling, as determined using a Nur77-GFP reporter, but were likely due to impaired induction and maintenance of signaling lymphocyte activation molecule 6 expression, a TCR costimulatory receptor required for NKT cell development. These data suggest that Pak2 controls thymic NKT cell development by providing a signal that links Egr2 to induce PLZF, in part by regulating signaling lymphocyte activation molecule 6 expression. PMID:26519537

  8. T Cell Receptor Signaling in the Control of Regulatory T Cell Differentiation and Function

    PubMed Central

    Li, Ming O.; Rudensky, Alexander Y.

    2016-01-01

    Regulatory T cells (TReg cells), a specialized T cell lineage, have a pivotal function in the control of self-tolerance and inflammatory responses. Recent studies have revealed a discrete mode of TCR signaling that regulates Treg cell differentiation, maintenance and function and that impacts on gene expression, metabolism, cell adhesion and migration of these cells. Here, we discuss the emerging understanding of TCR-guided differentiation of Treg cells in the context of their function in health and disease. PMID:27026074

  9. The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse.

    PubMed

    Finetti, Francesca; Patrussi, Laura; Galgano, Donatella; Cassioli, Chiara; Perinetti, Giuseppe; Pazour, Gregory J; Baldari, Cosima T

    2015-07-15

    IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11(+) endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR(+) endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis.

  10. The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

    PubMed Central

    Finetti, Francesca; Patrussi, Laura; Galgano, Donatella; Cassioli, Chiara; Perinetti, Giuseppe; Pazour, Gregory J.; Baldari, Cosima T.

    2015-01-01

    ABSTRACT IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11+ endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR+ endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis. PMID:26034069

  11. CD28 costimulatory signals in T lymphocyte activation: Emerging functions beyond a qualitative and quantitative support to TCR signalling.

    PubMed

    Porciello, Nicla; Tuosto, Loretta

    2016-04-01

    CD28 is one of the most important co-stimulatory receptors necessary for full T lymphocyte activation. By binding its cognate ligands, B7.1/CD80 or B7.2/CD86, expressed on the surface of professional antigen presenting cells (APC), CD28 initiates several signalling cascades, which qualitatively and quantitatively support T cell receptor (TCR) signalling. More recent data evidenced that human CD28 can also act as a TCR-independent signalling unit, by delivering specific signals, which regulate the expression of pro-inflammatory cytokine/chemokines. Despite the enormous progresses made in identifying the mechanisms and molecules involved in CD28 signalling properties, much remains to be elucidated, especially in the light of the functional differences observed between human and mouse CD28. In this review we provide an overview of the current mechanisms and molecules through which CD28 support TCR signalling and highlight recent findings on the specific signalling motifs that regulate the unique pro-inflammatory activity of human CD28.

  12. Two common structural motifs for TCR recognition by staphylococcal enterotoxins

    PubMed Central

    Rödström, Karin E. J.; Regenthal, Paulina; Bahl, Christopher; Ford, Alex; Baker, David; Lindkvist-Petersson, Karin

    2016-01-01

    Superantigens are toxins produced by Staphylococcus aureus, called staphylococcal enterotoxins (abbreviated SEA to SEU). They can cross-link the T cell receptor (TCR) and major histocompatibility complex class II, triggering a massive T cell activation and hence disease. Due to high stability and toxicity, superantigens are potential agents of bioterrorism. Hence, antagonists may not only be useful in the treatment of disease but also serve as countermeasures to biological warfare. Of particular interest are inhibitors against SEA and SEB. SEA is the main cause of food poisoning, while SEB is a common toxin manufactured as a biological weapon. Here, we present the crystal structures of SEA in complex with TCR and SEE in complex with the same TCR, complemented with computational alanine-scanning mutagenesis of SEA, SEB, SEC3, SEE, and SEH. We have identified two common areas that contribute to the general TCR binding for these superantigens. This paves the way for design of single antagonists directed towards multiple toxins. PMID:27180909

  13. Two common structural motifs for TCR recognition by staphylococcal enterotoxins.

    PubMed

    Rödström, Karin E J; Regenthal, Paulina; Bahl, Christopher; Ford, Alex; Baker, David; Lindkvist-Petersson, Karin

    2016-01-01

    Superantigens are toxins produced by Staphylococcus aureus, called staphylococcal enterotoxins (abbreviated SEA to SEU). They can cross-link the T cell receptor (TCR) and major histocompatibility complex class II, triggering a massive T cell activation and hence disease. Due to high stability and toxicity, superantigens are potential agents of bioterrorism. Hence, antagonists may not only be useful in the treatment of disease but also serve as countermeasures to biological warfare. Of particular interest are inhibitors against SEA and SEB. SEA is the main cause of food poisoning, while SEB is a common toxin manufactured as a biological weapon. Here, we present the crystal structures of SEA in complex with TCR and SEE in complex with the same TCR, complemented with computational alanine-scanning mutagenesis of SEA, SEB, SEC3, SEE, and SEH. We have identified two common areas that contribute to the general TCR binding for these superantigens. This paves the way for design of single antagonists directed towards multiple toxins. PMID:27180909

  14. Arthropathy, leucopenia and recurrent infection associated with a TcR gamma delta population.

    PubMed

    Hodges, E; Quin, C; Farrell, A M; Christmas, S; Sewell, H F; Doherty, M; Powell, R J; Smith, J L

    1995-10-01

    This report documents the presence of clonal gamma delta T-cell receptor (TcR) population in the blood of a patient who presented with an arthropathy of undetermined cause, leucopenia and splenomegaly. There was no evidence for lymphoid malignancy clinically or at post-mortem. The phenotype and genotype of the clonal T-cell population were not associated with the predominant TcR delta rearrangement found in peripheral blood gamma delta cells, but were similar to those found in gamma delta TcR cells infiltrating rheumatoid synovium. The data indicate the presence of a monoclonal population of gamma delta cells TcR cells which in the face of continued immunosuppression behaved benignly. The case may represent a cytomorphologically atypical example of the large granular lymphocytes, neutropenia and arthropathy syndrome/lymphoproliferative disease of granular lymphocytes and, although the patient's clinical features were not 'classical', rheumatoid arthritis (RA) may have been the underlying primary disorder.

  15. TCR engagement of CD4+CD8+ thymocytes in vitro induces early aspects of positive selection, but not apoptosis.

    PubMed

    Groves, T; Parsons, M; Miyamoto, N G; Guidos, C J

    1997-01-01

    Immature CD4/CD8 double-positive (DP) thymocytes expressing self MHC-restricted TCR are positively selected in response to TCR signals to survive and differentiate into functionally competent CD4 or CD8 single positive (SP) T cells. In contrast, DP precursors expressing autoreactive TCR are clonally deleted in response to TCR signals. We show here that in vitro TCR engagement of TCR(low) DP thymocytes rapidly triggers a variety of events considered to be hallmarks of positive selection in vivo. These include increased expression of CD5 and Bcl-2, termination of RAG-1 and pre-T(alpha) gene expression, and a switch in lck promoter usage. We also demonstrate that CD4- or CD28-mediated signals synergize with TCR signals to induce these outcomes. Finally, we show that the response of DP thymocytes to TCR engagement is selective in that clonal deletion, CD4/CD8 lineage commitment, and other events associated with maturation, such as changes in expression of Thy-1, HSA, MHC class I, and CD45-RB, were not induced. Thus, only subsets of maturational processes associated with positive selection in vivo were shown to be directly coupled to TCR signaling pathways at the DP stage. These observations support conclusions from in vivo systems suggesting that multiple, temporally separated TCR engagements are required to effect the entire spectrum of developmental changes associated with positive selection, and provide a conceptual and experimental framework for unraveling the complexity of positive selection.

  16. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

    PubMed

    Tomkowicz, Brian; Walsh, Eileen; Cotty, Adam; Verona, Raluca; Sabins, Nina; Kaplan, Fred; Santulli-Marotto, Sandy; Chin, Chen-Ni; Mooney, Jill; Lingham, Russell B; Naso, Michael; McCabe, Timothy

    2015-01-01

    TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

  17. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway

    PubMed Central

    Tomkowicz, Brian; Walsh, Eileen; Cotty, Adam; Verona, Raluca; Sabins, Nina; Kaplan, Fred; Santulli-Marotto, Sandy; Chin, Chen-Ni; Mooney, Jill; Lingham, Russell B.; Naso, Michael; McCabe, Timothy

    2015-01-01

    TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation. PMID:26492563

  18. Tight regulation of diacylglycerol-mediated signaling is critical for proper invariant NKT cell development

    PubMed Central

    Shen, Shudan; Wu, Jinhong; Srivatsan, Sruti; Gorentla, Balachandra; Shin, Jinwook; Xu, Li; Zhong, Xiao-Ping

    2011-01-01

    Type I natural killer T (NKT) cells, or iNKT cells, express a semi-invariant T cell receptor characterized by its unique V α 14-Jα 18 usage (iV α 14TCR). Upon interaction with glycolipid/CD1d complexes, the iV α 14TCRs transduce signals that are essential for iNKT selection and maturation. However, it remains unclear how these signals are regulated and how important such regulations are during iNKT development. Diacylglycerol (DAG) is an essential second messenger downstream of the TCR that activates the PKCθ-IKKα/β-NFκB pathway, known to be crucial for iNKT development, as well as the RasGRP1-Ras-Erk1/2 pathway in T cells. DAG kinases (DGKs) play an important role in controlling intracellular DAG concentration and thereby negatively regulate DAG signaling. Here we report that simultaneous absence of DAG kinase α and ζ causes severe defects in iNKT development, coincident with enhanced IKK-NFκB and Ras-Erk1/2 activation. Moreover, constitutive IKKβ and Ras activities also result in iNKT developmental defects. Thus, DAG-mediated signaling is not only essential but also needs to be tightly regulated for proper iNKT cell development. PMID:21775687

  19. Sialylation regulates peripheral tolerance in CD4+ T cells.

    PubMed

    Brennan, Patrick J; Saouaf, Sandra J; Van Dyken, Steve; Marth, Jamey D; Li, Bin; Bhandoola, Avinash; Greene, Mark I

    2006-05-01

    Decreased binding by the 6C10 auto-antibody serves as a unique marker for CD4+ T cell unresponsiveness after the induction of T cell tolerance in Vbeta8.1 TCR transgenic mice. We further define the nature of the epitope recognized by the 6C10 antibody to be a subset of Thy-1 bearing incompletely sialylated N-linked glycans, and furthermore, we demonstrate that tolerant CD4+ T cells have an increased degree of cell-surface sialylation. To test the significance of the altered glycosylation state identified by the 6C10 auto-antibody in the tolerant CD4+ T cell population, surface sialic acid was cleaved enzymatically. Treatment of purified peripheral CD4+ T cells with Vibrio cholerae sialidase (VCS) leads to increased 6C10 binding, significantly enhances proliferation in the tolerant CD4+ population and corrects defects in phosphotyrosine signaling observed in the tolerant CD4+ T cell. Furthermore, in vivo administration of VCS enhances proliferation in both tolerant and naive CD4+ T cell subsets. These studies suggest that sialylation of glycoproteins on the surface of the CD4+ T cell contributes to the regulation of T cell responsiveness in the tolerant state. PMID:16291658

  20. Activated PLC-γ1 is catalytically induced at LAT but activated PLC-γ1 is localized at both LAT- and TCR-containing complexes.

    PubMed

    Cruz-Orcutt, Noemi; Vacaflores, Aldo; Connolly, Sean F; Bunnell, Stephen C; Houtman, Jon C D

    2014-04-01

    Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex.

  1. Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death.

    PubMed

    Agea, E; Bistoni, O; Bini, P; Migliorati, G; Nicoletti, I; Bassotti, G; Riccardi, C; Bertotto, A; Spinozzi, F

    1995-08-01

    An optimal stimulation of CD4+ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells (APC). The intercellular adhesion molecule-1 (ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin (IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones (TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as Th0 (IL-4 plus interferon-gamma) or Th2 (IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4+ and CD8+ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones (with Th0- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7503404

  2. γδ T cell subsets play opposing roles in regulating experimental autoimmune encephalomyelitis.

    PubMed

    Blink, Sarah E; Caldis, Matthew W; Goings, Gwendolyn E; Harp, Christopher T; Malissen, Bernard; Prinz, Immo; Xu, Dan; Miller, Stephen D

    2014-07-01

    γδ T cells are resident in cerebrospinal fluid and central nervous system (CNS) lesions of multiple sclerosis (MS) patients, but as multifaceted cells exhibiting innate and adaptive characteristics, their function remains unknown. Previous studies in experimental autoimmune encephalomyelitis (EAE) are contradictory and identified these cells as either promoting or suppressing disease pathogenesis. This study examines distinct γδ T cell subsets during EAE and indicates they mediate differential functions in CNS inflammation and demyelination resulting in pathogenesis or protection. We identified two γδ subsets in the CNS, Vγ1(+) and Vγ4(+), with distinct cytokine profiles and tissue specificity. Anti-γδ T cell receptor (TCR) monoclonal antibody (mAb) administration results in activation and downregulation of surface TCR, rendering the cells undetectable, but with opposing effects: anti-Vγ4 treatment exacerbates disease whereas anti-Vγ1 treatment is protective. The Vγ4(+) subset produces multiple pro-inflammatory cytokines including high levels of IL-17, and accounts for 15-20% of the interleukin-17 (IL-17) producing cells in the CNS, but utilize a variant transcriptional program than CD4(+) Th17 cells. In contrast, the Vγ1 subset produces CCR5 ligands, which may promote regulatory T cell differentiation. γδ T cell subsets thus play distinct and opposing roles during EAE, providing an explanation for previous reports and suggesting selective targeting to optimize regulation as a potential therapy for MS.

  3. TCR usage, gene expression and function of two distinct FOXP3(+)Treg subsets within CD4(+)CD25(hi) T cells identified by expression of CD39 and CD45RO.

    PubMed

    Ye, Lingying; Goodall, Jane C; Zhang, Libin; Putintseva, Ekaterina V; Lam, Brian; Jiang, Lei; Liu, Wei; Yin, Jian; Lin, Li; Li, Ting; Wu, Xin; Yeo, Giles; Shugay, Mikhail; Chudakov, Dmitriy M; Gaston, Hill; Xu, Huji

    2016-03-01

    FOXP3+ regulatory T (Treg) cells are indispensable for immune homeostasis, but their study in humans is complicated by heterogeneity within Treg, the difficulty in purifying Tregs using surface marker expression (e.g. CD25) and the transient expression of FOXP3 by activated effector cells. Here, we report that expression of CD39 and CD45RO distinguishes three sub-populations within human CD4(+)CD25(hi) T cells. Initial phenotypic and functional analysis demonstrated that CD4(+)CD25(hi)CD39(+)CD45RO(+) cells had properties consistent with effector Treg, CD4(+)CD25(hi)CD39(-)CD45RO(-) cells were naïve Treg and CD4(+)CD25(hi)CD39(-)CD45RO(+) cells were predominantly non-Treg with effector T-cell function. Differences in these two newly identified Treg subsets were corroborated by studies of gene expression and TCR analysis. To apply this approach, we studied these two newly identified Treg subsets in ankylosing spondylitis, and showed impairment in both effector and naïve Treg. This work highlights the importance of discriminating Treg subsets to enable proper comparisons of immune regulatory capacity in healthy individuals and those with inflammatory disease.

  4. PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion.

    PubMed

    Tinoco, Roberto; Carrette, Florent; Barraza, Monique L; Otero, Dennis C; Magaña, Jonathan; Bosenberg, Marcus W; Swain, Susan L; Bradley, Linda M

    2016-05-17

    Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulatecell responses in the tumor microenvironment. PMID:27192578

  5. CDR3β sequence motifs regulate autoreactivity of human invariant NKT cell receptors.

    PubMed

    Chamoto, Kenji; Guo, Tingxi; Imataki, Osamu; Tanaka, Makito; Nakatsugawa, Munehide; Ochi, Toshiki; Yamashita, Yuki; Saito, Akiko M; Saito, Toshiki I; Butler, Marcus O; Hirano, Naoto

    2016-04-01

    Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid ligands presented by monomorphic CD1d. Human iNKT T cell receptor (TCR) is largely composed of invariant Vα24 (Vα24i) TCRα chain and semi-variant Vβ11 TCRβ chain, where complementarity-determining region (CDR)3β is the sole variable region. One of the characteristic features of iNKT cells is that they retain autoreactivity even after the thymic selection. However, the molecular features of human iNKT TCR CDR3β sequences that regulate autoreactivity remain unknown. Since the numbers of iNKT cells with detectable autoreactivity in peripheral blood is limited, we introduced the Vα24i gene into peripheral T cells and generated a de novo human iNKT TCR repertoire. By stimulating the transfected T cells with artificial antigen presenting cells (aAPCs) presenting self-ligands, we enriched strongly autoreactive iNKT TCRs and isolated a large panel of human iNKT TCRs with a broad range autoreactivity. From this panel of unique iNKT TCRs, we deciphered three CDR3β sequence motifs frequently encoded by strongly-autoreactive iNKT TCRs: a VD region with 2 or more acidic amino acids, usage of the Jβ2-5 allele, and a CDR3β region of 13 amino acids in length. iNKT TCRs encoding 2 or 3 sequence motifs also exhibit higher autoreactivity than those encoding 0 or 1 motifs. These data facilitate our understanding of the molecular basis for human iNKT cell autoreactivity involved in immune responses associated with human disease. PMID:26748722

  6. Structural interplay between germline and adaptive recognition determines TCR-peptide-MHC cross-reactivity

    PubMed Central

    Adams, Jarrett J.; Narayanan, Samanthi; Birnbaum, Michael E.; Sidhu, Sachdev S.; Blevins, Sydney J.; Gee, Marvin H.; Sibener, Leah V.; Baker, Brian M.; Kranz, David M.; Garcia, K. Christopher

    2015-01-01

    The T cell receptor - peptide-MHC interface is comprised of conserved and diverse regions, yet the relative contributions of each in shaping T cell recognition remain unclear. We isolated cross-reactive peptides with limited homology, allowing us to compare the structural properties of nine peptides for a single TCR-MHC pair. The TCR’s cross-reactivity is rooted in highly similar recognition of an apical ‘hotspot’ position in the peptide, while tolerating significant sequence variation at ancillary positions. Furthermore, we find a striking structural convergence onto a germline-mediated interaction between TCR CDR1α and the MHC α2 helix of twelve TCR-pMHC complexes. Our studies suggest that TCR-MHC germline-mediated constraints, together with a focus on a small peptide hotspot, may place limits on peptide antigen cross-reactivity. PMID:26523866

  7. Structural Features of the αβTCR Mechanotransduction Apparatus That Promote pMHC Discrimination

    PubMed Central

    Brazin, Kristine N.; Mallis, Robert J.; Das, Dibyendu Kumar; Feng, Yinnian; Hwang, Wonmuk; Wang, Jia-huai; Wagner, Gerhard; Lang, Matthew J.; Reinherz, Ellis L.

    2015-01-01

    The αβTCR was recently revealed to function as a mechanoreceptor. That is, it leverages mechanical energy generated during immune surveillance and at the immunological synapse to drive biochemical signaling following ligation by a specific foreign peptide–MHC complex (pMHC). Here, we review the structural features that optimize this transmembrane (TM) receptor for mechanotransduction. Specialized adaptations include (1) the CβFG loop region positioned between Vβ and Cβ domains that allosterically gates both dynamic T cell receptor (TCR)–pMHC bond formation and lifetime; (2) the rigid super β-sheet amalgams of heterodimeric CD3εγ and CD3εδ ectodomain components of the αβTCR complex; (3) the αβTCR subunit connecting peptides linking the extracellular and TM segments, particularly the oxidized CxxC motif in each CD3 heterodimeric subunit that facilitates force transfer through the TM segments and surrounding lipid, impacting cytoplasmic tail conformation; and (4) quaternary changes in the αβTCR complex that accompany pMHC ligation under load. How bioforces foster specific αβTCR-based pMHC discrimination and why dynamic bond formation is a primary basis for kinetic proofreading are discussed. We suggest that the details of the molecular rearrangements of individual αβTCR subunit components can be analyzed utilizing a combination of structural biology, single-molecule FRET, optical tweezers, and nanobiology, guided by insightful atomistic molecular dynamic studies. Finally, we review very recent data showing that the pre-TCR complex employs a similar mechanobiology to that of the αβTCR to interact with self-pMHC ligands, impacting early thymic repertoire selection prior to the CD4+CD8+ double positive thymocyte stage of development. PMID:26388869

  8. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

    PubMed

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-08-31

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

  9. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire

    PubMed Central

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D.; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-01-01

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability. PMID:20709959

  10. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

    PubMed

    Roy, Edwige; Togbe, Dieudonnée; Holdorf, Amy D; Trubetskoy, Dmitry; Nabti, Sabrina; Küblbeck, Günter; Klevenz, Alexandra; Kopp-Schneider, Annette; Leithäuser, Frank; Möller, Peter; Bladt, Friedhelm; Hämmerling, Günter; Arnold, Bernd; Pawson, Tony; Tafuri, Anna

    2010-08-31

    The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability. PMID:20709959

  11. Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing.

    PubMed

    Zvyagin, Ivan V; Pogorelyy, Mikhail V; Ivanova, Marina E; Komech, Ekaterina A; Shugay, Mikhail; Bolotin, Dmitry A; Shelenkov, Andrey A; Kurnosov, Alexey A; Staroverov, Dmitriy B; Chudakov, Dmitriy M; Lebedev, Yuri B; Mamedov, Ilgar Z

    2014-04-22

    Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation. PMID:24711416

  12. How many TCR clonotypes does a body maintain?

    PubMed Central

    Lythe, Grant; Callard, Robin E.; Hoare, Rollo L.; Molina-París, Carmen

    2016-01-01

    We consider the lifetime of a T cell clonotype, the set of T cells with the same T cell receptor, from its thymic origin to its extinction in a multiclonal repertoire. Using published estimates of total cell numbers and thymic production rates, we calculate the mean number of cells per TCR clonotype, and the total number of clonotypes, in mice and humans. When there is little peripheral division, as in a mouse, the number of cells per clonotype is small and governed by the number of cells with identical TCR that exit the thymus. In humans, peripheral division is important and a clonotype may survive for decades, during which it expands to comprise many cells. We therefore devise and analyse a computational model of homeostasis of a multiclonal population. Each T cell in the model competes for self pMHC stimuli, cells of any one clonotype only recognising a small fraction of the many subsets of stimuli. A constant mean total number of cells is maintained by a balance between cell division and death, and a stable number of clonotypes by a balance between thymic production of new clonotypes and extinction of existing ones. The number of distinct clonotypes in a human body may be smaller than the total number of naive T cells by only one order of magnitude. PMID:26546971

  13. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

    PubMed Central

    Ibiza, Sales; Pérez-Rodríguez, Andrea; Ortega, Ángel; Martínez-Ruiz, Antonio; Barreiro, Olga; García-Domínguez, Carlota A.; Víctor, Víctor M.; Esplugues, Juan V.; Rojas, José M.; Sánchez-Madrid, Francisco; Serrador, Juan M.

    2008-01-01

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys118, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys118 contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. PMID:18641128

  14. Identification of peptide-specific TCR genes by in vitro peptide stimulation and CDR3 length polymorphism analysis.

    PubMed

    Shao, Hongwei; Lin, Yanmei; Wang, Teng; Ou, Yusheng; Shen, Han; Tao, Changli; Wu, Fenglin; Zhang, Wenfeng; Bo, Huaben; Wang, Hui; Huang, Shulin

    2015-07-10

    Identification of TCR genes specific for tumor-associated antigens (TAAs) is necessary for TCR gene modification of T cells, which is applied in anti-tumor adoptive T cell therapy (ACT). The usual identification methods are based on isolating single peptide-responding T cells and cloning the TCR gene by in vitro expansion or by single-cell RT-PCR. However, the long and exacting in vitro culture period and demanding operational requirements restrict the application of these methods. Immunoscope is an effective tool that profiles a repertoire of TCRs and identifies significantly expanded clones through CDR3 length analysis. In this study, a survivin-derived mutant peptide optimized for HLA-A2 binding was selected to load DCs and activate T cells. The monoclonal expansion of TCRA and TCRB genes was separately identified by Immunoscope analysis and following sequence identification, the properly paired TCR genes were transferred into T cells. Peptide recognition and cytotoxicity assays indicated that TCR-modified PBMCs could respond to both the mutant and wild type peptides and lyse target cells. These results show that combining Immunoscope with in vitro peptide stimulation provides an alternative and superior method for identifying specific TCR genes, which represents a significant advance for the application of TCR gene-modified T cells. PMID:25890221

  15. Cross-Linking the TCR Complex Induces Apoptosis in CD4+8+ Thymocytes in the Presence of Cyclosporin A

    PubMed Central

    Poetschke, Heather L.; Klug, David B.; Walker, Dawn

    1996-01-01

    Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD+8+ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes. PMID:8828007

  16. The signaling symphony: T cell receptor tunes cytokine-mediated T cell differentiation.

    PubMed

    Huang, Weishan; August, Avery

    2015-03-01

    T cell development, differentiation, and maintenance are orchestrated by 2 key signaling axes: the antigen-specific TCR and cytokine-mediated signals. The TCR signals the recognition of self- and foreign antigens to control T cell homeostasis for immune tolerance and immunity, which is regulated by a variety of cytokines to determine T cell subset homeostasis and differentiation. TCR signaling can synergize with or antagonize cytokine-mediated signaling to fine tune T cell fate; however, the latter is less investigated. Murine models with attenuated TCR signaling strength have revealed that TCR signaling can function as regulatory feedback machinery for T cell homeostasis and differentiation in differential cytokine milieus, such as IL-2-mediated Treg development; IL-7-mediated, naïve CD8(+) T cell homeostasis; and IL-4-induced innate memory CD8(+) T cell development. In this review, we discuss the symphonic cross-talk between TCR and cytokine-mediated responses that differentially control T cell behavior, with a focus on the negative tuning by TCR activation on the cytokine effects.

  17. Development of CD8 alpha/beta + TCR alpha beta intestinal intraepithelial lymphocytes in athymic nu/nu mice and participation in regional immune responses.

    PubMed Central

    Emoto, M; Emoto, Y; Kaufmann, S H

    1996-01-01

    On the basis of the CD8 coreceptor expression, T-cell receptor (TCR)alpha beta-bearing intestinal intraepithelial lymphocytes (i-IEL) segregate into two populations. The CD8 alpha alpha + TCR alpha beta i-IEL develop thymus independently, whereas the CD8 alpha beta + TCR alpha beta i-IEL are generally considered to be thymus dependent. Flow cytometry analysis revealed a distinct population of CD8 alpha beta + TCR alpha beta i-IEL in individual athymic nu/nu mice. The i-IEL encompassing CD8 alpha beta + TCR alpha beta cells expressed potent cytolytic and interferon-gamma-producing activities. These findings demonstrate that CD8 alpha beta + TCR alpha beta i-IEL can develop in nu/nu mice independently from a functional thymus and suggest that these cells, directly or indirectly, perform biological functions in the gut. PMID:8881753

  18. Negative regulators of cell proliferation

    NASA Technical Reports Server (NTRS)

    Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Cell proliferation is governed by the influence of both mitogens and inhibitors. Although cell contact has long been thought to play a fundamental role in cell cycling regulation, and negative regulators have long been suspected to exist, their isolation and purification has been complicated by a variety of technical difficulties. Nevertheless, over recent years an ever-expanding list of putative negative regulators have emerged. In many cases, their biological inhibitory activities are consistent with density-dependent growth inhibition. Most likely their interactions with mitogenic agents, at an intracellular level, are responsible for either mitotic arrest or continued cell cycling. A review of naturally occurring cell growth inhibitors is presented with an emphasis on those factors shown to be residents of the cell surface membrane. Particular attention is focused on a cell surface sialoglycopeptide, isolated from intact bovine cerebral cortex cells, which has been shown to inhibit the proliferation of an unusually wide range of target cells. The glycopeptide arrest cells obtained from diverse species, both fibroblasts and epithelial cells, and a broad variety of transformed cells. Signal transduction events and a limited spectrum of cells that are refractory to the sialoglycopeptide have provided insight into the molecular events mediated by this cell surface inhibitor.

  19. Themis sets the signal threshold for positive and negative selection in T-cell development.

    PubMed

    Fu, Guo; Casas, Javier; Rigaud, Stephanie; Rybakin, Vasily; Lambolez, Florence; Brzostek, Joanna; Hoerter, John A H; Paster, Wolfgang; Acuto, Oreste; Cheroutre, Hilde; Sauer, Karsten; Gascoigne, Nicholas R J

    2013-12-19

    Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αβ-expressing 'single-positive' thymocytes from CD4(+)CD8αβ(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.

  20. Nuclear transfer nTreg model reveals fate-determining TCR-β and novel peripheral nTreg precursors

    PubMed Central

    Ku, Manching; Chang, Shih-En; Hernandez, Julio; Abadejos, Justin R.; Sabouri-Ghomi, Mohsen; Muenchmeier, Niklas J.; Schwarz, Anna; Valencia, Anna M.; Kirak, Oktay

    2016-01-01

    To study the development and function of “natural-arising” T regulatory (nTreg) cells, we developed a novel nTreg model on pure nonobese diabetic background using epigenetic reprogramming via somatic cell nuclear transfer. On RAG1-deficient background, we found that monoclonal FoxP3+ CD4+ Treg cells developed in the thymus in the absence of other T cells. Adoptive transfer experiments revealed that the thymic niche is not a limiting factor in nTreg development. In addition, we showed that the T-cell receptor (TCR) β-chain of our nTreg model was not only sufficient to bias T-cell development toward the CD4 lineage, but we also demonstrated that this TCR β-chain was able to provide stronger TCR signals. This TCR-β–driven mechanism would thus unify former per se contradicting hypotheses of TCR-dependent and -independent nTreg development. Strikingly, peripheral FoxP3− CD4+ T cells expressing the same TCR as this somatic cell nuclear transfer nTreg model had a reduced capability to differentiate into Th1 cells but were poised to differentiate better into induced nTreg cells, both in vitro and in vivo, representing a novel peripheral precursor subset of nTreg cells to which we refer to as pre-nTreg cells. PMID:27044095

  1. CD5-mediated inhibition of TCR signaling proceeds normally in the absence of SHP-1

    PubMed Central

    DONG, BAOXIA; SOMANI, ALLY-KHAN; LOVE, PAUL E.; ZHENG, XUAN; CHEN, XIEQUN; ZHANG, JINYI

    2016-01-01

    The CD5 transmembrane glycoprotein functions as a co-receptor in the signaling pathway linking T-cell antigen receptor (TCR) engagement to activation and differentiation. Although CD5 effects on TCR signaling have been shown to be primarily inhibitory, the underlying mechanisms remain unclear. In view of recent data revealing the ability of CD5 to associate with the SHP-1 tyrosine phosphatase, a protein that also downregulates TCR signaling, we examined the role of SHP-1 in modulating CD5 function using thymocytes from SHP-1-deficient viable motheaten (mev) mice. The results revealed the association of SHP-1 with CD5 to be markedly increased following TCR stimulation and indicated that this interaction was enhanced by and was dependent on CD5 tyrosine phosphorylation. However, there was no difference of the tyrosine phosphorylation status of CD5 between resting and TCR-stimulated cells in SHP-1-deficient compared to wild-type thymocytes. Lack of SHP-1 activity did not affect the levels of CD5 surface expression, CD5 co-immunoprecipitable tyrosine phosphatase activity and intracellular calcium increase following co-crosslinking of the TCR and CD5. Similarly, an analysis of T-cell thymocyte populations in mev mice expressing an H-Y transgene as well as a construct mediating T-cell restricted CD5 overexpression, revealed that the reduction in the positive selection conferred by CD5 overexpression was unaffected by SHP-1 deficiency. CD5 is not a SHP-1 substrate and SHP-1 is not required for and possibly not involved in the CD5-mediated modulation of TCR signaling. PMID:27221212

  2. Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease.

    PubMed

    Hwang, Sujin; Song, Ki-Duk; Lesourne, Renaud; Lee, Jan; Pinkhasov, Julia; Li, Liqi; El-Khoury, Dalal; Love, Paul E

    2012-09-24

    Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability. PMID:22945921

  3. αβ T cell receptor germline CDR regions moderate contact with MHC ligands and regulate peptide cross-reactivity

    PubMed Central

    Attaf, Meriem; Holland, Stephan J.; Bartok, Istvan; Dyson, Julian

    2016-01-01

    αβ T cells respond to peptide epitopes presented by major histocompatibility complex (MHC) molecules. The role of T cell receptor (TCR) germline complementarity determining regions (CDR1 and 2) in MHC restriction is not well understood. Here, we examine T cell development, MHC restriction and antigen recognition where germline CDR loop structure has been modified by multiple glycine/alanine substitutions. Surprisingly, loss of germline structure increases TCR engagement with MHC ligands leading to excessive loss of immature thymocytes. MHC restriction is, however, strictly maintained. The peripheral T cell repertoire is affected similarly, exhibiting elevated cross-reactivity to foreign peptides. Our findings are consistent with germline TCR structure optimising T cell cross-reactivity and immunity by moderating engagement with MHC ligands. This strategy may operate alongside co-receptor imposed MHC restriction, freeing germline TCR structure to adopt this novel role in the TCR-MHC interface. PMID:27775030

  4. Lead poisoning influences TCR-related gene expression patterns in peripheral blood T-lymphocytes of exposed workers.

    PubMed

    Niu, Yuzhe; Yu, Wei; Fang, Su; Liu, Sichu; Yang, Zhiqian; Liu, Weiwei; Chen, Shaohua; Yang, Lijian; Li, Bo; Li, Yangqiu

    2015-01-01

    Previous studies have shown that occupational lead (Pb) exposure might influence human T-lymphocyte function, including such as changes in T-cell receptor (TCR) Vβ and Vγ repertoire and in expression of the TCRζ gene. Thus, the study here further investigated expression of TCRζ-related factors and the FcεRIγ gene (whose product has a functional role complementary to the TCRζ chain) and the Elf-1 gene whose product is involved in regulation of TCR expression. Quantitative real-time RT-PCR was used to measure expression of TCRζ, FcεRIγ, and Elf-1 genes in peripheral blood mononuclear cells (PBMC) isolated from 17 Pb-exposed workers. Samples were collected before and after the workers had undergone chelation therapy regimens. Twenty-three healthy individuals served as controls. The results showed that TCRζ, FcεRIγ, and Elf-1 gene expression in Pb-exposed workers before chelation therapy was significantly lower than in PBMC from healthy individuals. After chelation therapy, expression of TCRζ appeared to trend toward normal levels; in comparison, lower expressions of FcεRIγ and Elf-1 persisted. In conclusion, the previously-documented impairment of T-lymphocyte functions and T- lymphocyte-mediated immune responses seen previously in response to occupational Pb exposure might be attributable, in part, to effects on TCR signaling pathways - including those related to TCRζ and FcεRIγ - and to any down-regulation of membrane TCRζ expression/activity that might be associated with Pb-induced effects on Elf-1 expression.

  5. Accurate detection of the tumor clone in peripheral T-cell lymphoma biopsies by flow cytometric analysis of TCR-Vβ repertoire.

    PubMed

    Salameire, Dimitri; Solly, Françoise; Fabre, Blandine; Lefebvre, Christine; Chauvet, Martine; Gressin, Rémy; Corront, Bernadette; Ciapa, Agnès; Pernollet, Martine; Plumas, Joël; Macintyre, Elizabeth; Callanan, Mary B; Leroux, Dominique; Jacob, Marie-Christine

    2012-09-01

    Multiparametric flow cytometry has proven to be a powerful method for detection and immunophenotypic characterization of clonal subsets, particularly in lymphoproliferative disorders of the B-cell lineage. Although in theory promising, this approach has not been comparably fulfilled in mature T-cell malignancies. Specifically, the T-cell receptor-Vβ repertoire analysis in blood can provide strong evidence of clonality, particularly when a single expanded Vß family is detected. The purpose of this study was to determine the relevance of this approach when applied to biopsies, at the site of tumor involvement. To this end, 30 peripheral T-cell lymphoma and 94 control biopsies were prospectively studied. Vβ expansions were commonly detected within CD4+ or CD8+ T cells (97% of peripheral T-cell lymphoma and 54% of non-peripheral T-cell lymphoma cases); thus, not differentiating malignant from reactive processes. Interestingly, we demonstrated that using a standardized evaluation, the detection of a high Vβ expansion was closely associated with diagnosis of peripheral T-cell lymphoma, with remarkable specificity (98%) and sensitivity (90%). This approach also identified eight cases of peripheral T-cell lymphoma that were not detectable by other forms of immunophenotyping. Moreover, focusing Vβ expression analysis to T-cell subsets with aberrant immunophenotypes, we demonstrated that the T-cell clone might be heterogeneous with regard to surface CD7 or CD10 expression (4/11 cases), providing indication on 'phenotypic plasticity'. Finally, among the wide variety of Vβ families, the occurrence of a Vβ17 expansion in five cases was striking. To our knowledge, this is the first report demonstrating the power of T-cell receptor-Vβ repertoire analysis by flow cytometry in biopsies as a basis for peripheral T-cell lymphoma diagnosis and precise T-cell clone identification and characterization.

  6. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

    SciTech Connect

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji; Jin, Aishun; Kishi, Hiroyuki; Muraguchi, Atsushi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

  7. Peptide length determines the outcome of TCR/peptide-MHCI engagement.

    PubMed

    Ekeruche-Makinde, Julia; Miles, John J; van den Berg, Hugo A; Skowera, Ania; Cole, David K; Dolton, Garry; Schauenburg, Andrea J A; Tan, Mai Ping; Pentier, Johanne M; Llewellyn-Lacey, Sian; Miles, Kim M; Bulek, Anna M; Clement, Mathew; Williams, Tamsin; Trimby, Andrew; Bailey, Mick; Rizkallah, Pierre; Rossjohn, Jamie; Peakman, Mark; Price, David A; Burrows, Scott R; Sewell, Andrew K; Wooldridge, Linda

    2013-02-14

    αβ-TCRs expressed at the CD8(+) T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8(+) T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8(+) T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8(+) T-cell clonotypes.

  8. Unraveling a Hotspot for TCR Recognition on HLA-A2: Evidence Against the Existence of Peptide-independent TCR Binding Determinants

    SciTech Connect

    Gagnon, Susan J.; Borbulevych, Oleg Y.; Davis-Harrison, Rebecca L.; Baxter, Tiffany K.; Clemens, John R.; Armstrong, Kathryn M.; Turner, Richard V.; Damirjian, Marale; Biddison, William E.; Baker, Brian M.

    2010-07-19

    T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 {alpha}1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.

  9. IRF4 Regulates the Ratio of T-Bet to Eomesodermin in CD8+ T Cells Responding to Persistent LCMV Infection

    PubMed Central

    Jangalwe, Sonal; Durost, Philip A.; Kenney, Laurie L.; Conley, James M.; Daniels, Keith; Brehm, Michael A.; Welsh, Raymond M.; Berg, Leslie J.

    2015-01-01

    CD8+ T cell exhaustion commonly occurs in chronic infections and cancers. During T cell exhaustion there is a progressive and hierarchical loss of effector cytokine production, up-regulation of inhibitory co-stimulatory molecules, and eventual deletion of antigen specific cells by apoptosis. A key factor that regulates T cell exhaustion is persistent TCR stimulation. Loss of this interaction results in restoration of CD8+ T cell effector functions in previously exhausted CD8+ T cells. TCR stimulation is also important for the differentiation of Eomeshi anti-viral CD8+ effector T cells from T-bethi precursors, both of which are required for optimal viral control. However, the molecular mechanisms regulating the differentiation of these two cell subsets and the relative ratios required for viral clearance have not been described. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8+ T cells by modulating levels of IRF4. Reduced IRF4 expression results in skewing of this ratio in the favor of Eomes, leading to lower proportions and numbers of T-bet+ Eomes- precursors and poor control of LCMV-clone 13 infection. Manipulation of this ratio in the favor of T-bet restores the differentiation of T-bet+ Eomes- precursors and the protective balance of T-bet to Eomes required for efficient viral control. These data highlight a critical role for IRF4 in regulating protective anti-viral CD8+ T cell responses by ensuring a balanced ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection. PMID:26714260

  10. Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation.

    PubMed

    D'Souza, Anthony D; Parikh, Neal; Kaech, Susan M; Shadel, Gerald S

    2007-12-01

    The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA copy number. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding amplification of mtDNA, consistent with a vital role for mitochondrial function for growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. Thus mitochondrial biogenesis is not under control of a single master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle's structure, composition, and function.

  11. Alpha beta T-cell development is not affected by inversion of TCR beta gene enhancer sequences: polar enhancement of gene expression regardless of enhancer orientation.

    PubMed

    Huang, Fang; Cabaud, Olivier; Verthuy, Christophe; Hueber, Anne-Odile; Ferrier, Pierre

    2003-08-01

    V(D)J recombination and expression of the T-cell receptor beta (TCRbeta) gene are required for the development of the alphabeta T lymphocyte lineage. These processes depend on a transcriptional enhancer (Ebeta) which acts preferentially on adjacent upstream sequences, and has little impact on the 5' distal and 3' proximal regions of the TCRbeta locus. Using knock-in mice, we show that alphabeta T-cell differentiation and TCRbeta gene recombination and expression are not sensitive to the orientation of Ebeta sequences. We discuss the implication of these results regarding the mode of enhancer function at this locus during T lymphocyte development.

  12. Computational spatiotemporal analysis identifies WAVE2 and Cofilin as joint regulators of costimulation-mediated T cell actin dynamics

    PubMed Central

    Roybal, Kole T.; Buck, Taráz E.; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J.; Ambler, Rachel; Tunbridge, Helen M.; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F.

    2016-01-01

    Fluorescence microscopy is one of the most important tools in cell biology research and it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells; however, given extensive cell-to-cell variation, methods do not currently exist to assemble these data into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. Here, we have developed one such method and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28 and we have determined how CD28 modulates actin dynamics. We imaged actin and eight core actin regulators under conditions where CD28 in the context of a strong TCR signal was engaged or blocked to yield over a thousand movies. Our computational analysis identified diminished recruitment of the activator of actin nucleation WAVE2 and the actin severing protein cofilin to F-actin as the dominant difference upon costimulation blockade. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics upon costimulation blockade. Thus we have developed and validated an approach to quantify protein distributions in time and space for analysis of complex regulatory systems. PMID:27095595

  13. Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

    PubMed Central

    Jahan, Akhee S.; Lestra, Maxime; Swee, Lee Kim; Fan, Ying; Lamers, Mart M.; Tafesse, Fikadu G.; Theile, Christopher S.; Spooner, Eric; Bruzzone, Roberto; Ploegh, Hidde L.; Sanyal, Sumana

    2016-01-01

    Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12−/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12−/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades. PMID:26811477

  14. Cross-Reactivity of TCR Repertoire: Current Concepts, Challenges, and Implication for Allotransplantation

    PubMed Central

    Degauque, Nicolas; Brouard, Sophie; Soulillou, Jean-Paul

    2016-01-01

    Being able to track donor reactive T cells during the course of organ transplantation is a key to improve the graft survival, to prevent graft dysfunction, and to adapt the immunosuppressive regimen. The attempts of transplant immunologists have been for long hampered by the large size of the alloreactive T cell repertoire. Understanding how self-TCR can interact with allogeneic MHC is a key to critically appraise the different assays available to analyze the TCR Vβ repertoire usage. In this report, we will review conceptually and experimentally the process of cross-reactivity. We will then highlight what can be learned from allotransplantation, a situation of artificial cross-reactivity. Finally, the low- and high-resolution techniques to characterize the TCR Vβ repertoire usage in transplantation will be critically discussed. PMID:27047489

  15. Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor.

    PubMed

    Casas, Javier; Brzostek, Joanna; Zarnitsyna, Veronika I; Hong, Jin-sung; Wei, Qianru; Hoerter, John A H; Fu, Guo; Ampudia, Jeanette; Zamoyska, Rose; Zhu, Cheng; Gascoigne, Nicholas R J

    2014-01-01

    The earliest molecular events in T-cell recognition have not yet been fully described, and the initial T-cell receptor (TCR)-triggering mechanism remains a subject of controversy. Here, using total internal reflection/Forster resonance energy transfer microscopy, we observe a two-stage interaction between TCR, CD8 and major histocompatibility complex (MHC)-peptide. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ-CD8 interactions require CD8-MHC binding. Lck can be found free or bound to the coreceptor. This work indicates that the initial TCR-triggering event is induced by free Lck. PMID:25427562

  16. An unusual association of Felty syndrome and TCR gamma delta lymphocytosis.

    PubMed Central

    Stanworth, S J; Green, L; Pumphrey, R S; Swinson, D R; Bhavnani, M

    1996-01-01

    Felty syndrome, comprised of neutropenia, rheumatoid arthritis and splenomegaly, occurs in approximately 1% of patients with rheumatoid arthritis. Up to one third of these patients have an increased number of large granular lymphocytes. The usual immunophenotype of these cells is CD3+, CD8+, CD57+, T cell receptor (TCR) alpha beta. A patient with Felty syndrome and large granular lymphocytosis, who had an unusual immunophenotype CD3+, CD4-, CD8-, TCR gamma delta, is described. Her neutropenia responded to treatment with granulocyte colony stimulating factor (G-CSF), which was given in order to raise her neutrophil count prior to bilateral knee replacement surgery. Thus, Felty syndrome with large granular lymphocytosis is a heterogeneous condition, one in which TCR gamma delta large granular lymphocytosis may be found, and also shows a response to treatment with G-CSF. PMID:8655718

  17. RIPK1 and RIPK3: critical regulators of inflammation and cell death.

    PubMed

    Newton, Kim

    2015-06-01

    RIPK1 and RIPK3 (receptor-interacting serine/threonine protein kinases 1/3) interact by virtue of their RIP homotypic interaction motifs to mediate a form of cell death called necroptosis, although mice lacking these kinases have very different phenotypes. RIPK1-deficient mice die soon after birth, whereas RIPK3-deficient mice are healthy. Necroptosis involves cell rupture and is triggered by tumor necrosis factor (TNF), Toll-like receptors (TLRs), or the T cell receptor (TCR) when pro-apoptotic caspase-8 is inhibited. Various mouse models of disease are ameliorated by RIPK3 deficiency, suggesting that necroptosis contributes to pathology. Genetic rescue experiments now reveal why RIPK3-deficient are viable but RIPK1-deficient mice are not. These and other experiments indicate unexpected complexity in the regulation of both apoptosis and necroptosis by RIPK1 and RIPK3.

  18. Upregulation of cAMP-specific PDE-4 activity following ligation of the TCR complex on thymocytes is blocked by selective inhibitors of protein kinase C and tyrosyl kinases.

    PubMed

    Michie, A M; Rena, G; Harnett, M M; Houslay, M D

    1998-01-01

    We have previously shown that the major cAMP phosphodiesterase (PDE) isoforms present in murine thymocytes are the cGMP-stimulated PDE activity (PDE-2) and the cAMP-specific PDE activity (PDE-4), and that these isoforms are differentially regulated following ligation of the TCR (Michie, A.M., Lobban, M. D., Mueller, T., Harnett, M. M., and Houslay, M.D. [1996] Cell. Signalling 8, 97-110). We show here that the anti-CD3-stimulated elevation in PDE-4 activity in murine thymocytes is dependent on protein tyrosine kinase and protein kinase C (PKC)-mediated signals as the TCR-coupled increase in PDE-4 activity can be abrogated by both the tyrosine kinase inhibitor, genistein, and the PKC selective inhibitors chelerythrine and staurosporine. Moreover, the PKC-activating phorbol ester, phorbol-12-myristate, 13-acetate (PMA) caused an increase in PDE-4 activity, similar to that observed in cells challenged with anti-CD3 monoclonal antibodies and which was not additive with cochallenge using anti-CD3 antibodies. Both the PMA- and the anti-CD3 antibody-mediated increases in PDE-4 activity were blocked by treatment with either cycloheximide or actinomycin D. Despite the upregulation of PDE-4 activity consequent to TCR ligation, intracellular cAMP levels increased on challenge of thymocytes with anti-CD3 antibody, indicating that adenylate cyclase activity was also increased by TCR ligation. It is suggested that the anti-CD3-mediated increase in PDE-4 activity was owing to a rapid PKC-dependent induction of PDE-4 activity following crosslinking of the TCR complex. This identifies "crosstalk" occurring between the PKA and PKC signaling pathways initiated by ligation of the antigen receptor in murine thymocytes. That both adenylate cyclase and PDE-4 activities were increased may indicate the presence of compartmentalized cAMP responses present in these cells. PMID:9515165

  19. Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency

    PubMed Central

    Wong, Gabriel K.; Millar, David; Penny, Sarah; Heather, James M.; Mistry, Punam; Buettner, Nico; Bryon, Jane; Huissoon, Aarnoud P.

    2016-01-01

    Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRβ sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21lo B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease. PMID:27481850

  20. A Carma1/MALT1-dependent, Bcl10-independent, pathway regulates antigen receptor-mediated mTOR signaling in T cells

    PubMed Central

    Hamilton, Kristia S.; Phong, Binh; Corey, Catherine; Cheng, Jing; Gorentla, Balachandra; Zhong, Xiaoping; Shiva, Sruti; Kane, Lawrence P.

    2015-01-01

    Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. These effects are mediated in part by the mTOR targets S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Most models place Akt upstream of the best-studied mTOR complex, mTORC1; however, studies have called into question whether Akt is necessary for this pathway, at least in T cells. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1 (Carma1)] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the coreceptor CD28. However, another common binding partner of Carma1 and MALT1, Bcl10, was not required for TCR-dependent activation of the mTOR pathway. Consistent with these findings, MALT1 activity was required for the proliferation of CD4+ T cells, but not early TCR-dependent activation events. Also consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR. PMID:24917592

  1. Positive and negative regulation of T-cell activation through kinases and phosphatases.

    PubMed Central

    Mustelin, Tomas; Taskén, Kjetil

    2003-01-01

    The sequence of events in T-cell antigen receptor (TCR) signalling leading to T-cell activation involves regulation of a number of protein tyrosine kinases (PTKs) and the phosphorylation status of many of their substrates. Proximal signalling pathways involve PTKs of the Src, Syk, Csk and Tec families, adapter proteins and effector enzymes in a highly organized tyrosine-phosphorylation cascade. In intact cells, tyrosine phosphorylation is rapidly reversible and generally of a very low stoichiometry even under induced conditions due to the fact that the enzymes removing phosphate from tyrosine-phosphorylated substrates, the protein tyrosine phosphatases (PTPases), have a capacity that is several orders of magnitude higher than that of the PTKs. It follows that a relatively minor change in the PTK/PTPase balance can have a major impact on net tyrosine phosphorylation and thereby on activation and proliferation of T-cells. This review focuses on the involvement of PTKs and PTPases in positive and negative regulation of T-cell activation, the emerging theme of reciprocal regulation of each type of enzyme by the other, as well as regulation of phosphotyrosine turnover by Ser/Thr phosphorylation and regulation of localization of signal components. PMID:12485116

  2. The negative regulators Foxj1 and Foxo3a are up-regulated by a peptide that inhibits systemic lupus erythematosus-associated T cell responses.

    PubMed

    Sela, Uri; Dayan, Molly; Hershkoviz, Rami; Cahalon, Liora; Lider, Ofer; Mozes, Edna

    2006-11-01

    A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorates systemic lupus erythematosus (SLE) in induced and spontaneous lupus models. Our objectives were to determine the effects of hCDR1 on TCR signaling and on its negative regulators, Foxj1 and Foxo3a. BALB/c mice were immunized with the SLE-inducing anti-DNA antibody, designated 16/6Id, and treated with hCDR1. hCDR1 treatment specifically inhibited IFN-gamma secretion by T cells in association with down-regulated T-bet expression and NF-kappaB activation; however, GATA-3 expression was not affected. Furthermore, TCR signaling (ZAP-70 phosphorylation) was inhibited, and the mRNA expression of the two modulators of Th1 activation, Foxj1 and Foxo3a, was significantly up-regulated. The latter were also elevated in SLE-afflicted (NZBxNZW)F1 mice that were treated with hCDR1. Addition of TGF-beta, which was elevated following treatment with hCDR1, to T cells from 16/6Id immunized mice, up-regulated Foxj1 and Foxo3a mRNA expression, similarly to hCDR1. In contrast, anti-TGF-beta antibodies added to hCDR1-treated T cells abrogated its effect. Thus, hCDR1 elevates TGF-beta, which contributes to the up-regulation of T cell Foxj1 and Foxo3a expression, leading to inhibition of NF-kappaB activation and IFN-gamma secretion, which is required for the maintenance of SLE. PMID:17051618

  3. Fine-tuning T cell receptor signaling to control T cell development.

    PubMed

    Fu, Guo; Rybakin, Vasily; Brzostek, Joanna; Paster, Wolfgang; Acuto, Oreste; Gascoigne, Nicholas R J

    2014-07-01

    T cell development from immature CD4(+)CD8(+) double-positive (DP) thymocytes to the mature CD4 or CD8 single-positive (SP) stage requires proper T cell receptor (TCR) signaling. The current working model of thymocyte development is that the strength of the TCR-mediated signal - from little-or-none, through intermediate, to strong - received by the immature cells determines whether they will undergo death by neglect, positive selection, or negative selection, respectively. In recent years, several developmentally regulated, stage-specifically expressed proteins and miRNAs have been found that act like fine-tuners for signal transduction and propagation downstream of the TCR. This allows them to govern thymocyte positive selection. Here, we summarize recent findings on these molecules and suggest new concepts of TCR positive-selection signaling.

  4. Fine-tuning T cell receptor signaling to control T cell development.

    PubMed

    Fu, Guo; Rybakin, Vasily; Brzostek, Joanna; Paster, Wolfgang; Acuto, Oreste; Gascoigne, Nicholas R J

    2014-07-01

    T cell development from immature CD4(+)CD8(+) double-positive (DP) thymocytes to the mature CD4 or CD8 single-positive (SP) stage requires proper T cell receptor (TCR) signaling. The current working model of thymocyte development is that the strength of the TCR-mediated signal - from little-or-none, through intermediate, to strong - received by the immature cells determines whether they will undergo death by neglect, positive selection, or negative selection, respectively. In recent years, several developmentally regulated, stage-specifically expressed proteins and miRNAs have been found that act like fine-tuners for signal transduction and propagation downstream of the TCR. This allows them to govern thymocyte positive selection. Here, we summarize recent findings on these molecules and suggest new concepts of TCR positive-selection signaling. PMID:24951034

  5. Comparative genomics of the human and mouse T cell receptor loci.

    PubMed

    Glusman, G; Rowen, L; Lee, I; Boysen, C; Roach, J C; Smit, A F; Wang, K; Koop, B F; Hood, L

    2001-09-01

    The availability of the complete genomic sequences of the human and mouse T cell receptor loci opens up new opportunities for understanding T cell receptors (TCRs) and their genes. The full complement of TCR gene segments is finally known and should prove a valuable resource for supporting functional studies. A rational nomenclature system has been implemented and is widely available through IMGT and other public databases. Systematic comparisons of the genomic sequences within each locus, between loci, and across species enable precise analyses of the various diversification mechanisms and some regulatory signals. The genomic landscape of the TCR loci provides fundamental insights into TCR evolution as highly localized and tightly regulated gene families.

  6. Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse.

    PubMed

    Soares, Helena; Henriques, Ricardo; Sachse, Martin; Ventimiglia, Leandro; Alonso, Miguel A; Zimmer, Christophe; Thoulouze, Maria-Isabel; Alcover, Andrés

    2013-10-21

    How the vesicular traffic of signaling molecules contributes to T cell receptor (TCR) signal transduction at the immunological synapse remains poorly understood. In this study, we show that the protein tyrosine kinase Lck, the TCRζ subunit, and the adapter LAT traffic through distinct exocytic compartments, which are released at the immunological synapse in a differentially regulated manner. Lck vesicular release depends on MAL protein. Synaptic Lck, in turn, conditions the calcium- and synaptotagmin-7-dependent fusion of LAT and TCRζ containing vesicles. Fusion of vesicles containing TCRζ and LAT at the synaptic membrane determines not only the nanoscale organization of phosphorylated TCRζ, ZAP70, LAT, and SLP76 clusters but also the presence of phosphorylated LAT and SLP76 in interacting signaling nanoterritories. This mechanism is required for priming IL-2 and IFN-γ production and may contribute to fine-tuning T cell activation breadth in response to different stimulatory conditions.

  7. Transglutaminase Regulation of Cell Function

    PubMed Central

    Kaartinen, Mari T.; Nurminskaya, Maria; Belkin, Alexey M.; Colak, Gozde; Johnson, Gail V. W.; Mehta, Kapil

    2014-01-01

    Transglutaminases (TGs) are multifunctional proteins having enzymatic and scaffolding functions that participate in regulation of cell fate in a wide range of cellular systems and are implicated to have roles in development of disease. This review highlights the mechanism of action of these proteins with respect to their structure, impact on cell differentiation and survival, role in cancer development and progression, and function in signal transduction. We also discuss the mechanisms whereby TG level is controlled and how TGs control downstream targets. The studies described herein begin to clarify the physiological roles of TGs in both normal biology and disease states. PMID:24692352

  8. Regulated cell death in AKI.

    PubMed

    Linkermann, Andreas; Chen, Guochun; Dong, Guie; Kunzendorf, Ulrich; Krautwald, Stefan; Dong, Zheng

    2014-12-01

    AKI is pathologically characterized by sublethal and lethal damage of renal tubules. Under these conditions, renal tubular cell death may occur by regulated necrosis (RN) or apoptosis. In the last two decades, tubular apoptosis has been shown in preclinical models and some clinical samples from patients with AKI. Mechanistically, apoptotic cell death in AKI may result from well described extrinsic and intrinsic pathways as well as ER stress. Central converging nodes of these pathways are mitochondria, which become fragmented and sensitized to membrane permeabilization in response to cellular stress, resulting in the release of cell death-inducing factors. Whereas apoptosis is known to be regulated, tubular necrosis was thought to occur by accident until recent work unveiled several RN subroutines, most prominently receptor-interacting protein kinase-dependent necroptosis and RN induced by mitochondrial permeability transition. Additionally, other cell death pathways, like pyroptosis and ferroptosis, may also be of pathophysiologic relevance in AKI. Combination therapy targeting multiple cell-death pathways may, therefore, provide maximal therapeutic benefits. PMID:24925726

  9. Regulated Cell Death in AKI

    PubMed Central

    Chen, Guochun; Dong, Guie; Kunzendorf, Ulrich; Krautwald, Stefan

    2014-01-01

    AKI is pathologically characterized by sublethal and lethal damage of renal tubules. Under these conditions, renal tubular cell death may occur by regulated necrosis (RN) or apoptosis. In the last two decades, tubular apoptosis has been shown in preclinical models and some clinical samples from patients with AKI. Mechanistically, apoptotic cell death in AKI may result from well described extrinsic and intrinsic pathways as well as ER stress. Central converging nodes of these pathways are mitochondria, which become fragmented and sensitized to membrane permeabilization in response to cellular stress, resulting in the release of cell death–inducing factors. Whereas apoptosis is known to be regulated, tubular necrosis was thought to occur by accident until recent work unveiled several RN subroutines, most prominently receptor-interacting protein kinase–dependent necroptosis and RN induced by mitochondrial permeability transition. Additionally, other cell death pathways, like pyroptosis and ferroptosis, may also be of pathophysiologic relevance in AKI. Combination therapy targeting multiple cell-death pathways may, therefore, provide maximal therapeutic benefits. PMID:24925726

  10. Cell Cycle Regulation and Melanoma.

    PubMed

    Xu, Wen; McArthur, Grant

    2016-06-01

    Dysregulation of cell cycle control is a hallmark of melanomagenesis. Agents targeting the G1-S and G2-M checkpoints, as well as direct anti-mitotic agents, have all shown promising preclinical activity in melanoma. However, in vivo, standalone single agents targeting cell cycle regulation have only demonstrated modest efficacy in unselected patients. The advent of specific CDK 4/6 inhibitors targeting the G1-S transition, with an improved therapeutic index, is a significant step forward. Potential synergy exists with the combination of CDK4/6 inhibitors with existing therapies targeting the MAPK pathway, particularly in subsets of metastatic melanomas such as NRAS and BRAF mutants. This reviews summaries of the latest developments in both preclinical and clinical data with cell cycle-targeted therapies in melanoma. PMID:27106898

  11. CD3ε recruits Numb to promote TCR degradation.

    PubMed

    Martin-Blanco, Nadia; Jiménez Teja, Daniel; Bretones, Gabriel; Borroto, Aldo; Caraballo, Michael; Screpanti, Isabella; León, Javier; Alarcón, Balbino; Canelles, Matilde

    2016-03-01

    Modulation of TCR signaling upon ligand binding is achieved by changes in the equilibrium between TCR degradation, recycling and synthesis; surprisingly, the molecular mechanism of such an important process is not fully understood. Here, we describe the role of a new player in the mediation of TCR degradation: the endocytic adaptor Numb. Our data show that Numb inhibition leads to abnormal intracellular distribution and defective TCR degradation in mature T lymphocytes. In addition, we find that Numb simultaneously binds to both Cbl and a site within CD3ε that overlaps with the Nck binding site. As a result, Cbl couples specifically to the CD3ε chain to mediate TCR degradation. The present study unveils a novel role of Numb that lies at the heart of TCR signaling initiation and termination.

  12. Stimulation through CD50 preferentially induces apoptosis of TCR1+ human peripheral blood lymphocytes.

    PubMed

    López-Briones, S; Portales-Pérez, D P; Baranda, L; de la Fuente, H; Rosenstein, Y; González-Amaro, R

    1998-01-01

    Apoptosis has an important role in several key immunological phenomena such as regulation of the immune response, and deletion of auto-reactive cells. This phenomenon is induced following the interaction of several cell membrane receptors with their respective ligands or after cell activation. We have studied the possible effect of signaling through CD50/ICAM-3 and CD69/AIM on apoptosis of peripheral blood lymphocytes. Apoptosis was assessed by both flow cytometry analysis (content of cell DNA and binding to annexin V), and detection of DNA fragmentation by agarose gel electrophoresis. We found that a stimulatory anti-CD50 mAb was able to induce a small but significant degree of apoptosis in resting peripheral blood mononuclear cells from most donors; this effect was dose-dependent and was evident as early as at 12 h, with a maximal induction at 48 h. Studies with T and non-T cells showed that only the former cell population was sensitive to the induction of apoptosis through CD50. Further experiments revealed that the anti-ICAM-3 mAb preferentially induced apoptosis of TCR gamma delta-bearing cells. In addition, we found a significant increase in Cai2+ in PBMC stimulated with an anti-CD50 mAb, suggesting the involvement of this signaling pathway in the induction of apoptosis through this adhesion receptor. In contrast, under our experimental conditions, stimulation through CD69 did not have any effect on the induction of apoptosis on either cultured T lymphoblasts or PMA-stimulated PBMC. Our findings suggest that the interaction of CD50 with its natural ligand LFA-1 results in the induction of apoptosis in a significant fraction of resting PBMC. This phenomenon may be involved in immune regulation, lymphocyte turnover and peripheral deletion of auto-reactive cells. PMID:9929740

  13. Designer T cells by T cell receptor replacement.

    PubMed

    Sommermeyer, Daniel; Neudorfer, Julia; Weinhold, Monika; Leisegang, Matthias; Engels, Boris; Noessner, Elfriede; Heemskerk, Mirjam H M; Charo, Jehad; Schendel, Dolores J; Blankenstein, Thomas; Bernhard, Helga; Uckert, Wolfgang

    2006-11-01

    T cell receptor (TCR) gene transfer is a convenient method to produce antigen-specific T cells for adoptive therapy. However, the expression of two TCR in T cells could impair their function or cause unwanted effects by mixed TCR heterodimers. With five different TCR and four different T cells, either mouse or human, we show that some TCR are strong--in terms of cell surface expression--and replace weak TCR on the cell surface, resulting in exchange of antigen specificity. Two strong TCR are co-expressed. A mouse TCR replaces human TCR on human T cells. Even though it is still poorly understood why some TCRalpha/beta combinations are preferentially expressed on T cells, our data suggest that, in the future, designer T cells with exclusive tumor reactivity can be generated by T cell engineering. PMID:17051621

  14. Negative regulation of mTOR activation by diacylglycerol kinases

    PubMed Central

    Gorentla, Balachandra K.; Wan, Chi-Keung

    2011-01-01

    The engagement of TCR induces T-cell activation, which initiates multiple characteristic changes such as increase in cell size, cell division, and the production of cytokines and other effector molecules. The mammalian target of rapamycin (mTOR) regulates protein synthesis, transcription, cell survival, and autophagy. Critical roles of mTOR in T-cell activation and effector/memory differentiation have been revealed using chemical inhibitors or by genetic ablation of mTOR in T cells. However, the connection between mTOR signaling and other signaling cascades downstream of TCR is unclear. We demonstrate that diacylglycerol (DAG) and TCR engagement activate signaling in both mTOR complexes 1 and 2 through the activation of the Ras–mitogen-activated protein kinase/extracellular signal–regulated kinase 1/2 (Mek1/2)–extracellular signal–regulated kinase 1/2 (Erk1/2)–activator protein 1 (AP-1), known collectively as the Ras-Mek1/2-Erk1/2-AP-1 pathway. Deficiency of RasGRP1 or inhibition of Mek1/2 activity drastically decreases TCR-induced mTOR activation, whereas constitutively active Ras or Mek1 promotes mTOR activation. Although constitutively active Akt promotes TCR-induced mTOR activation, such activation is attenuated by Mek1/2 inhibition. We demonstrated further that DAG kinases (DGKs) α and ζ, which terminate DAG-mediated signaling, synergistically inhibit TCR-induced mTOR activation by inhibiting the Ras-Mek1/2-Erk/12 pathway. These observations provide novel insights into the regulation of mTOR activation. PMID:21310925

  15. microRNA-mediated regulation of mTOR complex components facilitates discrimination between activation and anergy in CD4 T cells

    PubMed Central

    Marcais, Antoine; Blevins, Rory; Graumann, Johannes; Feytout, Amelie; Dharmalingam, Gopuraja; Carroll, Thomas; Amado, Inês F.; Bruno, Ludovica; Lee, Keunwook; Walzer, Thierry; Mann, Matthias; Freitas, Antonio A.; Boothby, Mark; Fisher, Amanda G.

    2014-01-01

    T cell receptor (TCR) signals can elicit full activation with acquisition of effector functions or a state of anergy. Here, we ask whether microRNAs affect the interpretation of TCR signaling. We find that Dicer-deficient CD4 T cells fail to correctly discriminate between activating and anergy-inducing stimuli and produce IL-2 in the absence of co-stimulation. Excess IL-2 production by Dicer-deficient CD4 T cells was sufficient to override anergy induction in WT T cells and to restore inducible Foxp3 expression in Il2-deficient CD4 T cells. Phosphorylation of Akt on S473 and of S6 ribosomal protein was increased and sustained in Dicer-deficient CD4 T cells, indicating elevated mTOR activity. The mTOR components Mtor and Rictor were posttranscriptionally deregulated, and the microRNAs Let-7 and miR-16 targeted the Mtor and Rictor mRNAs. Remarkably, returning Mtor and Rictor to normal levels by deleting one allele of Mtor and one allele of Rictor was sufficient to reduce Akt S473 phosphorylation and to reduce co-stimulation–independent IL-2 production in Dicer-deficient CD4 T cells. These results show that microRNAs regulate the expression of mTOR components in T cells, and that this regulation is critical for the modulation of mTOR activity. Hence, microRNAs contribute to the discrimination between T cell activation and anergy. PMID:25311506

  16. TCR backscattering characterization for microwave remote sensing

    NASA Astrophysics Data System (ADS)

    Riccio, Giovanni; Gennarelli, Claudio

    2014-05-01

    A Trihedral Corner Reflector (TCR) is formed by three mutually orthogonal metal plates of various shapes and is a very important scattering structure since it exhibits a high monostatic Radar Cross Section (RCS) over a wide angular range. Moreover it is a handy passive device with low manufacturing costs and robust geometric construction, the maintenance of its efficiency is not difficult and expensive, and it can be used in all weather conditions (i.e., fog, rain, smoke, and dusty environment). These characteristics make it suitable as reference target and radar enhancement device for satellite- and ground-based microwave remote sensing techniques. For instance, TCRs have been recently employed to improve the signal-to-noise ratio of the backscattered signal in the case of urban ground deformation monitoring [1] and dynamic survey of civil infrastructures without natural corners as the Musmeci bridge in Basilicata, Italy [2]. The region of interest for the calculation of TCR's monostatic RCS is here confined to the first quadrant containing the boresight direction. The backscattering term is presented in closed form by evaluating the far-field scattering integral involving the contributions related to the direct illumination and the internal bouncing mechanisms. The Geometrical Optics (GO) laws allow one to determine the field incident on each TCR plate and the patch (integration domain) illuminated by it, thus enabling the use of a Physical Optics (PO) approximation for the corresponding surface current densities to consider for integration on each patch. Accordingly, five contributions are associated to each TCR plate: one contribution is due to the direct illumination of the whole internal surface; two contributions originate by the impinging rays that are simply reflected by the other two internal surfaces; and two contributions are related to the impinging rays that undergo two internal reflections. It is useful to note that the six contributions due to the

  17. Lymphokine-dependent proliferation of T-lymphoid cells: regulated responsiveness and role in vivo.

    PubMed

    Boothby, M; Mora, A L; Stephenson, L M

    2001-01-01

    The discovery of lymphokines stemmed from their ability to promote T-lymphocyte proliferation in vitro. Even after 20 years of intensive investigation, crucial aspects remain to be clarified about the role of specific lymphokines in T-cell proliferation and the biochemical mechanisms by which they play these roles, particularly in vivo. The present review focuses on conventional populations of TCR(alpha)beta T cells. Older findings and new insights into the function of specific lymphokines in T-lymphocyte proliferation in vivo are summarized along with unanswered questions raised by these observations. Vital contributions of lymphokines to clonal proliferation arise from two processes: the protection of cells against apoptosis and the activation of cell cycling. Findings are underscored indicating that the activity of a particular lymphokine depends on the subset of T cells (CD4 vs. CD8; naive vs. memory) to which it binds, and that point to potential pitfalls of extrapolating from tissue culture-adapted models to the regulation of T cells in vivo. After summaries of signaling mechanisms related to the proliferative activity of lymphokines, recent findings are highlighted suggesting that such signaling is a regulated and plastic process rather than one fixed schema of action. PMID:12058862

  18. Hypertonic stress regulates T cell function via pannexin-1 hemichannels and P2X receptors

    PubMed Central

    Woehrle, Tobias; Yip, Linda; Manohar, Monali; Sumi, Yuka; Yao, Yongli; Chen, Yu; Junger, Wolfgang G.

    2010-01-01

    Hypertonic saline (HS) resuscitation increases T cell function and inhibits posttraumatic T cell anergy, which can reduce immunosuppression and sepsis in trauma patients. We have previously shown that HS induces the release of cellular ATP and enhances T cell function. However, the mechanism by which HS induces ATP release and the subsequent regulation of T cell function by ATP remain poorly understood. In the present study, we show that inhibition of the gap junction hemichannel pannexin-1 (Panx1) blocks ATP release in response to HS, and HS exposure triggers significant changes in the expression of all P2X-type ATP receptors in Jurkat T cells. Blocking or silencing of Panx1 or of P2X1, P2X4, or P2X7 receptors blunts HS-induced p38 MAPK activation and the stimulatory effects of HS on TCR/CD28-induced IL-2 gene transcription. Moreover, treatment with HS or agonists of P2X receptors overcomes T cell suppression induced by the anti-inflammatory cytokine IL-10. These findings indicate that Panx1 hemichannels facilitate ATP release in response to hypertonic stress and that P2X1, P2X4, and P2X7 receptor activation enhances T cell function. We conclude that HS and P2 receptor agonists promote T cell function and thus, could be used to improve T cell function in trauma patients. PMID:20884646

  19. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    NASA Astrophysics Data System (ADS)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  20. Stem cell regulation: Implications when differentiated cells regulate symmetric stem cell division.

    PubMed

    Høyem, Marte Rørvik; Måløy, Frode; Jakobsen, Per; Brandsdal, Bjørn Olav

    2015-09-01

    We use a mathematical model to show that if symmetric stem cell division is regulated by differentiated cells, then changes in the population dynamics of the differentiated cells can lead to changes in the population dynamics of the stem cells. More precisely, the relative fitness of the stem cells can be affected by modifying the death rate of the differentiated cells. This result is interesting because stem cells are less sensitive than differentiated cells to environmental factors, such as medical therapy. Our result implies that stem cells can be manipulated indirectly by medical treatments that target the differentiated cells. PMID:25997796

  1. β-arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism

    PubMed Central

    Fernández-Arenas, Elena; Calleja, Enrique; Martínez-Martín, Nadia; Gharbi, Severine I; Navajas, Rosana; García-Medel, Noel; Penela, Petronila; Alcamí, Antonio; Mayor, Federico; Albar, Juan P; Alarcón, Balbino

    2014-01-01

    T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that β-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the β-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of β-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal. PMID:24502978

  2. The large ectodomains of CD45 and CD148 regulate their segregation from and inhibition of ligated T-cell receptor.

    PubMed

    Cordoba, Shaun-Paul; Choudhuri, Kaushik; Zhang, Hao; Bridge, Marcus; Basat, Alp Bugra; Dustin, Michael L; van der Merwe, P Anton

    2013-05-23

    T-cell receptor (TCR) triggering results in a cascade of intracellular tyrosine phosphorylation events that ultimately leads to T-cell activation. It is dependent on changes in the relative activities of membrane-associated tyrosine kinases and phosphatases near the engaged TCR. CD45 and CD148 are transmembrane tyrosine phosphatases with large ectodomains that have activatory and inhibitory effects on TCR triggering. This study investigates whether and how the ectodomains of CD45 and CD148 modulate their inhibitory effect on TCR signaling. Expression in T cells of forms of these phosphatases with truncated ectodomains inhibited TCR triggering. In contrast, when these phosphatases were expressed with large ectodomains, they had no inhibitory effect. Imaging studies revealed that truncation of the ectodomains enhanced colocalization of these phosphatases with ligated TCR at the immunological synapse. Our results suggest that the large ectodomains of CD45 and CD148 modulate their inhibitory effect by enabling their passive, size-based segregation from ligated TCR, supporting the kinetic-segregation model of TCR triggering.

  3. Structure and diversity of the TCR alpha-chain in a teleost fish.

    PubMed

    Partula, S; de Guerra, A; Fellah, J S; Charlemagne, J

    1996-07-01

    T cell receptor beta-chain genes are well characterized in representatives of most vertebrate phyla, from sharks to mammals, but the molecular structure of complete TCR alpha-chains has not yet been established in cold-blooded vertebrates. We used a PCR approach to isolate cDNAs encoding putative teleost fish (Oncorhynchus mykiss, rainbow trout) TCR alpha-chains. Eight V alpha segments were identified, belonging to six different families, and the best amino acid sequence identity scores for these trout V alpha were all provided by mammalian V alpha or V delta sequences. Twenty-four (60.1 %) of the 39 analyzed V alpha segments belong to the V alpha 2 family, which has limited homology with mammalian V alpha/delta sequences and with the human V pre-B sequence. A total of 32 different J alpha segments were identified from 40 J alpha regions sequenced, suggesting that a large repertoire of J alpha segments is a characteristic of most vertebrates. The structural properties of the TCR alpha-chain complementarity-determining region 3 loop are well conserved between trout and mammals, suggesting that this region has been under continuous selective pressure in jawed vertebrate evolution. The trout C alpha segment has conserved N-terminal and transmembrane domains, but the C alpha intercysteine distance contains only 40 residues, significantly smaller as compared with mammals (49-56 residues). The conserved features of teleost fish TCR beta- and alpha-chains with their mammalian equivalents suggest that TCR-alpha beta receptors were still present in the common Devonian ancestors of modern teleost fish and mammals, about 450 million years ago. PMID:8683116

  4. Arrested rearrangement of TCR V[beta] genes in thymocytes from children with x-linked severe combined immunodeficiency disease

    SciTech Connect

    Sleasman, J.W.; Harville, T.O.; White, G.B.; Barrett, D.J. ); George, J.F. ); Goodenow, M.M. Univ. of Alabama, Birmingham, AL )

    1994-07-01

    Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R [gamma]-chain. Because TCR-[beta] gene recombination is a pivotal initial event in T lymphocyte onteogeny within the thymus, the authors hypothesized that a failure to express normal IL-2R[gamma] could lead to impaired TCR-[beta] gene recombination in early thymic development. PCR was used to determine the status of TCR-[beta] gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-[beta] gene rearrangement, that of D[beta] to J[beta] recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V[beta] to DJ[beta] gene rearrangements were undetectable in the same samples. Both D[beta] to J[beta] and V[beta] to DJ[beta] TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. The authors conclude that TCR[beta]-chain gene rearrangement is arrested in children with X-linked SCID. The results suggest a causative relationship between the failure of TCR [beta]-chain gene arrangements to proceed beyond DJ[beta] rearrangements and the production of a nonfunctional IL-2R [gamma]-chain. 45 refs., 3 figs.

  5. Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

    PubMed Central

    D’Souza, Anthony D.; Parikh, Neal; Kaech, Susan M.; Shadel, Gerald S.

    2009-01-01

    The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA levels. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding increase of mtDNA copy number, indicating the vital role for mitochondrial function for the growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. We propose that mitochondrial biogenesis is not under control of a master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function. PMID:17890163

  6. αβ TCR-mediated recognition: relevance to tumor-antigen discovery and cancer immunotherapy

    PubMed Central

    Reinherz, Ellis L.

    2015-01-01

    αβ T lymphocytes sense perturbations in host cellular body components induced by infectious pathogens, oncogenic transformation or chemical or physical damage. Millions-billions of these lymphocytes are generated through T-lineage development in the thymus, each endowed with a clonally-restricted surface T-cell receptor (TCR). An individual TCR has the capacity to recognize a distinct “foreign” peptide among the myriad of antigens that the mammalian host must be capable of detecting. TCRs explicitly distinguish foreign from self peptides bound to major histocompatibility complex (MHC) molecules. This is a daunting challenge, given that the MHC-linked peptidome consists of thousands of distinct peptides with a relevant non-self target antigen often embedded at low number, among orders of magnitude higher frequency self-peptides. In this Masters of Immunology article, I shall review how TCR structure and attendant mechanobiology involving non-linear responses impact sensitivity as well as specificity to meet this requirement. Assessment of human tumor-cell display using state of the art mass spectrometry physical detection methods that quantify epitope copy number can help inform as to requisite T-cell functional avidity affording protection and/or therapeutic immunity. Future rational CD8 cytotoxic T cell-based vaccines may follow, targeting virally-induced cancers, other non-viral immunogenic tumors, and potentially even non-immunogenic tumors whose peptide display can be purposely altered by MHC-binding drugs to stimulate immune attack. PMID:25847967

  7. Cancer regression and neurologic toxicity following anti-MAGE-A3 TCR gene therapy

    PubMed Central

    Morgan, Richard A.; Chinnasamy, Nachimuthu; Abate-Daga, Daniel D; Gros, Alena; Robbins, Paul F.; Zheng, Zhili; Feldman, Steven A.; Yang, James C.; Sherry, Richard M.; Phan, Giao Q.; Hughes, Marybeth S.; Kammula, Udai S.; Miller, Akemi D.; Hessman, Crystal J.; Stewart, Ashley A.; Restifo, Nicholas P.; Quezado, Martha M.; Alimchandani, Meghna; Rosenberg, Avi Z.; Nath, Avindra; Wang, Tongguang; Bielekova, Bibiana; Wuest, Simone C.; Nirmala, Akula; McMahon, Francis J.; Wilde, Susanne; Mosetter, Barbara; Schendel, Dolores J.; Laurencot, Carolyn M.; Rosenberg, Steven A

    2013-01-01

    Nine cancer patients were treated with adoptive cell therapy using autologous anti-MAGE-A3 TCR engineered T cells. Five patients experienced clinical regression of their cancers including two on-going responders. Beginning 1–2 days post-infusion, three patients (#’s 5, 7, and 8) experienced mental status changes, and two patients (5 and 8) lapsed into comas and subsequently died. Magnetic resonance imagining analysis of patients 5 and 8 demonstrated periventricular leukomalacia, and examination of their brains at autopsy revealed necrotizing leukoencephalopathy with extensive white matter defects associated with infiltration of CD3+/CD8+ T cells. Patient 7, developed Parkinson-like symptoms, which resolved over 4 weeks and fully recovered. Immunohistochemical staining of patient and normal brain samples demonstrated rare positively staining neurons with an antibody that recognizes multiple MAGE-A family members. The TCR used in this study recognized epitopes in MAGE-A3/A9/A12. Molecular assays of human brain samples using Q-RT-PCR, Nano string quantitation, and deep-sequencing indicated that MAGE -A12 was expressed in human brain (and possibly MAGE-A1, MAGE-A8, and MAGE-A9). This previously unrecognized expression of MAGE-A12 in human brain was possibly the initiating event of a TCR-mediated inflammatory response that resulted in neuronal cell destruction and raises caution for clinical applications targeting MAGE-A family members with highly active immunotherapies. PMID:23377668

  8. The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4+ T cells

    PubMed Central

    Bertin, Samuel; Aoki-Nonaka, Yukari; de Jong, Petrus Rudolf; Stanwood, Shawna R.; Srikanth, Sonal; Lee, Jihyung; To, Keith; Abramson, Lior; Yu, Timothy; Han, Tiffany; Touma, Ranim; Li, Xiangli; González-Navajas, José M.; Herdman, Scott; Corr, Maripat; Fu, Guo; Dong, Hui; Gwack, Yousang; Franco, Alessandra; Jefferies, Wilfred A.; Raz, Eyal

    2016-01-01

    TRPV1 is a Ca2+-permeable channel mostly studied as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here, we demonstrate that TRPV1 is functionally expressed in CD4+ T cells where it acts as a non-store-operated Ca2+ channel and contributes to T cell receptor (TCR)-induced Ca2+ influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promotes colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4+ T cells recapitulates the phenotype of murine Trpv1−/− CD4+ T cells. These findings suggest that TRPV1 inhibition could represent a new therapeutic strategy to restrain proinflammatory T cell responses. PMID:25282159

  9. How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire.

    PubMed

    Blevins, Sydney J; Pierce, Brian G; Singh, Nishant K; Riley, Timothy P; Wang, Yuan; Spear, Timothy T; Nishimura, Michael I; Weng, Zhiping; Baker, Brian M

    2016-03-01

    How T-cell receptors (TCRs) can be intrinsically biased toward MHC proteins while simultaneously display the structural adaptability required to engage diverse ligands remains a controversial puzzle. We addressed this by examining αβ TCR sequences and structures for evidence of physicochemical compatibility with MHC proteins. We found that human TCRs are enriched in the capacity to engage a polymorphic, positively charged "hot-spot" region that is almost exclusive to the α1-helix of the common human class I MHC protein, HLA-A*0201 (HLA-A2). TCR binding necessitates hot-spot burial, yielding high energetic penalties that must be offset via complementary electrostatic interactions. Enrichment of negative charges in TCR binding loops, particularly the germ-line loops encoded by the TCR Vα and Vβ genes, provides this capacity and is correlated with restricted positioning of TCRs over HLA-A2. Notably, this enrichment is absent from antibody genes. The data suggest a built-in TCR compatibility with HLA-A2 that biases receptors toward, but does not compel, particular binding modes. Our findings provide an instructional example for how structurally pliant MHC biases can be encoded within TCRs. PMID:26884163

  10. How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire

    PubMed Central

    Blevins, Sydney J.; Pierce, Brian G.; Singh, Nishant K.; Riley, Timothy P.; Wang, Yuan; Spear, Timothy T.; Nishimura, Michael I.; Weng, Zhiping; Baker, Brian M.

    2016-01-01

    How T-cell receptors (TCRs) can be intrinsically biased toward MHC proteins while simultaneously display the structural adaptability required to engage diverse ligands remains a controversial puzzle. We addressed this by examining αβ TCR sequences and structures for evidence of physicochemical compatibility with MHC proteins. We found that human TCRs are enriched in the capacity to engage a polymorphic, positively charged “hot-spot” region that is almost exclusive to the α1-helix of the common human class I MHC protein, HLA-A*0201 (HLA-A2). TCR binding necessitates hot-spot burial, yielding high energetic penalties that must be offset via complementary electrostatic interactions. Enrichment of negative charges in TCR binding loops, particularly the germ-line loops encoded by the TCR Vα and Vβ genes, provides this capacity and is correlated with restricted positioning of TCRs over HLA-A2. Notably, this enrichment is absent from antibody genes. The data suggest a built-in TCR compatibility with HLA-A2 that biases receptors toward, but does not compel, particular binding modes. Our findings provide an instructional example for how structurally pliant MHC biases can be encoded within TCRs. PMID:26884163

  11. Materials as stem cell regulators

    NASA Astrophysics Data System (ADS)

    Murphy, William L.; McDevitt, Todd C.; Engler, Adam J.

    2014-06-01

    The stem cell/material interface is a complex, dynamic microenvironment in which the cell and the material cooperatively dictate one another's fate: the cell by remodelling its surroundings, and the material through its inherent properties (such as adhesivity, stiffness, nanostructure or degradability). Stem cells in contact with materials are able to sense their properties, integrate cues via signal propagation and ultimately translate parallel signalling information into cell fate decisions. However, discovering the mechanisms by which stem cells respond to inherent material characteristics is challenging because of the highly complex, multicomponent signalling milieu present in the stem cell environment. In this Review, we discuss recent evidence that shows that inherent material properties may be engineered to dictate stem cell fate decisions, and overview a subset of the operative signal transduction mechanisms that have begun to emerge. Further developments in stem cell engineering and mechanotransduction are poised to have substantial implications for stem cell biology and regenerative medicine.

  12. Materials as stem cell regulators

    PubMed Central

    Murphy, William L.; McDevitt, Todd C.; Engler, Adam J.

    2014-01-01

    The stem cell/material interface is a complex, dynamic microenvironment in which the cell and the material cooperatively dictate one another's fate: the cell by remodelling its surroundings, and the material through its inherent properties (such as adhesivity, stiffness, nanostructure or degradability). Stem cells in contact with materials are able to sense their properties, integrate cues via signal propagation and ultimately translate parallel signalling information into cell fate decisions. However, discovering the mechanisms by which stem cells respond to inherent material characteristics is challenging because of the highly complex, multicomponent signalling milieu present in the stem cell environment. In this Review, we discuss recent evidence that shows that inherent material properties may be engineered to dictate stem cell fate decisions, and overview a subset of the operative signal transduction mechanisms that have begun to emerge. Further developments in stem cell engineering and mechanotransduction are poised to have substantial implications for stem cell biology and regenerative medicine. PMID:24845994

  13. Regulation of T-plastin expression by promoter hypomethylation in primary cutaneous T-cell lymphoma.

    PubMed

    Jones, Christine L; Ferreira, Silvia; McKenzie, Robert C T; Tosi, Isabella; Caesar, Jacqueline A; Bagot, Martine; Whittaker, Sean J; Mitchell, Tracey J

    2012-08-01

    T-plastin (PLS3) is an actin-bundling protein normally expressed in epithelial cells but absent in cells of hematopoietic origin. Aberrant PLS3 expression has been demonstrated in lymphocytes from Sézary syndrome (SS) patients and has been proposed as a biomarker for SS; however, the mechanism underlying dysregulation of PLS3 has not been determined. In this study, PLS3 mRNA expression was demonstrated in 21/35 (60%) SS patients and in 3/8 (38%) mycosis fungoides patients, all of whom had clonal blood involvement. No evidence for PLS3 mutations within coding or promoter regions was found, but significant hypomethylation of CpG dinucleotides 95-99 within the PLS3 CpG island was observed and this was restricted to the PLS3+ population. A polyclonal antibody specific to PLS3 was raised to examine coexpression of PLS3 with a panel of T-cell differentiation markers. All PLS3+ cells were CD3+CD4+ and CD26-, suggesting that loss of CD26 is consistently associated with gain of PLS3, whereas all other markers were distributed heterogeneously. However, a patient-specific TCR copy number assay also demonstrated heterogeneity in PLS3 expression in tumor cell populations. Importantly, our findings demonstrate PLS3 expression in the majority of SS patients and provide insight into the molecular regulation of PLS3 expression in CTCL.

  14. Conformational Melding Permits a Conserved Binding Geometry in TCR Recognition of Foreign and Self Molecular Mimics

    SciTech Connect

    Borbulevych, Oleg Y.; Piepenbrink, Kurt H.; Baker, Brian M.

    2012-03-16

    Molecular mimicry between foreign and self Ags is a mechanism of TCR cross-reactivity and is thought to contribute to the development of autoimmunity. The {alpha}{beta} TCR A6 recognizes the foreign Ag Tax from the human T cell leukemia virus-1 when presented by the class I MHC HLA-A2. In a possible link with the autoimmune disease human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis, A6 also recognizes a self peptide from the neuronal protein HuD in the context of HLA-A2. We found in our study that the complexes of the HuD and Tax epitopes with HLA-A2 are close but imperfect structural mimics and that in contrast with other recent structures of TCRs with self Ags, A6 engages the HuD Ag with the same traditional binding mode used to engage Tax. Although peptide and MHC conformational changes are needed for recognition of HuD but not Tax and the difference of a single hydroxyl triggers an altered TCR loop conformation, TCR affinity toward HuD is still within the range believed to result in negative selection. Probing further, we found that the HuD-HLA-A2 complex is only weakly stable. Overall, these findings help clarify how molecular mimicry can drive self/nonself cross-reactivity and illustrate how low peptide-MHC stability can permit the survival of T cells expressing self-reactive TCRs that nonetheless bind with a traditional binding mode.

  15. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling

    PubMed Central

    Dik, Willem A.; Pike-Overzet, Karin; Weerkamp, Floor; de Ridder, Dick; de Haas, Edwin F.E.; Baert, Miranda R.M.; van der Spek, Peter; Koster, Esther E.L.; Reinders, Marcel J.T.; van Dongen, Jacques J.M.; Langerak, Anton W.; Staal, Frank J.T.

    2005-01-01

    To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin− cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Dδ2-Dδ3 rearrangement occurs at the most immature CD34+CD38−CD1a− stage. TCRB rearrangement starts at the CD34+CD38+CD1a− stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTα) expression pattern show that human TCRβ-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before. PMID:15928199

  16. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  17. Allelic exclusion of TCR α-chains upon severe restriction of Vα repertoire.

    PubMed

    Rybakin, Vasily; Westernberg, Luise; Fu, Guo; Kim, Hee-Ok; Ampudia, Jeanette; Sauer, Karsten; Gascoigne, Nicholas R J

    2014-01-01

    Development of thymocytes through the positive selection checkpoint requires the rearrangement and expression of a suitable T cell receptor (TCR) α-chain that can pair with the already-expressed β-chain to make a TCR that is selectable. That is, it must have sufficient affinity for self MHC-peptide to induce the signals required for differentiation, but not too strong so as to induce cell death. Because both alleles of the α-chain continue to rearrange until a positively-selectable heterodimer is formed, thymocytes and T cells can in principle express dual α-chains. However, cell-surface expression of two TCRs is comparatively rare in mature T cells because of post-transcriptional regulatory mechanisms termed "phenotypic allelic exclusion". We produced mice transgenic for a rearranged β-chain and for two unrearranged α-chains on a genetic background where endogenous α-chains could not be rearranged. Both Vα3.2 and Vα2 containing α-chains were efficiently positively selected, to the extent that a population of dual α-chain-bearing cells was not distinguishable from single α-chain-expressors. Surprisingly, Vα3.2-expressing cells were much more frequent than the Vα2 transgene-expressing cells, even though this Vα3.2-Vβ5 combination can reconstitute a known selectable TCR. In accord with previous work on the Vα3 repertoire, T cells bearing Vα3.2 expressed from the rearranged minilocus were predominantly selected into the CD8+ T cell subpopulation. Because of the dominance of Vα3.2 expression over Vα2 expressed from the miniloci, the peripheral T cell population was predominantly CD8+ cells. PMID:25500569

  18. Positive selection of T cells: rescue from programmed cell death and differentiation require continual engagement of the T cell receptor

    PubMed Central

    1995-01-01

    Positive selection of T cells is a complex developmental process generating long-lived, functionally mature CD4+CD8- and CD4-CD8+ cells from short-lived, immature CD4+CD8+ precursors. The process is initiated in the thymus by interaction of the alpha beta TCR with molecules encoded by the MHC, occurs without cell division, and involves rescue from programmed cell death (PCD), as well as induction of differentiation and maturation of selected precursors. It is unclear whether development of small, positively selected CD4+CD8+ thymocytes (characterized by up-regulated levels of TCR and CD69 molecules) depends on further interactions with MHC molecules and, if so, whether such interactions are required for survival, for maturation, or for both. The involvement of the TCR and/or CD4/CD8 coreceptors in transmitting additional signals is also unknown. We have examined these questions by analyzing survival and differentiation of early (CD4+CD8+TCRhi) and later (CD4-CD8+TCRhi) postselection stages of thymocytes from normal and bcl-2 transgenic mice expressing transgenic, class I MHC-restricted TCR, upon intrathymic transfer into recipients that lacked ligands either for both the TCR and CD8 coreceptor, or for the TCR only. The results provide direct evidence that induction of differentiation of CD4+CD8+ thymocytes by recognition of MHC molecules does not rescue them from PCD and is insufficient to activate the entire maturation program. Both processes require continual engagement of the TCR by positively selecting MHC molecules that, at least in the case of class I MHC-restricted CD4-CD8+ T cells, cannot be substituted by the engagement of coreceptor alone. PMID:7759993

  19. Regulated cell death and adaptive stress responses.

    PubMed

    Galluzzi, Lorenzo; Bravo-San Pedro, José Manuel; Kepp, Oliver; Kroemer, Guido

    2016-06-01

    Eukaryotic cells react to potentially dangerous perturbations of the intracellular or extracellular microenvironment by activating rapid (transcription-independent) mechanisms that attempt to restore homeostasis. If such perturbations persist, cells may still try to cope with stress by activating delayed and robust (transcription-dependent) adaptive systems, or they may actively engage in cellular suicide. This regulated form of cell death can manifest with various morphological, biochemical and immunological correlates, and constitutes an ultimate attempt of stressed cells to maintain organismal homeostasis. Here, we dissect the general organization of adaptive cellular responses to stress, their intimate connection with regulated cell death, and how the latter operates for the preservation of organismal homeostasis.

  20. Cell competition in vertebrate organ size regulation.

    PubMed

    Penzo-Méndez, Alfredo I; Stanger, Ben Z

    2014-01-01

    The study of animal organ size determination has provided evidence of the existence of organ-intrinsic mechanisms that 'sense' and adjust organ growth. Cell competition, a form of cell interaction that equalizes cell population growth, has been proposed to play a role in organ size regulation. Cell competition involves a cell-context dependent response triggered by perceived differences in cell growth and/or proliferation rates, resulting in apoptosis in growth-disadvantaged cells and compensatory expansion of the more 'fit' cells. The mechanisms that allow cells to compare growth are not yet understood, but a number of genes and pathways have been implicated in cell competition. These include Myc, the members of the Hippo, JAK/STAT and WNT signaling pathways, and the Dlg/Lgl/Scrib and the Crb/Std/PatJ membrane protein complexes. Cell competition was initially characterized in the Drosophila imaginal disc, but several recent studies have shown that cell competition occurs in mouse embryonic stem cells and in the embryonic epiblast, where it plays a role in the regulation of early embryo size. In addition, competition-like behavior has been described in the adult mouse liver and the hematopoietic stem cell compartment. These data indicate that cell competition plays a more universal role in organ size regulation. In addition, as some authors have suggested that similar types of competitive behavior may operate in during tumorigenesis, there may be additional practical reasons for understanding this fundamental process of intercellular communication.

  1. Alternative splicing regulation and cell lineage differentiation.

    PubMed

    Liu, Huan; He, Ling; Tang, Liling

    2012-11-01

    The alternative splicing of precursor mRNA is an essential mechanism for protein diversity. It plays important biological roles, such as proliferation, differentiation and development of cells. Furthermore, alternative splicing participates in the pathogenesis of diseases, including cancer. Thus, in-depth understanding of splicing regulation is of great significance. Regulation of alternative splicing is an extraordinary complicated process in which several signal molecules are at work. Besides the cis-elements and trans-factors, several lines of evidences suggest that other molecules, structures or process also regulate splicing, such as RNA structures, transcription and transcription factors, chromatin and protein. Meanwhile, increasing body of evidence shows that alternative splicing correlated closely to stem cell lineage differentiation. It means that there is a fundamental role for splicing in controlling regulatory program required for cell lineage differentiation. This review systematically sums up the regulation of alternative splicing and summarizes the splicing events during cell lineage differentiation of stem cells.

  2. Turned on by danger: activation of CD1d-restricted invariant natural killer T cells.

    PubMed

    Lawson, Victoria

    2012-09-01

    CD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, β-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)-antigen-CD1d complex show how docking between CD1d-antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3β loop determines iNKT TCR affinity for ligand-CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails. Ligands loaded into CD1d on the cell surface promote Th2 responses, whereas ligands with long hydrophobic tails are loaded endosomally and promote Th1 responses. This information is informing the design of synthetic iNKT-cell antigens. The iNKT cells may be activated by exogenous antigen, or by a combination of dendritic cell-derived interleukin-12 and iNKT TCR-self-antigen-CD1d engagement. The iNKT-cell activation is further modulated by recent foreign or self-antigen encounter. Activation of dendritic cells through pattern recognition receptors alters their antigen presentation and cytokine production, strongly influencing iNKT-cell activation. In a range of bacterial infections, dendritic cell-dependent innate activation of iNKT cells through interleukin-12 is the dominant influence on their activity.

  3. Small GTPases as regulators of cell division

    PubMed Central

    Militello, Rodrigo; Colombo, María I.

    2013-01-01

    The superfamily of small GTPases serves as a signal transducer to regulate a diverse array of cellular functions. The members of this superfamily are structurally and functionally classified into at least 5 groups (Ras, Rho/Rac, Rab, Arf, and Ran) and they are involved in the control of cell proliferation and differentiation, regulation of the actin cytoskeleton, membrane trafficking, and nuclear transport. It is widely reported that members of the Rab family participate in the control of intracellular membrane trafficking through the interaction with specific effector molecules. However, many Rabs and other small GTPases have also been shown to function in cell division. In this review, we discuss current knowledge about Rab proteins regulating different stages of the cell cycle, such as the congregation and segregation of chromosomes (during metaphase) and the final stage of cell division known as cytokinesis, in which a cell is cleaved originating 2 daughter cells. PMID:24265858

  4. Metabolic regulation of natural killer cells.

    PubMed

    Finlay, David K

    2015-08-01

    Natural killer (NK) cells have key roles in anti-viral and anti-tumour immune responses. Recent research demonstrates that cellular metabolism is an important determinant for the function of pro-inflammatory immune cells, including activated NK cells. The mammalian target of rapamcyin (mTOR) complex 1 (mTORC1) has been identified as a key metabolic regulator that promotes glycolytic metabolism in multiple immune cell subsets. Glycolysis is integrally linked to pro-inflammatory immune responses such that activated NK cells and effector T-cell subsets are reliant on sufficient glucose availability for maximal effector function. This article will discuss the regulation of cellular metabolism in NK cells as compared with that of T lymphocytes and discuss the implications for NK cell responses to viral infection and cancer.

  5. T cell receptor (TCR)-transgenic CD8 lymphocytes rendered insensitive to transforming growth factor beta (TGFβ) signaling mediate superior tumor regression in an animal model of adoptive cell therapy

    PubMed Central

    2012-01-01

    Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFβ), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFβ by transduction with a TGFβ dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer. PMID:22713761

  6. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

    PubMed

    Sutton, Vivien R; Brennan, Amelia J; Ellis, Sarah; Danne, Jill; Thia, Kevin; Jenkins, Misty R; Voskoboinik, Ilia; Pejler, Gunnar; Johnstone, Ricky W; Andrews, Daniel M; Trapani, Joseph A

    2016-03-01

    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.

  7. Regulation of Th2 Cell Immunity by Dendritic Cells.

    PubMed

    Na, Hyeongjin; Cho, Minkyoung; Chung, Yeonseok

    2016-02-01

    Th2 cell immunity is required for host defense against helminths, but it is detrimental in allergic diseases in humans. Unlike Th1 cell and Th17 cell subsets, the mechanism by which dendritic cells modulate Th2 cell responses has been obscure, in part because of the inability of dendritic cells to provide IL-4, which is indispensable for Th2 cell lineage commitment. In this regard, immune cells other than dendritic cells, such as basophils and innate lymphoid cells, have been suggested as Th2 cell inducers. More recently, multiple independent researchers have shown that specialized subsets of dendritic cells mediate Th2 cell responses. This review will discuss the current understanding related to the regulation of Th2 cell responses by dendritic cells and other immune cells. PMID:26937227

  8. Antigen presenting cells - diversity, differentiation, and regulation

    SciTech Connect

    Schook, L.B. ); Tew, J.G. )

    1988-01-01

    This book contains 35 papers. Some of the titles are: DNA-mediated gene transfer as a tool for analyzing Ia structure-function relationships and antigen presentation; Regulation of immune-associated genes during macrophage differentiation; Presentation of arsonate-tyrosine to cloned T-cells by L-Cells transfected with class II genes; and The duration of class II MHC glycoprotein expression by mononuclear phagocytes is regulated by the Bcg gene.

  9. Anisosmotic cell volume regulation: a comparative view.

    PubMed

    Chamberlin, M E; Strange, K

    1989-08-01

    A variety of organisms and cell types spanning the five taxonomic kingdoms are exposed, either naturally or through experimental means, to osmotic stresses. A common physiological response to these challenges is maintenance of cell volume through changes in the concentration of intracellular inorganic and organic solutes, collectively termed osmolytes. Research on the mechanisms by which the concentration of these solutes is regulated has proceeded along several experimental lines. Extensive studies on osmotically activated ion transport pathways have been carried out in vertebrate cells and tissues. Much of our knowledge on organic osmolytes has come from investigations on invertebrates, bacteria, and protists. The relative simplicity of bacterial genetics has provided a powerful and elegant tool to explore the modifications of gene expression during volume regulation. An implication of this diverse experimental approach is that phylogenetically divergent organisms employ uniquely adapted mechanisms of cell volume regulation. Given the probability that changes in extracellular osmolality were physiological stresses faced by the earliest organisms, it is more likely that cell volume regulation proceeds by highly conserved physiological processes. We review volume regulation from a comparative perspective, drawing examples from all five taxonomic kingdoms. Specifically, we discuss the role of inorganic and organic solutes in volume maintenance and the mechanisms by which the concentrations of these osmolytes are regulated. In addition, the processes that may transduce volume perturbations into regulatory responses, such as stretch activation of ion channels, intracellular signaling, and genomic regulation, are discussed. Throughout this review we emphasize areas we feel are important for future research.

  10. A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells.

    PubMed

    Epel, Malka; Ellenhorn, Joshua D; Diamond, Don J; Reiter, Yoram

    2002-11-01

    Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy. PMID:12384808

  11. Biophysical regulation of stem cell differentiation.

    PubMed

    Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

    2013-06-01

    Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

  12. Physiology of cell volume regulation in vertebrates.

    PubMed

    Hoffmann, Else K; Lambert, Ian H; Pedersen, Stine F

    2009-01-01

    The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adaptive (e.g., altered expression of osmolyte transporters and heat shock proteins) measures and, in most cases, activation of volume regulatory osmolyte transport. After acute swelling, cell volume is regulated by the process of regulatory volume decrease (RVD), which involves the activation of KCl cotransport and of channels mediating K(+), Cl(-), and taurine efflux. Conversely, after acute shrinkage, cell volume is regulated by the process of regulatory volume increase (RVI), which is mediated primarily by Na(+)/H(+) exchange, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) channels. Here, we review in detail the current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species, upon changes in cell volume. We also discuss the nature of the upstream elements in volume sensing in vertebrate organisms. Importantly, cell volume impacts on a wide array of physiological processes, including transepithelial transport; cell migration, proliferation, and death; and changes in cell volume function as specific signals regulating these processes. A discussion of this issue concludes the review. PMID:19126758

  13. Regulation of cell volume by glycosaminoglycans.

    PubMed

    Joerges, Jelena; Schulz, Tobias; Wegner, Jeannine; Schumacher, Udo; Prehm, Peter

    2012-01-01

    Cell volume is regulated by a delicate balance between ion distribution across the plasma membrane and the osmotic properties of intra- and extracellular components. Using a fluorescent calcein indicator, we analysed the effects of glycosaminoglycans on the cell volume of hyaluronan producing fibroblasts and hyaluronan deficient HEK cells over a time period of 30 h. Exogenous glycosaminoglycans induced cell blebbing after 2 min and swelling of fibroblasts to about 110% of untreated cell volume at low concentrations which decreased at higher concentrations. HEK cells did not show cell blebbing and responded by shrinking to 65% of untreated cell volume. Heparin induced swelling of both fibroblasts and HEK cells. Hyaluronidase treatment or inhibition of hyaluronan export led to cell shrinkage indicating that the hyaluronan coat maintained fibroblasts in a swollen state. These observations were explained by the combined action of the Donnan effect and molecular crowding. PMID:21928313

  14. Extensive TCR junctional diversity of V gamma 9/V delta 2 clones from human female reproductive tissues.

    PubMed

    Christmas, S E; Brew, R; Thornton, S M; Deniz, G; Flanagan, B F

    1995-09-01

    Panels of gamma delta T cell clones bearing the V gamma 9/V delta 2 form of TCR were derived from human first trimester decidualized endometrium and cervix. Seventy-three percent of these clones expressed the human mucosal lymphocyte Ag HML-1 compared with only 14% of PBL V gamma 9/V delta 2 clones, indicating that most clones were derived from the tissue itself rather than contaminating peripheral blood. All 13 clones isolated expressed V gamma 9JPC gamma 1- and V delta 2(D)J delta 1-encoded receptors; TCR gamma and delta junctional regions from most of these were sequenced and analyzed, together with the TCR-delta junctional region of a sequence obtained from bulk CD3+ decidual leukocytes. There was considerable junctional diversity of both gamma- and delta-chains with a similar extent of germline V and J gene trimming and N-region nucleotide addition to that found in PBL V gamma 9/V delta 2 cells. Eight of eleven TCR-delta junctional sequences contained a strongly hydrophobic amino acid in position 97, as has been found in > 90% o V gamma 9/V delta 2 clones. Thymic V gamma 9/V delta 2 cells show much less junctional diversity and less pronounced selection at residue 97 of the delta-chain. Thus, unlike the mouse, gamma delta T cells from human female reproductive tissues exhibit extensive TCR junctional as well as combinatorial diversity. This suggests that V gamma 9/V delta 2 cells in these human tissues have undergone selective but diverse peripheral expansion in response to antigenic stimuli in a similar manner to those in peripheral blood.

  15. Deadenylation and its regulation in eukaryotic cells.

    PubMed

    Zhang, Xiaokan; Kleiman, Frida E; Devany, Emral

    2014-01-01

    Messenger RNA deadenylation is a process that allows rapid regulation of gene expression in response to different cellular conditions. The change of the mRNA poly(A) tail length by the activation of deadenylation might regulate gene expression by affecting mRNA stability, mRNA transport, or translation initiation. Activation of deadenylation processes are highly regulated and associated with different cellular conditions such as cancer, development, mRNA surveillance, DNA damage response, and cell differentiation. In the last few years, new technologies for studying deadenylation have been developed. Here we overview concepts related to deadenylation and its regulation in eukaryotic cells. We also describe some of the most commonly used protocols to study deadenylation in eukaryotic cells.

  16. Cell Cycle Regulation of DNA Replication

    PubMed Central

    Sclafani, R. A.; Holzen, T. M.

    2008-01-01

    Eukaryotic DNA replication is regulated to ensure all chromosomes replicate once and only once per cell cycle. Replication begins at many origins scattered along each chromosome. Except for budding yeast, origins are not defined DNA sequences and probably are inherited by epigenetic mechanisms. Initiation at origins occurs throughout the S phase according to a temporal program that is important in regulating gene expression during development. Most replication proteins are conserved in evolution in eukaryotes and archaea, but not in bacteria. However, the mechanism of initiation is conserved and consists of origin recognition, assembly of pre-replication (pre-RC) initiative complexes, helicase activation, and replisome loading. Cell cycle regulation by protein phosphorylation ensures that pre-RC assembly can only occur in G1 phase, whereas helicase activation and loading can only occur in S phase. Checkpoint regulation maintains high fidelity by stabilizing replication forks and preventing cell cycle progression during replication stress or damage. PMID:17630848

  17. Collecting duct intercalated cell function and regulation.

    PubMed

    Roy, Ankita; Al-bataineh, Mohammad M; Pastor-Soler, Núria M

    2015-02-01

    Intercalated cells are kidney tubule epithelial cells with important roles in the regulation of acid-base homeostasis. However, in recent years the understanding of the function of the intercalated cell has become greatly enhanced and has shaped a new model for how the distal segments of the kidney tubule integrate salt and water reabsorption, potassium homeostasis, and acid-base status. These cells appear in the late distal convoluted tubule or in the connecting segment, depending on the species. They are most abundant in the collecting duct, where they can be detected all the way from the cortex to the initial part of the inner medulla. Intercalated cells are interspersed among the more numerous segment-specific principal cells. There are three types of intercalated cells, each having distinct structures and expressing different ensembles of transport proteins that translate into very different functions in the processing of the urine. This review includes recent findings on how intercalated cells regulate their intracellular milieu and contribute to acid-base regulation and sodium, chloride, and potassium homeostasis, thus highlighting their potential role as targets for the treatment of hypertension. Their novel regulation by paracrine signals in the collecting duct is also discussed. Finally, this article addresses their role as part of the innate immune system of the kidney tubule. PMID:25632105

  18. Regulation of cell-cell fusion by nanotopography

    NASA Astrophysics Data System (ADS)

    Padmanabhan, Jagannath; Augelli, Michael J.; Cheung, Bettina; Kinser, Emily R.; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J.; Li, Rui; Schwarz, Udo D.; Schroers, Jan; Kyriakides, Themis R.

    2016-09-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions.

  19. Regulation of cell-cell fusion by nanotopography

    PubMed Central

    Padmanabhan, Jagannath; Augelli, Michael J.; Cheung, Bettina; Kinser, Emily R.; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J.; Li, Rui; Schwarz, Udo D.; Schroers, Jan; Kyriakides, Themis R.

    2016-01-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions. PMID:27615159

  20. Regulation of cell-cell fusion by nanotopography.

    PubMed

    Padmanabhan, Jagannath; Augelli, Michael J; Cheung, Bettina; Kinser, Emily R; Cleary, Barnett; Kumar, Priyanka; Wang, Renhao; Sawyer, Andrew J; Li, Rui; Schwarz, Udo D; Schroers, Jan; Kyriakides, Themis R

    2016-01-01

    Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions. PMID:27615159

  1. A subset of gamma delta T-cell receptor-positive cells produce T-helper type-2 cytokines and regulate mouse skin graft rejection following portal venous pretransplant preimmunization.

    PubMed Central

    Gorczynski, R M; Chen, Z; Hoang, Y; Rossi-Bergman, B

    1996-01-01

    C3H/HeJ mice received B10.BR skin grafts following portal or lateral tail vein infusion of irradiated B10.BR spleen cells. Thereafter mice were injected with anti-alpha beta or anti-gamma delta T-cell receptor (TCR) monoclonal antibody (mAb). Anti-gamma delta TCR mAb abolished the increased graft survival afforded by portal venous (p.v.) immunization, and reversed the bias towards expression of mRNA for type-2 cytokines [interleukin-4 (IL-4), IL-10] seen in lymphoid tissue of p.v.-immunized mice. When gamma delta TCR+ and alpha beta TCR+ cells were isolated from the intestinal epithelial compartment (IEL), liver or Peyer's Patch (PP) of p.v.-immunized mice, the gamma delta TCR+ cells were found to be enriched in cells producing type-2 cytokines on rechallenge with irradiated B10.BR cells in vitro. gamma delta TCR+ cells from p.v.-immunized mice were further expanded in vitro with anti-CD3 and cytokines (combined IL-2 and IL-4). Following expansion these cells were capable of adoptively transferring increased B10.BR skin graft survival to naive mice, and continued to show a bias in type-2 cytokine synthesis after allostimulation in vitro. When gamma delta TCR chain expression was assessed in cells taken from p.v.-immunized mice, or in cells expanded in culture, our data suggest that p.v. immunization leads to oligoclonal, not polyclonal, expansion of those gamma delta TCR+ cells involved in inhibition of graft rejection. Images Figure 2 Figure 3 Figure 4 PMID:8778022

  2. Glial Cell Regulation of Rhythmic Behavior

    PubMed Central

    Jackson, F. Rob; Ng, Fanny S.; Sengupta, Sukanya; You, Samantha; Huang, Yanmei

    2015-01-01

    Brain glial cells, in particular astrocytes and microglia, secrete signaling molecules that regulate glia–glia or glia–neuron communication and synaptic activity. While much is known about roles of glial cells in nervous system development, we are only beginning to understand the physiological functions of such cells in the adult brain. Studies in vertebrate and invertebrate models, in particular mice and Drosophila, have revealed roles of glia–neuron communication in the modulation of complex behavior. This chapter emphasizes recent evidence from studies of rodents and Drosophila that highlight the importance of glial cells and similarities or differences in the neural circuits regulating circadian rhythms and sleep in the two models. The chapter discusses cellular, molecular, and genetic approaches that have been useful in these models for understanding how glia–neuron communication contributes to the regulation of rhythmic behavior. PMID:25707272

  3. Regulation of cell proliferation by G proteins.

    PubMed

    Dhanasekaran, N; Tsim, S T; Dermott, J M; Onesime, D

    1998-09-17

    G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the alpha-subunits as well as the betagamma-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Of the 17 alpha-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in fibroblast cells. Activating mutations in G alpha(s), G alpha(i)2, and G alpha12 have been correlated with different types of tumors. In addition, the ability of the betagamma-subunits to activate mitogenic pathways in different cell-types has been defined. The present review briefly summarizes the diverse and novel signaling pathways regulated by the alpha- as well as the betagamma-subunits of G proteins in regulating cell proliferation. PMID:9779986

  4. Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

    NASA Astrophysics Data System (ADS)

    Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

    1987-10-01

    The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

  5. Roles of sucrose in guard cell regulation.

    PubMed

    Daloso, Danilo M; Dos Anjos, Leticia; Fernie, Alisdair R

    2016-08-01

    The control of stomatal aperture involves reversible changes in the concentration of osmolytes in guard cells. Sucrose has long been proposed to have an osmolytic role in guard cells. However, direct evidence for such a role is lacking. Furthermore, recent evidence suggests that sucrose may perform additional roles in guard cells. Here, we provide an update covering the multiple roles of sucrose in guard cell regulation, highlighting the knowledge accumulated regarding spatiotemporal differences in the synthesis, accumulation, and degradation of sucrose as well as reviewing the role of sucrose as a metabolic connector between mesophyll and guard cells. Analysis of transcriptomic data from previous studies reveals that several genes encoding sucrose and hexose transporters and genes involved in gluconeogenesis, sucrose and trehalose metabolism are highly expressed in guard cells compared with mesophyll cells. Interestingly, this analysis also showed that guard cells have considerably higher expression of C4 -marker genes than mesophyll cells. We discuss the possible roles of these genes in guard cell function and the role of sucrose in stomatal opening and closure. Finally, we provide a perspective for future experiments which are required to fill gaps in our understanding of both guard cell metabolism and stomatal regulation.

  6. The tyrosine phosphatase PTPN22 discriminates weak self peptides from strong agonist TCR signals.

    PubMed

    Salmond, Robert J; Brownlie, Rebecca J; Morrison, Vicky L; Zamoyska, Rose

    2014-09-01

    T cells must be tolerant of self antigens to avoid autoimmunity but responsive to foreign antigens to provide protection against infection. We found that in both naive T cells and effector T cells, the tyrosine phosphatase PTPN22 limited signaling via the T cell antigen receptor (TCR) by weak agonists and self antigens while not impeding responses to strong agonist antigens. T cells lacking PTPN22 showed enhanced formation of conjugates with antigen-presenting cells pulsed with weak peptides, which led to activation of the T cells and their production of inflammatory cytokines. This effect was exacerbated under conditions of lymphopenia, with the formation of potent memory T cells in the absence of PTPN22. Our data address how loss-of-function PTPN22 alleles can lead to the population expansion of effector and/or memory T cells and a predisposition to human autoimmunity. PMID:25108421

  7. Regulation of pancreatic beta-cell mass.

    PubMed

    Bouwens, Luc; Rooman, Ilse

    2005-10-01

    Beta-cell mass regulation represents a critical issue for understanding diabetes, a disease characterized by a near-absolute (type 1) or relative (type 2) deficiency in the number of pancreatic beta cells. The number of islet beta cells present at birth is mainly generated by the proliferation and differentiation of pancreatic progenitor cells, a process called neogenesis. Shortly after birth, beta-cell neogenesis stops and a small proportion of cycling beta cells can still expand the cell number to compensate for increased insulin demands, albeit at a slow rate. The low capacity for self-replication in the adult is too limited to result in a significant regeneration following extensive tissue injury. Likewise, chronically increased metabolic demands can lead to beta-cell failure to compensate. Neogenesis from progenitor cells inside or outside islets represents a more potent mechanism leading to robust expansion of the beta-cell mass, but it may require external stimuli. For therapeutic purposes, advantage could be taken from the surprising differentiation plasticity of adult pancreatic cells and possibly also from stem cells. Recent studies have demonstrated that it is feasible to regenerate and expand the beta-cell mass by the application of hormones and growth factors like glucagon-like peptide-1, gastrin, epidermal growth factor, and others. Treatment with these external stimuli can restore a functional beta-cell mass in diabetic animals, but further studies are required before it can be applied to humans. PMID:16183912

  8. Tip cells: master regulators of tubulogenesis?

    PubMed

    Weavers, Helen; Skaer, Helen

    2014-07-01

    The normal development of an organ depends on the coordinated regulation of multiple cell activities. Focusing on tubulogenesis, we review the role of specialised cells or groups of cells that are selected from within tissue primordia and differentiate at the outgrowing tips or leading edge of developing tubules. Tip or leading cells develop distinctive patterns of gene expression that enable them to act both as sensors and transmitters of intercellular signalling. This enables them to explore the environment, respond to both tissue intrinsic signals and extrinsic cues from surrounding tissues and to regulate the behaviour of their neighbours, including the setting of cell fate, patterning cell division, inducing polarity and promoting cell movement and cell rearrangements by neighbour exchange. Tip cells are also able to transmit mechanical tension to promote tissue remodelling and, by interacting with the extracellular matrix, they can dictate migratory pathways and organ shape. Where separate tubular structures fuse to form networks, as in the airways of insects or the vascular system of vertebrates, specialised fusion tip cells act to interconnect disparate elements of the developing network. Finally, we consider their importance in the maturation of mature physiological function and in the development of disease.

  9. Identification of PGAM5 as a Mammalian Protein Histidine Phosphatase that Plays a Central Role to Negatively Regulate CD4(+) T Cells.

    PubMed

    Panda, Saswati; Srivastava, Shekhar; Li, Zhai; Vaeth, Martin; Fuhs, Stephen R; Hunter, Tony; Skolnik, Edward Y

    2016-08-01

    Whereas phosphorylation of serine, threonine, and tyrosine is exceedingly well characterized, the role of histidine phosphorylation in mammalian signaling is largely unexplored. Here we show that phosphoglycerate mutase family 5 (PGAM5) functions as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic histidine on nucleoside diphosphate kinase B (NDPK-B). By dephosphorylating NDPK-B, PGAM5 negatively regulates CD4(+) T cells by inhibiting NDPK-B-mediated histidine phosphorylation and activation of the K(+) channel KCa3.1, which is required for TCR-stimulated Ca(2+) influx and cytokine production. Using recently developed monoclonal antibodies that specifically recognize phosphorylation of nitrogens at the N1 (1-pHis) or N3 (3-pHis) positions of the imidazole ring, we detect for the first time phosphoisoform-specific regulation of histidine-phosphorylated proteins in vivo, and we link these modifications to TCR signaling. These results represent an important step forward in studying the role of histidine phosphorylation in mammalian biology and disease. PMID:27453048

  10. Multiplex matrix network analysis of protein complexes in the human TCR signalosome.

    PubMed

    Smith, Stephen E P; Neier, Steven C; Reed, Brendan K; Davis, Tessa R; Sinnwell, Jason P; Eckel-Passow, Jeanette E; Sciallis, Gabriel F; Wieland, Carilyn N; Torgerson, Rochelle R; Gil, Diana; Neuhauser, Claudia; Schrum, Adam G

    2016-08-02

    Multiprotein complexes transduce cellular signals through extensive interaction networks, but the ability to analyze these networks in cells from small clinical biopsies is limited. To address this, we applied an adaptable multiplex matrix system to physiologically relevant signaling protein complexes isolated from a cell line or from human patient samples. Focusing on the proximal T cell receptor (TCR) signalosome, we assessed 210 pairs of PiSCES (proteins in shared complexes detected by exposed surface epitopes). Upon stimulation of Jurkat cells with superantigen-loaded antigen-presenting cells, this system produced high-dimensional data that enabled visualization of network activity. A comprehensive analysis platform generated PiSCES biosignatures by applying unsupervised hierarchical clustering, principal component analysis, an adaptive nonparametric with empirical cutoff analysis, and weighted correlation network analysis. We generated PiSCES biosignatures from 4-mm skin punch biopsies from control patients or patients with the autoimmune skin disease alopecia areata. This analysis distinguished disease patients from the controls, detected enhanced basal TCR signaling in the autoimmune patients, and identified a potential signaling network signature that may be indicative of disease. Thus, generation of PiSCES biosignatures represents an approach that can provide information about the activity of protein signaling networks in samples including low-abundance primary cells from clinical biopsies.

  11. Epigenetic Mechanisms Regulating Mesenchymal Stem Cell Differentiation

    PubMed Central

    Pérez-Campo, Flor M.; Riancho, José A.

    2015-01-01

    Human Mesenchymal Stem Cells (hMSCs) have emerged in the last few years as one of the most promising therapeutic cell sources and, in particular, as an important tool for regenerative medicine of skeletal tissues. Although they present a more restricted potency than Embryonic Stem (ES) cells, the use of hMCS in regenerative medicine avoids many of the drawbacks characteristic of ES cells or induced pluripotent stem cells. The challenge in using these cells lies into developing precise protocols for directing cellular differentiation to generate a specific cell lineage. In order to achieve this goal, it is of the upmost importance to be able to control de process of fate decision and lineage commitment. This process requires the coordinate regulation of different molecular layers at transcriptional, posttranscriptional and translational levels. At the transcriptional level, switching on and off different sets of genes is achieved not only through transcriptional regulators, but also through their interplay with epigenetic modifiers. It is now well known that epigenetic changes take place in an orderly way through development and are critical in the determination of lineage-specific differentiation. More importantly, alteration of these epigenetic changes would, in many cases, lead to disease generation and even tumour formation. Therefore, it is crucial to elucidate how epigenetic factors, through their interplay with transcriptional regulators, control lineage commitment in hMSCs. PMID:27019612

  12. Recruitment of Slp-76 to the Membrane and Glycolipid-Enriched Membrane Microdomains Replaces the Requirement for Linker for Activation of T Cells in T Cell Receptor Signaling

    PubMed Central

    Boerth, Nancy J.; Sadler, Jeffrey J.; Bauer, Daniel E.; Clements, James L.; Gheith, Shereen M.; Koretzky, Gary A.

    2000-01-01

    Two hematopoietic-specific adapters, src homology 2 domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224–244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cγ1 phosphorylation, extracellular signal–regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224–244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane. PMID:11015445

  13. TCR transgenic mice reveal the impact of type 1 diabetes loci on early and late disease checkpoints.

    PubMed

    Hillhouse, Erin E; Liston, Adrian; Collin, Roxanne; Desautels, Eric; Goodnow, Christopher C; Lesage, Sylvie

    2016-08-01

    Linkage analysis studies for autoimmune diabetes have revealed multiple non-major histocompatibility complex (MHC) chromosomal regions linked to disease susceptibility. To date, more than 20 insulin-dependent diabetes (Idd) loci linked to diabetes susceptibility have been identified in NOD mice and validated via congenic breeding. Importantly, evidence suggests that Idd loci may regulate at least two pathological steps during autoimmune diabetes development, namely the onset of insulitis and the transition from insulitis to overt diabetes. Here we assess the role of various non-MHC Idd diabetes-resistance loci, which have been validated in the non-transgenic setting, on autoimmune diabetes progression in the transgenic setting. Specifically, we generated multiple Idd congenic strains in the 3A9-TCR:insHEL NOD.H2(k) transgenic model and monitored their diabetes incidence. We show that 3A9-TCR:insHEL NOD.H2(k) mice congenic for Idd3 or Idd5 display a reduction in diabetes development, whereas mice congenic for Idd9 or Idd13 exhibit an increase, in comparison with 3A9-TCR:insHEL NOD.H2(k) mice. These results suggest that the presence of the 3A9-TCR and hen egg lysosyme transgenes can offset the regulatory function of certain diabetes-resistance genetic variants contained within the Idd loci, including Idd9 and Idd13. We propose the antigen-specific 3A9-TCR:insHEL transgenic model as a useful tool for the study of the genetics of autoimmune diabetes development. PMID:27046082

  14. Cell Polarity As A Regulator of Cancer Cell Behavior Plasticity

    PubMed Central

    Muthuswamy, Senthil K; Xue, Bin

    2013-01-01

    Cell polarization is an evolutionarily conserved process that facilitates asymmetric distribution of organelles and proteins, is an evolutionarily conserved property that is modified dynamically during physiological processes such as cell division, migration, and morphogenesis. The plasticity with which cells change their behavior and phenotype in response to cell intrinsic and extrinsic cues is an essential feature of normal physiology. In disease states such as cancer, cells lose their ability to behave normally in response to physiological cues. A molecular understanding of mechanisms that alter the behavior of cancer cells is limited. Cell polarity proteins are an recognized class of molecules that can receive and interpret both intrinsic and extrinsic signals to modulate cell behavior. In this review, we discuss how cell polarity proteins regulate a diverse array of biological processes and how they can contribute to alterations in the behavior of cancer cells. PMID:22881459

  15. Stem cell technologies: regulation, patents and problems.

    PubMed

    Then, Shih-Ning

    2004-11-01

    Human embryonic stem cell research promises to deliver in the future a whole range of therapeutic treatments, but currently governments in different jurisdictions must try to regulate this burgeoning area. Part of the problem has been, and continues to be, polarised community opinion on the use of human embryonic stem cells for research. This article compares the approaches of the Australian, United Kingdom and United States governments in regulating human embryonic stem cell research. To date, these governments have approached the issue through implementing legislation or policy to control research. Similarly, the three jurisdictions have viewed the patentability of human embryonic stem cell technologies in their own ways with different policies being adopted by the three patent offices. This article examines these different approaches and discusses the inevitable concerns that have been raised due to the lack of a universal approach in relation to the regulation of research; the patenting of stem cell technologies; and the effects patents granted are having on further human embryonic stem cell research.

  16. Regulation of cell division in higher plants

    SciTech Connect

    Jacobs, T.W.

    1992-01-01

    Cell division is arguably the most fundamental of all developmental processes. In higher plants, mitotic activity is largely confined to foci of patterned cell divisions called meristems. From these perpetually embryonic tissues arise the plant's essential organs of light capture, support, protection and reproduction. Once an adequate understanding of plant cell mitotic regulation is attained, unprecedented opportunities will ensue for analyzing and genetically controlling diverse aspects of development, including plant architecture, leaf shape, plant height, and root depth. The mitotic cycle in a variety of model eukaryotic systems in under the control of a regulatory network of striking evolutionary conservation. Homologues of the yeast cdc2 gene, its catalytic product, p34, and the cyclin regulatory subunits of the MPF complex have emerged as ubiquitous mitotic regulators. We have cloned cdc2-like and cyclin genes from pea. As in other eukaryotic model systems, p34 of Pisum sativum is a subunit of a high molecular weight complex which binds the fission yeast p13 protein and displays histone H1 kinase activity in vitro. Our primary objective in this study is to gain baseline information about the regulation of this higher plant cell division control complex in non-dividing, differentiated cells as well as in synchronous and asynchronous mitotic cells. We are investigating cdc2 and cyclin expression at the levels of protein abundance, protein phosphorylation and quaternary associations.

  17. Regulation of T cell receptor expression in immature CD4+CD8+ thymocytes by p56lck tyrosine kinase: basis for differential signaling by CD4 and CD8 in immature thymocytes expressing both coreceptor molecules

    PubMed Central

    1993-01-01

    Signals transduced through the T cell antigen receptor (TCR) are modulated by the src family tyrosine kinase p56lck (lck), which associates in mature T cells with the coreceptor molecules CD4 and CD8. Here we describe a novel function of lck in immature CD4+CD8+ thymocytes, that of regulating TCR expression. Activation of lck in immature CD4+CD8+ thymocytes by intrathymic engagement of CD4 maintains low TCR expression by causing most TCR components to be retained and degraded within the endoplasmic reticulum. Importantly, activation of lck in immature CD4+CD8+ thymocytes results from engagement of surface CD4 molecules, but not surface CD8 molecules, despite the nearly fourfold greater surface expression of CD8 than CD4. The competence of CD4 to activate lck in CD4+CD8+ thymocytes relates to the fact that a relatively large fraction of surface CD4 molecules (25-50%) are associated with intracellular lck molecules, whereas only 2% of surface CD8 molecules are associated with lck. The amount of lck associated with CD4 in CD4+CD8+ thymocytes is diminished by chronic CD4 engagement in the thymus, as activated lck molecules subsequently dissociate from CD4. Indeed, the amount of lck associated with CD4 in CD4+CD8+ thymocytes is markedly increased in major histocompatibility complex (MHC) class II- mice that lack the intrathymic ligand for CD4 and in which surface CD4 molecules are consequently not engaged. Thus, the present study demonstrates that (a) activation of lck in CD4+CD8+ thymocytes regulates distribution and expression of TCR components; (b) unlike CD4 molecules, CD8 molecules on CD4+CD8+ thymocytes cannot efficiently activate lck despite their significantly greater surface expression; and (c) the amount of lck associated with CD4 in the CD4+CD8+ thymocytes is inversely related to the extent of CD4 engagement by MHC class II molecules in the thymus. PMID:8228817

  18. Matrix regulators in neural stem cells functions

    PubMed Central

    Wade, Anna; McKinney, Andrew; Phillips, Joanna J.

    2014-01-01

    Background Neural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism. Scope of Review In this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior. Major Conclusions The NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC niche provides temporal and spatial regulation of NSPC behaviors. General Significance The factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. PMID:24447567

  19. Redox regulation in cancer stem cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reactive oxygen species (ROS) and ROS-dependent (redox regulation) signaling pathways and transcriptional activities are thought to be critical in stem cell self-renewal and differentiation during growth and organogenesis. Aberrant ROS burst and dysregulation of those ROS-dependent cellular processe...

  20. LAPTM5 promotes lysosomal degradation of intracellular CD3ζ but not of cell surface CD3ζ.

    PubMed

    Kawai, Yohei; Ouchida, Rika; Yamasaki, Sho; Dragone, Leonard; Tsubata, Takeshi; Wang, Ji-Yang

    2014-07-01

    The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.

  1. Cell cycle regulation of hematopoietic stem or progenitor cells.

    PubMed

    Hao, Sha; Chen, Chen; Cheng, Tao

    2016-05-01

    The highly regulated process of blood production is achieved through the hierarchical organization of hematopoietic stem cell (HSC) subsets and their progenies, which differ in self-renewal and differentiation potential. Genetic studies in mice have demonstrated that cell cycle is tightly controlled by the complex interplay between extrinsic cues and intrinsic regulatory pathways involved in HSC self-renewal and differentiation. Deregulation of these cellular programs may transform HSCs or hematopoietic progenitor cells (HPCs) into disease-initiating stem cells, and can result in hematopoietic malignancies such as leukemia. While previous studies have shown roles for some cell cycle regulators and related signaling pathways in HSCs and HPCs, a more complete picture regarding the molecular mechanisms underlying cell cycle regulation in HSCs or HPCs is lacking. Based on accumulated studies in this field, the present review introduces the basic components of the cell cycle machinery and discusses their major cellular networks that regulate the dormancy and cell cycle progression of HSCs. Knowledge on this topic would help researchers and clinicians to better understand the pathogenesis of relevant blood disorders and to develop new strategies for therapeutic manipulation of HSCs.

  2. Triiodothyronine regulates cell growth and survival in renal cell cancer.

    PubMed

    Czarnecka, Anna M; Matak, Damian; Szymanski, Lukasz; Czarnecka, Karolina H; Lewicki, Slawomir; Zdanowski, Robert; Brzezianska-Lasota, Ewa; Szczylik, Cezary

    2016-10-01

    Triiodothyronine plays an important role in the regulation of kidney cell growth, differentiation and metabolism. Patients with renal cell cancer who develop hypothyreosis during tyrosine kinase inhibitor (TKI) treatment have statistically longer survival. In this study, we developed cell based model of triiodothyronine (T3) analysis in RCC and we show the different effects of T3 on renal cell cancer (RCC) cell growth response and expression of the thyroid hormone receptor in human renal cell cancer cell lines from primary and metastatic tumors along with human kidney cancer stem cells. Wild-type thyroid hormone receptor is ubiquitously expressed in human renal cancer cell lines, but normalized against healthy renal proximal tube cell expression its level is upregulated in Caki-2, RCC6, SKRC-42, SKRC-45 cell lines. On the contrary the mRNA level in the 769-P, ACHN, HKCSC, and HEK293 cells is significantly decreased. The TRβ protein was abundant in the cytoplasm of the 786-O, Caki-2, RCC6, and SKRC-45 cells and in the nucleus of SKRC-42, ACHN, 769-P and cancer stem cells. T3 has promoting effect on the cell proliferation of HKCSC, Caki-2, ASE, ACHN, SK-RC-42, SMKT-R2, Caki-1, 786-0, and SK-RC-45 cells. Tyrosine kinase inhibitor, sunitinib, directly inhibits proliferation of RCC cells, while thyroid hormone receptor antagonist 1-850 (CAS 251310‑57-3) has less significant inhibitory impact. T3 stimulation does not abrogate inhibitory effect of sunitinib. Renal cancer tumor cells hypostimulated with T3 may be more responsive to tyrosine kinase inhibition. Moreover, some tumors may be considered as T3-independent and present aggressive phenotype with thyroid hormone receptor activated independently from the ligand. On the contrary proliferation induced by deregulated VHL and or c-Met pathways may transgress normal T3 mediated regulation of the cell cycle. PMID:27632932

  3. "Natural Regulators": NK Cells as Modulators of T Cell Immunity.

    PubMed

    Schuster, Iona S; Coudert, Jerome D; Andoniou, Christopher E; Degli-Esposti, Mariapia A

    2016-01-01

    Natural killer (NK) cells are known as frontline responders capable of rapidly mediating a response upon encountering transformed or infected cells. Recent findings indicate that NK cells, in addition to acting as innate effectors, can also regulate adaptive immune responses. Here, we review recent studies on the immunoregulatory function of NK cells with a specific focus on their ability to affect the generation of early, as well as long-term antiviral T cell responses, and their role in modulating immune pathology and disease. In addition, we summarize the current knowledge of the factors governing regulatory NK cell responses and discuss origin, tissue specificity, and open questions about the classification of regulatory NK cells as classical NK cells versus group 1 innate lymphoid cells. PMID:27379097

  4. Regulation of mammalian brain cell volume.

    PubMed

    Law, R O

    1994-02-01

    Maintenance of brain cell volume is of crucial importance for normal central nervous system (CNS) function. This review considers volume regulation primarily in response to disturbances of body fluid osmolality. Brain cells counter the tendency to swell or shrink by appropriate adjustment of their internal osmotic potential. This is achieved by loss or uptake of inorganic ions and low molecular weight organic solutes (osmolytes). The latter comprise mainly amino acids, myoinositol, choline, and methylamines. Taurine may be of particular importance in volume control, especially in young animals. Brain cell volume regulation, however, is only one contributory factor to maintenance of constant brain volume (water content), and operates in parallel with important alterations in bulk fluid and electrolyte movement across the blood-brain barrier and between the interstitium and cerebrospinal fluid, which themselves moderate the requirement for transient alteration in cell volume during acute osmotic imbalance. Although altered cerebral content of inorganic ions and osmolytes are usually regarded as responses, respectively, to acute and chronic osmotic disturbances, osmolytes (especially taurine) may also participate in short-term cell volume regulation. PMID:8301256

  5. Autophagic regulation of smooth muscle cell biology

    PubMed Central

    Salabei, Joshua K.; Hill, Bradford G.

    2014-01-01

    Autophagy regulates the metabolism, survival, and function of numerous cell types, including those comprising the cardiovascular system. In the vasculature, changes in autophagy have been documented in atherosclerotic and restenotic lesions and in hypertensive vessels. The biology of vascular smooth muscle cells appears particularly sensitive to changes in the autophagic program. Recent evidence indicates that stimuli or stressors evoked during the course of vascular disease can regulate autophagic activity, resulting in modulation of VSMC phenotype and viability. In particular, certain growth factors and cytokines, oxygen tension, and pharmacological drugs have been shown to trigger autophagy in smooth muscle cells. Importantly, each of these stimuli has a redox component, typically associated with changes in the abundance of reactive oxygen, nitrogen, or lipid species. Collective findings support the hypothesis that autophagy plays a critical role in vascular remodeling by regulating smooth muscle cell phenotype transitions and by influencing the cellular response to stress. In this graphical review, we summarize current knowledge on the role of autophagy in the biology of the smooth muscle cell in (patho)physiology. PMID:25544597

  6. Biomechanical regulation of mesenchymal cell function

    PubMed Central

    Tschumperlin, Daniel J.; Liu, Fei; Tager, Andrew M.

    2016-01-01

    Purpose of review Cells of mesenchymal origin are strongly influenced by their biomechanical environment. They also help to shape tissue architecture and reciprocally influence tissue mechanical environments through their capacity to deposit, remodel, and resorb extracellular matrix and to promote tissue vascularization. Although mechanical regulation of cell function and tissue remodeling has long been appreciated in other contexts, the purpose of this review is to highlight the increasing appreciation of its importance in fibrosis and hypertrophic scarring. Recent findings Experiments in both animal and cellular model systems have demonstrated pivotal roles for the biomechanical environment in regulating myofibroblast differentiation and contraction, endothelial barrier function and angiogenesis, and mesenchymal stem cell fate decisions. Through these studies, a better understanding of the molecular mechanisms transducing the biomechanical environment is emerging, with prominent and interacting roles recently identified for key network components including transforming growth factor-β/SMAD, focal adhesion kinase, MRTFs, Wnt/β-catenin and YAP/TAZ signaling pathways. Summary Progress in understanding biomechanical regulation of mesenchymal cell function is leading to novel approaches for improving clinical outcomes in fibrotic diseases and wound healing. These approaches include interventions aimed at modifying the tissue biomechanical environment, and efforts to target mesenchymal cell activation by, and reciprocal interactions with, the mechanical environment. PMID:23114589

  7. The endocytosis and signaling of the γδ T cell coreceptor WC1 are regulated by a dileucine motif.

    PubMed

    Hsu, Haoting; Baldwin, Cynthia L; Telfer, Janice C

    2015-03-01

    WC1 proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WC1 cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens.

  8. Mitochondrial Regulation of Cell Cycle and Proliferation

    PubMed Central

    Antico Arciuch, Valeria Gabriela; Elguero, María Eugenia; Poderoso, Juan José

    2012-01-01

    Abstract Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly, defective organelles contribute to cell transformation and cancer, diabetes, and neurodegenerative diseases. Most cell and transcriptional effects of mitochondria depend on the modulation of respiratory rate and on the production of hydrogen peroxide released into the cytosol. The mitochondrial oxidative rate has to remain depressed for cell proliferation; even in the presence of O2, energy is preferentially obtained from increased glycolysis (Warburg effect). In response to stress signals, traffic of pro- and antiapoptotic mitochondrial proteins in the intermembrane space (B-cell lymphoma-extra large, Bcl-2-associated death promoter, Bcl-2 associated X-protein and cytochrome c) is modulated by the redox condition determined by mitochondrial O2 utilization and mitochondrial nitric oxide metabolism. In this article, we highlight the traffic of the different canonical signaling pathways to mitochondria and the contributions of organelles to redox regulation of kinases. Finally, we analyze the dynamics of the mitochondrial population in cell cycle and apoptosis. Antioxid. Redox Signal. 16, 1150–1180. PMID:21967640

  9. Epigenetic Regulation of Hematopoietic Stem Cells.

    PubMed

    Sharma, Shilpa; Gurudutta, Gangenahalli

    2016-05-30

    Hematopoietic stem cells are endowed with a distinct potential to bolster self-renewal and to generate progeny that differentiate into mature cells of myeloid and lymphoid lineages. Both hematopoietic stem cells and mature cells have the same genome, but their gene expression is controlled by an additional layer of epigenetics such as DNA methylation and post-translational histone modifications, enabling each cell-type to acquire various forms and functions. Until recently, several studies have largely focussed on the transcription factors andniche factors for the understanding of the molecular mechanisms by which hematopoietic cells replicate and differentiate. Several lines of emerging evidence suggest that epigenetic modifications eventually result in a defined chromatin structure and an "individual" gene expression pattern, which play an essential role in the regulation of hematopoietic stem cell self-renewal and differentiation. Distinct epigenetic marks decide which sets of genes may be expressed and which genes are kept silent. Epigenetic mechanisms are interdependent and ensure lifelong production of blood and bone marrow, thereby contributing to stem cell homeostasis. The epigenetic analysis of hematopoiesis raises the exciting possibility that chromatin structure is dynamic enough for regulated expression of genes. Though controlled chromatin accessibility plays an essential role in maintaining blood homeostasis; mutations in chromatin impacts on the regulation of genes critical to the development of leukemia. In this review, we explored the contribution of epigenetic machinery which has implications for the ramification of molecular details of hematopoietic self-renewal for normal development and underlying events that potentially co-operate to induce leukemia.

  10. Extrinsic regulation of satellite cell specification

    PubMed Central

    2010-01-01

    Cellular commitment during vertebrate embryogenesis is controlled by an interplay of intrinsic regulators and morphogenetic signals. These mechanisms recruit a subset of cells in the developing organism to become the ancestors of skeletal muscle. Signals that control progression through the myogenic lineage converge on a battery of hierarchically organized transcription factors which modulate the cells to either remain in a primitive state or allow their commitment and differentiation into skeletal muscle fibers. A small population of cells will retain a largely unspecified state throughout development. Such stem cells, in conjunction with more committed myogenic progenitors, form a heterogeneous population that colonizes adult skeletal muscle as satellite cells. The satellite cell pool is responsible for the remarkable regenerative capacity of skeletal muscle. Similar to their counterparts during embryonic development, satellite cells are capable of self-renewal and can give rise to myogenic progeny. Impaired satellite cell homeostasis has been associated with numerous muscular disorders. Due to intense research efforts in the past two decades, the complex biology of muscle stem cells has now revealed some of its secrets and new avenues for the development of therapeutic molecules have emerged. In the present review we focus on the extrinsic mechanisms that control self-renewal, specification and differentiation of satellite cells and their significance for the development of biologic drugs. PMID:20804582

  11. Autophagy in immune cell regulation and dysregulation.

    PubMed

    Chaturvedi, Akanksha; Pierce, Susan K

    2009-09-01

    Autophagy is an ancient pathway required for cell and tissue homeostasis and differentiation. Initially thought to be a process leading to cell death, autophagy is currently viewed as a beneficial catabolic process that promotes cell survival under starvation conditions by sequestering components of the cytoplasm, including misfolded proteins, protein aggregates, and damaged organelles, and targeting them for lysosome-mediated degradation. In this way, autophagy plays a role in maintaining a balance between degradation and recycling of cellular material. The importance of autophagy is underscored by the fact that malfunctioning of this pathway results in neurodegeneration, cancer, susceptibility to microbial infection, and premature aging. Autophagy occurs in almost all cell types, including immune cells. Recent advances in the field suggest that autophagy plays a central role in regulating the immune system at multiple levels. In this review, we focus on recent developments in the area of autophagy-mediated modulation of immune responses. PMID:19671376

  12. Cell cycle checkpoint regulators reach a zillion

    PubMed Central

    Yasutis, Kimberly M.; Kozminski, Keith G.

    2013-01-01

    Entry into mitosis is regulated by a checkpoint at the boundary between the G2 and M phases of the cell cycle (G2/M). In many organisms, this checkpoint surveys DNA damage and cell size and is controlled by both the activation of mitotic cyclin-dependent kinases (Cdks) and the inhibition of an opposing phosphatase, protein phosphatase 2A (PP2A). Misregulation of mitotic entry can often lead to oncogenesis or cell death. Recent research has focused on discovering the signaling pathways that feed into the core checkpoint control mechanisms dependent on Cdk and PP2A. Herein, we review the conserved mechanisms of the G2/M transition, including recently discovered upstream signaling pathways that link cell growth and DNA replication to cell cycle progression. Critical consideration of the human, frog and yeast models of mitotic entry frame unresolved and emerging questions in this field, providing a prediction of signaling molecules and pathways yet to be discovered. PMID:23598718

  13. Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion

    PubMed Central

    Álvarez-González, Begoña; Meili, Ruedi; Firtel, Richard; Bastounis, Effie; del Álamo, Juan C.; Lasheras, Juan C.

    2014-01-01

    Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during

  14. Regulated Hyaluronan Synthesis by Vascular Cells

    PubMed Central

    Viola, Manuela; Karousou, Evgenia; D'Angelo, Maria Luisa; Caon, Ilaria; De Luca, Giancarlo; Passi, Alberto; Vigetti, Davide

    2015-01-01

    Cellular microenvironment plays a critical role in several pathologies including atherosclerosis. Hyaluronan (HA) content often reflects the progression of this disease in promoting vessel thickening and cell migration. HA synthesis is regulated by several factors, including the phosphorylation of HA synthase 2 (HAS2) and other covalent modifications including ubiquitination and O-GlcNAcylation. Substrate availability is important in HA synthesis control. Specific drugs reducing the UDP precursors are able to reduce HA synthesis whereas the hexosamine biosynthetic pathway (HBP) increases the concentration of HA precursor UDP-N-acetylglucosamine (UDP-GlcNAc) leading to an increase of HA synthesis. The flux through the HBP in the regulation of HA biosynthesis in human aortic vascular smooth muscle cells (VSMCs) was reported as a critical aspect. In fact, inhibiting O-GlcNAcylation reduced HA production whereas increased O-GlcNAcylation augmented HA secretion. Additionally, O-GlcNAcylation regulates HAS2 gene expression resulting in accumulation of its mRNA after induction of O-GlcNAcylation with glucosamine treatments. The oxidized LDLs, the most common molecules related to atherosclerosis outcome and progression, are also able to induce a strong HA synthesis when they are in contact with vascular cells. In this review, we present recent described mechanisms involved in HA synthesis regulation and their role in atherosclerosis outcome and development. PMID:26448750

  15. MHC class I molecules with Superenhanced CD8 binding properties bypass the requirement for cognate TCR recognition and nonspecifically activate CTLs.

    PubMed

    Wooldridge, Linda; Clement, Mathew; Lissina, Anna; Edwards, Emily S J; Ladell, Kristin; Ekeruche, Julia; Hewitt, Rachel E; Laugel, Bruno; Gostick, Emma; Cole, David K; Debets, Reno; Berrevoets, Cor; Miles, John J; Burrows, Scott R; Price, David A; Sewell, Andrew K

    2010-04-01

    CD8(+) CTLs are essential for effective immune defense against intracellular microbes and neoplasia. CTLs recognize short peptide fragments presented in association with MHC class I (MHCI) molecules on the surface of infected or dysregulated cells. Ag recognition involves the binding of both TCR and CD8 coreceptor to a single ligand (peptide MHCI [pMHCI]). The TCR/pMHCI interaction confers Ag specificity, whereas the pMHCI/CD8 interaction mediates enhanced sensitivity to Ag. Striking biophysical differences exist between the TCR/pMHCI and pMHCI/CD8 interactions; indeed, the pMHCI/CD8 interaction can be >100-fold weaker than the cognate TCR/pMHCI interaction. In this study, we show that increasing the strength of the pMHCI/CD8 interaction by approximately 15-fold results in nonspecific, cognate Ag-independent pMHCI tetramer binding at the cell surface. Furthermore, pMHCI molecules with superenhanced affinity for CD8 activate CTLs in the absence of a specific TCR/pMHCI interaction to elicit a full range of effector functions, including cytokine/chemokine release, degranulation and proliferation. Thus, the low solution binding affinity of the pMHCI/CD8 interaction is essential for the maintenance of CTL Ag specificity.

  16. Crystallization and preliminary X-ray structural studies of a Melan-A pMHC–TCR complex

    SciTech Connect

    Yuan, Fang; Georgiou, Theonie; Hillon, Theresa; Gostick, Emma; Price, David A.; Sewell, Andrew K.; Moysey, Ruth; Gavarret, Jessie; Vuidepot, Annelise; Sami, Malkit; Bell, John I.; Gao, George F.; Rizkallah, Pierre J.; Jakobsen, Bent K.

    2007-09-01

    A preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR–pMHC complex crystals were cryocooled and used for data collection. Melanocytes are specialized pigmented cells that are found in all healthy skin tissue. In certain individuals, diseased melanocytes can form malignant tumours, melanomas, which cause the majority of skin-cancer-related deaths. The melanoma-associated antigenic peptides are presented on cell surfaces via the class I major histocompatibility complex (MHC). Among the melanoma-associated antigens, the melanoma self-antigen A/melanoma antigen recognized by T cells (Melan-A/MART-1) has attracted attention because of its wide expression in primary and metastatic melanomas. Here, a preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR–pMHC complex crystals were cryocooled and used for data collection. Diffraction data showed that these crystals belonged to space group P4{sub 1}/P4{sub 3}, with unit-cell parameters a = b = 120.4, c = 81.6 Å. A complete data set was collected to 3.1 Å and the structure is currently being analysed.

  17. The A2 gene of alcelaphine herpesvirus-1 is a transcriptional regulator affecting cytotoxicity in virus-infected T cells but is not required for malignant catarrhal fever induction in rabbits.

    PubMed

    Parameswaran, Nevi; Dewals, Benjamin G; Giles, Tom C; Deppmann, Christopher; Blythe, Martin; Vanderplasschen, Alain; Emes, Richard D; Haig, David

    2014-08-01

    Alcelaphine herpesvirus-1 (AlHV-1) causes malignant catarrhal fever (MCF). The A2 gene of AlHV-1 is a member of the bZIP transcription factor family. We wished to determine whether A2 is a virulence gene or not and whether it is involved in pathogenesis by interference with host transcription pathways. An A2 gene knockout (A2ΔAlHV-1) virus, revertant (A2revAlHV-1) virus, and wild-type virus (wtAlHV-1) were used to infect three groups of rabbits. A2ΔAlHV-1-infected rabbits succumbed to MCF, albeit with a delayed onset compared to the control groups, so A2 is not a critical virulence factor. Differential gene transcription analysis by RNAseq and qRT-PCR validation of a selection of these was performed in infected large granular lymphocyte (LGL) T cells obtained in culture from the MCF-affected animals. A2 was involved in the transcriptional regulation of immunological, cell cycle and apoptosis pathways. In particular, there was a bias towards γδ T cell receptor (TCR) expression and downregulation of αβ TCR. TCR signalling, apoptosis, cell cycle, IFN-γ and NFAT pathways were affected. Of particular interest was partial inhibition of the cytotoxicity-associated pathways involving perforin and the granzymes A and B in the A2ΔAlHV-1-infected LGLs compared to controls. In functional assays, A2ΔAlHV-1-infected LGLs were significantly less cytotoxic than wtAlHV-1- and A2revAlHV-1-infected LGLs using rabbit corneal epithelial cells (SIRC) as targets. This implies that A2 is involved in a pathway enhancing the expression of LGL cytotoxicity. This is important as virus-infected T cell cytotoxicity in vivo has been suggested as a potential mechanism of disease induction in MCF.

  18. The A2 gene of alcelaphine herpesvirus-1 is a transcriptional regulator affecting cytotoxicity in virus-infected T cells but is not required for malignant catarrhal fever induction in rabbits.

    PubMed

    Parameswaran, Nevi; Dewals, Benjamin G; Giles, Tom C; Deppmann, Christopher; Blythe, Martin; Vanderplasschen, Alain; Emes, Richard D; Haig, David

    2014-08-01

    Alcelaphine herpesvirus-1 (AlHV-1) causes malignant catarrhal fever (MCF). The A2 gene of AlHV-1 is a member of the bZIP transcription factor family. We wished to determine whether A2 is a virulence gene or not and whether it is involved in pathogenesis by interference with host transcription pathways. An A2 gene knockout (A2ΔAlHV-1) virus, revertant (A2revAlHV-1) virus, and wild-type virus (wtAlHV-1) were used to infect three groups of rabbits. A2ΔAlHV-1-infected rabbits succumbed to MCF, albeit with a delayed onset compared to the control groups, so A2 is not a critical virulence factor. Differential gene transcription analysis by RNAseq and qRT-PCR validation of a selection of these was performed in infected large granular lymphocyte (LGL) T cells obtained in culture from the MCF-affected animals. A2 was involved in the transcriptional regulation of immunological, cell cycle and apoptosis pathways. In particular, there was a bias towards γδ T cell receptor (TCR) expression and downregulation of αβ TCR. TCR signalling, apoptosis, cell cycle, IFN-γ and NFAT pathways were affected. Of particular interest was partial inhibition of the cytotoxicity-associated pathways involving perforin and the granzymes A and B in the A2ΔAlHV-1-infected LGLs compared to controls. In functional assays, A2ΔAlHV-1-infected LGLs were significantly less cytotoxic than wtAlHV-1- and A2revAlHV-1-infected LGLs using rabbit corneal epithelial cells (SIRC) as targets. This implies that A2 is involved in a pathway enhancing the expression of LGL cytotoxicity. This is important as virus-infected T cell cytotoxicity in vivo has been suggested as a potential mechanism of disease induction in MCF. PMID:24732177

  19. T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR-binding parameters.

    PubMed

    Brodovitch, Alexandre; Shenderov, Eugene; Cerundolo, Vincenzo; Bongrand, Pierre; Pierres, Anne; van der Merwe, Philip Anton

    2015-06-01

    T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self-peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide-MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN-γ production by T cells, and 1G4-pMHC dissociation rates measured in soluble phase or on surface-bound molecules, displayed six- to sevenfold variation among pMHCs. When T cells were dropped onto pMHC-coated surfaces, rapid spreading occurred after a 2-min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all-or-none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.

  20. Regulation of gene expression in autoimmune disease loci and the genetic basis of proliferation in CD4+ effector memory T cells.

    PubMed

    Hu, Xinli; Kim, Hyun; Raj, Towfique; Brennan, Patrick J; Trynka, Gosia; Teslovich, Nikola; Slowikowski, Kamil; Chen, Wei-Min; Onengut, Suna; Baecher-Allan, Clare; De Jager, Philip L; Rich, Stephen S; Stranger, Barbara E; Brenner, Michael B; Raychaudhuri, Soumya

    2014-06-01

    Genome-wide association studies (GWAS) and subsequent dense-genotyping of associated loci identified over a hundred single-nucleotide polymorphism (SNP) variants associated with the risk of rheumatoid arthritis (RA), type 1 diabetes (T1D), and celiac disease (CeD). Immunological and genetic studies suggest a role for CD4-positive effector memory T (CD+ TEM) cells in the pathogenesis of these diseases. To elucidate mechanisms of autoimmune disease alleles, we investigated molecular phenotypes in CD4+ effector memory T cells potentially affected by these variants. In a cohort of genotyped healthy individuals, we isolated high purity CD4+ TEM cells from peripheral blood, then assayed relative abundance, proliferation upon T cell receptor (TCR) stimulation, and the transcription of 215 genes within disease loci before and after stimulation. We identified 46 genes regulated by cis-acting expression quantitative trait loci (eQTL), the majority of which we detected in stimulated cells. Eleven of the 46 genes with eQTLs were previously undetected in peripheral blood mononuclear cells. Of 96 risk alleles of RA, T1D, and/or CeD in densely genotyped loci, eleven overlapped cis-eQTLs, of which five alleles completely explained the respective signals. A non-coding variant, rs389862A, increased proliferative response (p=4.75 × 10-8). In addition, baseline expression of seventeen genes in resting cells reliably predicted proliferative response after TCR stimulation. Strikingly, however, there was no evidence that risk alleles modulated CD4+ TEM abundance or proliferation. Our study underscores the power of examining molecular phenotypes in relevant cells and conditions for understanding pathogenic mechanisms of disease variants. PMID:24968232

  1. Nuclear myosin I regulates cell membrane tension.

    PubMed

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-08-02

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.

  2. Nuclear myosin I regulates cell membrane tension

    PubMed Central

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  3. Nuclear myosin I regulates cell membrane tension.

    PubMed

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel

    2016-01-01

    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  4. Ulk4 Regulates Neural Stem Cell Pool.

    PubMed

    Liu, Min; Guan, Zhenlong; Shen, Qin; Flinter, Frances; Domínguez, Laura; Ahn, Joo Wook; Collier, David A; O'Brien, Timothy; Shen, Sanbing

    2016-09-01

    The size of neural stem cell (NSC) pool at birth determines the starting point of adult neurogenesis. Aberrant neurogenesis is associated with major mental illness, in which ULK4 is proposed as a rare risk factor. Little is known about factors regulating the NSC pool, or function of the ULK4. Here, we showed that Ulk4(tm1a/tm1a) mice displayed a dramatically reduced NSC pool at birth. Ulk4 was expressed in a cell cycle-dependent manner and peaked in G2/M phases. Targeted disruption of the Ulk4 perturbed mid-neurogenesis and significantly reduced cerebral cortex in postnatal mice. Pathway analyses of dysregulated genes in Ulk4(tm1a/tm1a) mice revealed Ulk4 as a key regulator of cell cycle and NSC proliferation, partially through regulation of the Wnt signaling. In addition, we identified hemizygous deletion of ULK4 gene in 1.2/1,000 patients with pleiotropic symptoms including severe language delay and learning difficulties. ULK4, therefore, may significantly contribute to neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Stem Cells 2016;34:2318-2331.

  5. Ulk4 Regulates Neural Stem Cell Pool.

    PubMed

    Liu, Min; Guan, Zhenlong; Shen, Qin; Flinter, Frances; Domínguez, Laura; Ahn, Joo Wook; Collier, David A; O'Brien, Timothy; Shen, Sanbing

    2016-09-01

    The size of neural stem cell (NSC) pool at birth determines the starting point of adult neurogenesis. Aberrant neurogenesis is associated with major mental illness, in which ULK4 is proposed as a rare risk factor. Little is known about factors regulating the NSC pool, or function of the ULK4. Here, we showed that Ulk4(tm1a/tm1a) mice displayed a dramatically reduced NSC pool at birth. Ulk4 was expressed in a cell cycle-dependent manner and peaked in G2/M phases. Targeted disruption of the Ulk4 perturbed mid-neurogenesis and significantly reduced cerebral cortex in postnatal mice. Pathway analyses of dysregulated genes in Ulk4(tm1a/tm1a) mice revealed Ulk4 as a key regulator of cell cycle and NSC proliferation, partially through regulation of the Wnt signaling. In addition, we identified hemizygous deletion of ULK4 gene in 1.2/1,000 patients with pleiotropic symptoms including severe language delay and learning difficulties. ULK4, therefore, may significantly contribute to neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Stem Cells 2016;34:2318-2331. PMID:27300315

  6. Study of Redirected Autologous T Cells Engineered to Contain Anti-CD19 Attached to TCR and 4-1BB Signaling Domains in Patients With Chemotherapy Resistant or Refractory Acute Lymphoblastic Leukemia

    ClinicalTrials.gov

    2016-09-27

    Patients With B Cell ALL, Relapsed or Refractory, With no Available; Curative Treatment Options (Such as Autologous or Allogeneic Stem Cell; Transplantation) Who Have Limited Prognosis (> 12 Weeks Survival Expectancy); With Currently Available Therapies.

  7. Antigen-specific immunomodulation for type 1 diabetes by novel recombinant antibodies directed against diabetes-associates auto-reactive T cell epitope.

    PubMed

    Dahan, Rony; Gebe, John A; Preisinger, Anton; James, Eddie A; Tendler, Mark; Nepom, Gerald T; Reiter, Yoram

    2013-12-01

    The trimolecular complex composed of autoreactive T-cell receptor, MHC class II, and an autoantigenic peptide plays a central role in the activation of pathogenic Islet-specific CD4+ T cells in type 1 diabetes (T1D). We isolated and characterized novel antibodies against autoreactive T-cell epitopes associated with T1D. Our antibodies mimic the specificity of the T-cell receptor (TCR), while binding MHC class II/peptide complexes in an autoantigen peptide specific, MHC-restricted manner. The isolated TCR-like antibodies were directed against the minimal T-cell epitope GAD-555-567 in the context of the HLA-DR4-diabetic-associated molecule. A representative high-affinity TCR-like antibody clone (G3H8) enabled the detection of intra- and extra-cellular DR4/GAD-555-567 complexes in antigen presenting cells. I561M single mutation at the central position (P5) of the GAD-555-567 peptide abolished the binding of G3H8 to the DR4/GAD complex, demonstrating its high fine TCR-like specificity. The G3H8 TCR-like antibody significantly inhibited GAD-555-567 specific, DR4 restricted T-cell response in vitro and in vivo in HLA-DR4 transgenic mice. Our findings constitute a proof-of-concept for the utility of TCR-like antibodies as antigen-specific immunomodulation agents for regulating pathogenic T-cells and suggest that TCR-like antibodies targeting autoreactive MHC class II epitopes are valuable research tools that enable studies related to antigen presentation as well as novel therapeutic agents that may be used to modulate autoimmune disorders such as T1D. PMID:24090977

  8. Antigen-specific immunomodulation for type 1 diabetes by novel recombinant antibodies directed against diabetes-associates auto-reactive T cell epitope.

    PubMed

    Dahan, Rony; Gebe, John A; Preisinger, Anton; James, Eddie A; Tendler, Mark; Nepom, Gerald T; Reiter, Yoram

    2013-12-01

    The trimolecular complex composed of autoreactive T-cell receptor, MHC class II, and an autoantigenic peptide plays a central role in the activation of pathogenic Islet-specific CD4+ T cells in type 1 diabetes (T1D). We isolated and characterized novel antibodies against autoreactive T-cell epitopes associated with T1D. Our antibodies mimic the specificity of the T-cell receptor (TCR), while binding MHC class II/peptide complexes in an autoantigen peptide specific, MHC-restricted manner. The isolated TCR-like antibodies were directed against the minimal T-cell epitope GAD-555-567 in the context of the HLA-DR4-diabetic-associated molecule. A representative high-affinity TCR-like antibody clone (G3H8) enabled the detection of intra- and extra-cellular DR4/GAD-555-567 complexes in antigen presenting cells. I561M single mutation at the central position (P5) of the GAD-555-567 peptide abolished the binding of G3H8 to the DR4/GAD complex, demonstrating its high fine TCR-like specificity. The G3H8 TCR-like antibody significantly inhibited GAD-555-567 specific, DR4 restricted T-cell response in vitro and in vivo in HLA-DR4 transgenic mice. Our findings constitute a proof-of-concept for the utility of TCR-like antibodies as antigen-specific immunomodulation agents for regulating pathogenic T-cells and suggest that TCR-like antibodies targeting autoreactive MHC class II epitopes are valuable research tools that enable studies related to antigen presentation as well as novel therapeutic agents that may be used to modulate autoimmune disorders such as T1D.

  9. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  10. Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor–mediated immune responses but not in TCR signaling

    PubMed Central

    Kawagoe, Tatsukata; Sato, Shintaro; Jung, Andreas; Yamamoto, Masahiro; Matsui, Kosuke; Kato, Hiroki; Uematsu, Satoshi; Takeuchi, Osamu; Akira, Shizuo

    2007-01-01

    Interleukin-1 receptor–associated kinase 4 (IRAK-4) was reported to be essential for the Toll-like receptor (TLR)– and T cell receptor (TCR)–mediated signaling leading to the activation of nuclear factor κB (NF-κB). However, the importance of kinase activity of IRAK family members is unclear. In this study, we investigated the functional role of IRAK-4 activity in vivo by generating mice carrying a knockin mutation (KK213AA) that abrogates its kinase activity. IRAK-4KN/KN mice were highly resistant to TLR-induced shock response. The cytokine production in response to TLR ligands was severely impaired in IRAK-4KN/KN as well as IRAK-4−/− macrophages. The IRAK-4 activity was essential for the activation of signaling pathways leading to mitogen-activated protein kinases. TLR-induced IRAK-4/IRAK-1–dependent and –independent pathways were involved in early induction of NF-κB–regulated genes in response to TLR ligands such as tumor necrosis factor α and IκBζ. In contrast to a previous paper (Suzuki, N., S. Suzuki, D.G. Millar, M. Unno, H. Hara, T. Calzascia, S. Yamasaki, T. Yokosuka, N.J. Chen, A.R. Elford, et al. 2006. Science. 311:1927–1932), the TCR signaling was not impaired in IRAK-4−/− and IRAK-4KN/KN mice. Thus, the kinase activity of IRAK-4 is essential for the regulation of TLR-mediated innate immune responses. PMID:17485511

  11. Invariant NKT cells: regulation and function during viral infection.

    PubMed

    Juno, Jennifer A; Keynan, Yoav; Fowke, Keith R

    2012-01-01

    Natural killer T cells (NKT cells) represent a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT), express a highly restricted T cell receptor (TCR) and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV) infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation. In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses.

  12. Invariant NKT cells: regulation and function during viral infection.

    PubMed

    Juno, Jennifer A; Keynan, Yoav; Fowke, Keith R

    2012-01-01

    Natural killer T cells (NKT cells) represent a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT), express a highly restricted T cell receptor (TCR) and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV) infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation. In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses. PMID:22916008

  13. Regulation of satellite cell function in sarcopenia.

    PubMed

    Alway, Stephen E; Myers, Matthew J; Mohamed, Junaith S

    2014-01-01

    The mechanisms contributing to sarcopenia include reduced satellite cell (myogenic stem cell) function that is impacted by the environment (niche) of these cells. Satellite cell function is affected by oxidative stress, which is elevated in aged muscles, and this along with changes in largely unknown systemic factors, likely contribute to the manner in which satellite cells respond to stressors such as exercise, disuse, or rehabilitation in sarcopenic muscles. Nutritional intervention provides one therapeutic strategy to improve the satellite cell niche and systemic factors, with the goal of improving satellite cell function in aging muscles. Although many elderly persons consume various nutraceuticals with the hope of improving health, most of these compounds have not been thoroughly tested, and the impacts that they might have on sarcopenia and satellite cell function are not clear. This review discusses data pertaining to the satellite cell responses and function in aging skeletal muscle, and the impact that three compounds: resveratrol, green tea catechins, and β-Hydroxy-β-methylbutyrate have on regulating satellite cell function and therefore contributing to reducing sarcopenia or improving muscle mass after disuse in aging. The data suggest that these nutraceutical compounds improve satellite cell function during rehabilitative loading in animal models of aging after disuse (i.e., muscle regeneration). While these compounds have not been rigorously tested in humans, the data from animal models of aging provide a strong basis for conducting additional focused work to determine if these or other nutraceuticals can offset the muscle losses, or improve regeneration in sarcopenic muscles of older humans via improving satellite cell function. PMID:25295003

  14. Targeting cell cycle regulators in hematologic malignancies.

    PubMed

    Aleem, Eiman; Arceci, Robert J

    2015-01-01

    Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed. PMID:25914884

  15. Targeting cell cycle regulators in hematologic malignancies

    PubMed Central

    Aleem, Eiman; Arceci, Robert J.

    2015-01-01

    Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed. PMID:25914884

  16. Flavonoids: from cell cycle regulation to biotechnology.

    PubMed

    Woo, Ho-Hyung; Jeong, Byeong Ryong; Hawes, Martha C

    2005-03-01

    Flavonoids have been proposed to play diverse roles in plant growth and development, including defense, symbiosis, pollen development and male fertility, polar auxin transport, and protection against ultraviolet radiation. Recently, a new role in cell cycle regulation has emerged. Genetic alteration of glucuronide metabolism by altered expression of a Pisum sativum UDP-glucuronosyltransferase (PsUGT1) results in an altered cell cycle in pea, alfalfa, and Arabidopsis. In alfalfa, altered expression of PsUGT1 results in accumulation of a flavonoid-like compound that suppresses growth of cultured cells. The results are consistent with the hypothesis that PsUGT1 functions by controlling cellular levels of a factor controlling cell cycle (FCC). PMID:15834800

  17. T-cell-receptor engagement and tumor ICAM-1 up-regulation are required to by-pass low susceptibility of melanoma cells to autologous CTL-mediated lysis.

    PubMed

    Anichini, A; Mortarini, R; Alberti, S; Mantovani, A; Parmiani, G

    1993-04-01

    Tumor-specific and non-specific CD3+, TcR alpha beta+, CD8+ cytotoxic T-cell (CTL) clones, isolated from tumor-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) of a melanoma patient and allogeneic LAK cells, were used to investigate the requirements for bypassing the low lysability of some melanoma clones derived from an s.c. metastasis from which highly lysable clones were also obtained. Cytofluorimetric analysis showed that all melanoma clones expressed ICAM-1, although to different extents, reaching a 10-fold difference in fluorescence units, while HLA class-I antigens were similarly expressed. The differences in expression of ICAM-1 among tumor clones correlated with differences in lysability, by both specific and non-specific CTL, but were not large enough to affect lymphocyte-tumor conjugate formation. Cytokine- or gene-transfer-mediated up-regulation of ICAM-1 did not induce de novo lysis of ICAM-1low tumor cells; however, it markedly enhanced a low level of killing of the same cells by tumor-specific, TcR-dependent and HLA-restricted CTL clones but not by non-specific, TcR-independent effectors. In addition, lysis of melanoma clones by any effector was similarly inhibited by anti-ICAM-1 and anti-LFA-1 antibodies. This indicates that by-pass of low lysability of ICAM-1low melanoma clones by CTL clones, after ICAM-1 up-regulation, is possible only if simultaneous LFA-1 and TcR engagement takes place. In addition, these results suggest that the constitutive high level of expression of ICAM-1 on the subset of ICAM-1high melanoma cells must be only one of the factors contributing to the high lysability of these cells by any effector.

  18. Glycolytic regulation of cell rearrangement in angiogenesis.

    PubMed

    Cruys, Bert; Wong, Brian W; Kuchnio, Anna; Verdegem, Dries; Cantelmo, Anna Rita; Conradi, Lena-Christin; Vandekeere, Saar; Bouché, Ann; Cornelissen, Ivo; Vinckier, Stefan; Merks, Roeland M H; Dejana, Elisabetta; Gerhardt, Holger; Dewerchin, Mieke; Bentley, Katie; Carmeliet, Peter

    2016-01-01

    During vessel sprouting, endothelial cells (ECs) dynamically rearrange positions in the sprout to compete for the tip position. We recently identified a key role for the glycolytic activator PFKFB3 in vessel sprouting by regulating cytoskeleton remodelling, migration and tip cell competitiveness. It is, however, unknown how glycolysis regulates EC rearrangement during vessel sprouting. Here we report that computational simulations, validated by experimentation, predict that glycolytic production of ATP drives EC rearrangement by promoting filopodia formation and reducing intercellular adhesion. Notably, the simulations correctly predicted that blocking PFKFB3 normalizes the disturbed EC rearrangement in high VEGF conditions, as occurs during pathological angiogenesis. This interdisciplinary study integrates EC metabolism in vessel sprouting, yielding mechanistic insight in the control of vessel sprouting by glycolysis, and suggesting anti-glycolytic therapy for vessel normalization in cancer and non-malignant diseases. PMID:27436424

  19. Glycolytic regulation of cell rearrangement in angiogenesis

    PubMed Central

    Cruys, Bert; Wong, Brian W.; Kuchnio, Anna; Verdegem, Dries; Cantelmo, Anna Rita; Conradi, Lena-Christin; Vandekeere, Saar; Bouché, Ann; Cornelissen, Ivo; Vinckier, Stefan; Merks, Roeland M. H.; Dejana, Elisabetta; Gerhardt, Holger; Dewerchin, Mieke; Bentley, Katie; Carmeliet, Peter

    2016-01-01

    During vessel sprouting, endothelial cells (ECs) dynamically rearrange positions in the sprout to compete for the tip position. We recently identified a key role for the glycolytic activator PFKFB3 in vessel sprouting by regulating cytoskeleton remodelling, migration and tip cell competitiveness. It is, however, unknown how glycolysis regulates EC rearrangement during vessel sprouting. Here we report that computational simulations, validated by experimentation, predict that glycolytic production of ATP drives EC rearrangement by promoting filopodia formation and reducing intercellular adhesion. Notably, the simulations correctly predicted that blocking PFKFB3 normalizes the disturbed EC rearrangement in high VEGF conditions, as occurs during pathological angiogenesis. This interdisciplinary study integrates EC metabolism in vessel sprouting, yielding mechanistic insight in the control of vessel sprouting by glycolysis, and suggesting anti-glycolytic therapy for vessel normalization in cancer and non-malignant diseases. PMID:27436424

  20. Patz1 regulates embryonic stem cell identity.

    PubMed

    Ow, Jin Rong; Ma, Hui; Jean, Angela; Goh, Ziyi; Lee, Yun Hwa; Chong, Yew Mei; Soong, Richie; Fu, Xin-Yuan; Yang, Henry; Wu, Qiang

    2014-05-15

    Embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of blastocysts are pluripotent. Pluripotency is maintained by a transcriptional network in which Oct4 and Nanog are master regulators. Notably, several zinc finger transcription factors have important roles in this network. Patz1, a BTB/POZ-domain-containing zinc finger protein, is expressed at higher levels in the ICM relative to the trophectoderm. However, its function in pluripotency has been poorly studied. Here, we show that Patz1 is an important regulator of pluripotency in ESCs. Patz1 RNAi, chromatin immunoprecipitation (ChIP), and reporter assays indicate that Patz1 directly regulates Pou5f1 and Nanog. Global transcriptome changes upon Patz1 knockdown largely involve upregulation of apoptotic genes and downregulation of cell cycle and cellular metabolism genes. Patz1 ChIP sequencing further identified more than 5,000 binding sites of Patz1 in mouse genome, from which two binding motifs were extracted. Further, gene ontology analysis of genes associated with the binding sites displays enrichment for proximity to developmental genes. In addition, embryoid body assays suggest that Patz1 represses developmental genes. Together, these results propose that Patz1 is important for ESC pluripotency. PMID:24380431

  1. Auxin regulation of cell polarity in plants.

    PubMed

    Pan, Xue; Chen, Jisheng; Yang, Zhenbiao

    2015-12-01

    Auxin is well known to control pattern formation and directional growth at the organ/tissue levels via the nuclear TIR1/AFB receptor-mediated transcriptional responses. Recent studies have expanded the arena of auxin actions as a trigger or key regulator of cell polarization and morphogenesis. These actions require non-transcriptional responses such as changes in the cytoskeleton and vesicular trafficking, which are commonly regulated by ROP/Rac GTPase-dependent pathways. These findings beg for the question about the nature of auxin receptors that regulate these responses and renew the interest in ABP1 as a cell surface auxin receptor, including the work showing auxin-binding protein 1 (ABP1) interacts with the extracellular domain of the transmembrane kinase (TMK) receptor-like kinases in an auxin-dependent manner, as well as the debate on this auxin binding protein discovered about 40 years ago. This review highlights recent work on the non-transcriptional auxin signaling mechanisms underscoring cell polarity and shape formation in plants.

  2. Auxin regulation of cell polarity in plants.

    PubMed

    Pan, Xue; Chen, Jisheng; Yang, Zhenbiao

    2015-12-01

    Auxin is well known to control pattern formation and directional growth at the organ/tissue levels via the nuclear TIR1/AFB receptor-mediated transcriptional responses. Recent studies have expanded the arena of auxin actions as a trigger or key regulator of cell polarization and morphogenesis. These actions require non-transcriptional responses such as changes in the cytoskeleton and vesicular trafficking, which are commonly regulated by ROP/Rac GTPase-dependent pathways. These findings beg for the question about the nature of auxin receptors that regulate these responses and renew the interest in ABP1 as a cell surface auxin receptor, including the work showing auxin-binding protein 1 (ABP1) interacts with the extracellular domain of the transmembrane kinase (TMK) receptor-like kinases in an auxin-dependent manner, as well as the debate on this auxin binding protein discovered about 40 years ago. This review highlights recent work on the non-transcriptional auxin signaling mechanisms underscoring cell polarity and shape formation in plants. PMID:26599954

  3. Regulating functional cell fates in CD8 T cells

    PubMed Central

    Rao, Rajesh; Li, Qingsheng; Kesterson, Joshua; Eppolito, Cheryl; Mischo, Axel; Singhal, Pankaj

    2016-01-01

    The attributes of specificity and memory enable CD8+ T cells to provide long-lasting protection against a variety of challenges. Although, the importance of CD8+ T cells for protection against intracellular infections and transformation is well-established, the functional type; effector phenotypes (Tc1, Tc2, Tc17 and/or Tcreg) and/or memory (effector or central), of CD8+ T cells most desirable for tumor immunity is not established. To determine the tumor efficacy of various effector types and/or memory CD8 T cells, it is imperative to better understand intrinsic and extrinsic factors that regulate CD8+ T cell differentiation and use this information to generate and test distinct functional cell types in tumor models. The focus of our laboratory investigations is to identify the extrinsic factors such as antigen strength, co-stimulatory molecules, cytokines, and small molecule modifiers that regulate intrinsic programs for various effector and/or memory cell fate in antigen specific CD8 T cells. The use of this information to generate immunity in murine tumor models has facilitated development of new adoptive cell transfer (ACT) as well as immunization strategies for cancer treatment. PMID:19859830

  4. The small GTPase RhoH is an atypical regulator of haematopoietic cells

    PubMed Central

    Fueller, Florian; Kubatzky, Katharina F

    2008-01-01

    Rho GTPases are a distinct subfamily of the superfamily of Ras GTPases. The best-characterised members are RhoA, Rac and Cdc42 that regulate many diverse actions such as actin cytoskeleton reorganisation, adhesion, motility as well as cell proliferation, differentiation and gene transcription. Among the 20 members of that family, only Rac2 and RhoH show an expression restricted to the haematopoietic lineage. RhoH was first discovered in 1995 as a fusion transcript with the transcriptional repressor LAZ3/BCL6. It was therefore initially named translation three four (TTF) but later on renamed RhoH due to its close relationship to the Ras/Rho family of GTPases. Since then, RhoH has been implicated in human cancer as the gene is subject to somatic hypermutation and by the detection of RHOH as a translocation partner for LAZ3/BCL6 or other genes in human lymphomas. Underexpression of RhoH is found in hairy cell leukaemia and acute myeloid leukaemia. Some of the amino acids that are crucial for GTPase activity are mutated in RhoH so that the protein is a GTPase-deficient, so-called atypical Rho GTPase. Therefore other mechanisms of regulating RhoH activity have been described. These include regulation at the mRNA level and tyrosine phosphorylation of the protein's unique ITAM-like motif. The C-terminal CaaX box of RhoH is mainly a target for farnesyl-transferase but can also be modified by geranylgeranyl-transferase. Isoprenylation of RhoH and changes in subcellular localisation may be an additional factor to fine-tune signalling. Little is currently known about its signalling, regulation or interaction partners. Recent studies have shown that RhoH negatively influences the proliferation and homing of murine haematopoietic progenitor cells, presumably by acting as an antagonist for Rac1. In leukocytes, RhoH is needed to keep the cells in a resting, non-adhesive state, but the exact mechanism has yet to be elucidated. RhoH has also been implicated as a regulatory molecule

  5. Tolerance and autoimmunity to a gastritogenic peptide in TCR transgenic mice.

    PubMed

    Alderuccio, F; Cataldo, V; van Driel, I R; Gleeson, P A; Toh, B H

    2000-03-01

    The catalytic alpha and glycoprotein beta subunits of the gastric H/K ATPase are major molecular targets in human and mouse autoimmune gastritis. We have previously shown that the H/K ATPase beta subunit is required for the initiation of mouse gastritis and identified a gastritogenic H/K ATPase beta subunit peptide (H/Kbeta253-277). Here we report the generation of MHC class II-restricted TCR transgenic mice using V(alpha)9 and V(beta)8.3 TCR chains with specificity for the gastritogenic H/Kbeta253-277 peptide. We found an 8-fold reduction in CD4(+) T cells in the thymus of the transgenic mice. Despite the reduction in intrathymic CD4(+) T cells, V(beta)8. 3-expressing T cells comprised the majority (>90%) of peripheral spleen and lymph node T cells. These peripheral T cells retained their capacity to proliferate in vitro to the H/Kbeta253-277 peptide. Using the responsive T cells, we have restricted the gastritogenic T cell epitope to H/Kbeta261-274. Despite the capacity of the peripheral T cells to proliferate in vitro to the peptide, the majority ( approximately 80%, 13 of 16) of transgenic mice remained free of gastritis while a minority (20%, three of 16) spontaneously developed an invasive and destructive gastritis. Our results confirm that H/Kbeta261-274 is a gastritogenic peptide. The data also suggest that CD4 T cell tolerance to the gastritogenic peptide in the transgenic mice is maintained by a combination of intrathymic and peripheral tolerance mechanisms.

  6. The Timing of T Cell Priming and Cycling.

    PubMed

    Obst, Reinhard

    2015-01-01

    The proliferation of specific lymphocytes is the central tenet of the clonal selection paradigm. Antigen recognition by T cells triggers a series of events that produces expanded clones of differentiated effector cells. TCR signaling events are detectable within seconds and minutes and are likely to continue for hours and days in vivo. Here, I review the work done on the importance of TCR signals in the later part of the expansion phase of the primary T cell response, primarily regarding the regulation of the cell cycle in CD4(+) and CD8(+) cells. The results suggest a degree of programing by early signals for effector differentiation, particularly in the CD8(+) T cell compartment, with optimal expansion supported by persistent antigen presentation later on. Differences to CD4(+) T cell expansion and new avenues toward a molecular understanding of cell cycle regulation in lymphocytes are discussed. PMID:26594213

  7. Redox Regulation in Cancer Stem Cells

    PubMed Central

    Ding, Shijie; Li, Chunbao; Cheng, Ninghui; Cui, Xiaojiang; Xu, Xinglian; Zhou, Guanghong

    2015-01-01

    Reactive oxygen species (ROS) and ROS-dependent (redox regulation) signaling pathways and transcriptional activities are thought to be critical in stem cell self-renewal and differentiation during growth and organogenesis. Aberrant ROS burst and dysregulation of those ROS-dependent cellular processes are strongly associated with human diseases including many cancers. ROS levels are elevated in cancer cells partially due to their higher metabolism rate. In the past 15 years, the concept of cancer stem cells (CSCs) has been gaining ground as the subpopulation of cancer cells with stem cell-like properties and characteristics have been identified in various cancers. CSCs possess low levels of ROS and are responsible for cancer recurrence after chemotherapy or radiotherapy. Unfortunately, how CSCs control ROS production and scavenging and how ROS-dependent signaling pathways contribute to CSCs function remain poorly understood. This review focuses on the role of redox balance, especially in ROS-dependent cellular processes in cancer stem cells (CSCs). We updated recent advances in our understanding of ROS generation and elimination in CSCs and their effects on CSC self-renewal and differentiation through modulating signaling pathways and transcriptional activities. The review concludes that targeting CSCs by manipulating ROS metabolism/dependent pathways may be an effective approach for improving cancer treatment. PMID:26273424

  8. Regulation of the transcriptional program by DNA methylation during human αβ T-cell development

    PubMed Central

    Rodriguez, Ramon M.; Suarez-Alvarez, Beatriz; Mosén-Ansorena, David; García-Peydró, Marina; Fuentes, Patricia; García-León, María J.; Gonzalez-Lahera, Aintzane; Macias-Camara, Nuria; Toribio, María L.; Aransay, Ana M.; Lopez-Larrea, Carlos

    2015-01-01

    Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis. PMID:25539926

  9. Wnt pathway regulation of intestinal stem cells.

    PubMed

    Mah, Amanda T; Yan, Kelley S; Kuo, Calvin J

    2016-09-01

    Wnt signalling is involved in multiple aspects of embryonic development and adult tissue homeostasis, notably via controlling cellular proliferation and differentiation. Wnt signalling is subject to stringent positive and negative regulation to promote proper development and homeostasis yet avoid aberrant growth. Such multi-layer regulation includes post-translational modification and processing of Wnt proteins themselves, R-spondin (Rspo) amplification of Wnt signalling, diverse receptor families, and intracellular and extracellular antagonists and destruction and transcription complexes. In the gastrointestinal tract, Wnt signalling is crucial for development and renewal of the intestinal epithelium. Intestinal stem cells (ISCs) undergo symmetric division and neutral drift dynamics to renew the intestinal epithelium. Sources of Wnts and Wnt amplifers such as R-spondins are beginning to be elucidated as well as their functional contribution to intestinal homeostasis. In this review we focus on regulation of ISCs and intestinal homeostasis by the Wnt/Rspo pathway, the potential cellular sources of Wnt signalling regulators and highlight potential future areas of study. PMID:27581568

  10. Nck recruitment to the TCR required for ZAP70 activation during thymic development.

    PubMed

    Borroto, Aldo; Arellano, Irene; Dopfer, Elaine P; Prouza, Marek; Suchànek, Miloslav; Fuentes, Manuel; Orfao, Alberto; Schamel, Wolfgang W; Alarcón, Balbino

    2013-02-01

    The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3ε after TCR engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4(+)CD8(+) thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired tyrosine phosphorylation of CD3ζ, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic differences in both knockin mice. PMID:23267019

  11. Shaping the T-cell repertoire: a matter of life and death.

    PubMed

    Wiegers, G Jan; Kaufmann, Manuel; Tischner, Denise; Villunger, Andreas

    2011-01-01

    Thymocyte selection aims to shape a T-cell repertoire that, on the one hand, is able to recognize and respond to foreign peptides and, on the other hand, tolerizes the presence of self-peptides in the periphery. Deletion of T cells or their precursors that fail to fulfill these criteria is mainly mediated by the Bcl-2-regulated apoptosis pathway. Absence of T-cell receptor (TCR)-mediated signals or hyperactivation of the TCR by high-affinity self-peptide-major histocompatibility complexes can both trigger apoptotic cell death in developing thymocytes. Notably, TCR-signaling strength also defines survival and outgrowth of the fittest antigen-specific T-cell clones in the periphery. TCR threshold activity leading to such drastically opposing signaling outcomes (life or death) is modulated in part by cytokines and other factors, such as glucocorticoids, that fine-tune the Bcl-2 rheostat, thereby impacting on cell survival. This review aims to highlight the role of Bcl-2-regulated cell death for clonal T-cell selection. PMID:21060321

  12. The regulation of hematopoietic stem cell populations

    PubMed Central

    Mayani, Hector

    2016-01-01

    Evidence presented over the last few years indicates that the hematopoietic stem cell (HSC) compartment comprises not just one but a number of different cell populations. Based on HSCs’ proliferation and engraftment potential, it has been suggested that there are two classes of HSC, with long- and short-term engraftment potential. HSC heterogeneity seems to involve differentiation capacities as well, since it has been shown that some HSC clones are able to give rise to both myeloid and lymphoid progeny, whereas others are lymphoid deficient. It has been recognized that HSC function depends on intrinsic cell regulators, which are modulated by external signals. Among the former, we can include transcription factors and non-coding RNAs as well as epigenetic modifiers. Among the latter, cytokines and extracellular matrix molecules have been implicated. Understanding the elements and mechanisms that regulate HSC populations is of significant relevance both in biological and in clinical terms, and research in this area still has to face several complex and exciting challenges. PMID:27408695

  13. Cell cycle regulation of human WEE1.

    PubMed Central

    McGowan, C H; Russell, P

    1995-01-01

    WEE1 kinase negatively regulates entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of CDC2/cyclin B kinase. We report here an investigation of human WEE1. Endogenous WEE1 migrates as an approximately 94 kDa protein in SDS-PAGE, substantially larger than the 49 kDa protein encoded by the original human WEE1 cDNA clone that was truncated at the 5'-end. Antibody depletion experiments demonstrate that WEE1 accounts for most of the activity that phosphorylates CDC2 on Tyr15 in an in vitro assay of HeLa cell lysates, hence it is likely to have an important role in the mitotic control of human cells. WEE1 activity was not found to be elevated in HeLa cells arrested in S phase, suggesting that unreplicated DNA does not delay M phase by hyperactivating WEE1. WEE1 activity is strongly suppressed during M phase, suggesting that negative regulation of WEE1 could be part of the mechanism by which activation of CDC2/cyclin B kinase is promoted during the G2/M transition. M phase WEE1 is re-activated in samples prepared in the absence of protein phosphatase inhibitors, demonstrating that WEE1 is inhibited by a mechanism that requires protein phosphorylation. Images PMID:7774574

  14. Matricellular protein Cfl1 regulates cell differentiation.

    PubMed

    Tian, Xiuyun; Lin, Xiaorong

    2013-11-01

    Like higher eukaryotic cells in tissues, microbial cells in a community act in concert in response to environmental stimuli. They coordinate gene expression and their physiological and morphological states through intercellular communication mediated by matricellular signals. The adhesion protein Cfl1 was recently shown to be a matricellular signal in regulating morphogenesis and biofilm formation in the eukaryotic microbe Cryptococcus neoformans. Cfl1 is naturally highly expressed in the hyphal subpopulation during the mating colony development. Some Cfl1 proteins are cleaved and released to the ECM (extracellular matrix). The released exogenous Cfl1 activates Cryptococcus cells to express their endogenous Cfl1, to undergo filamentation, and to form structured biofilm colonies. In this study, we demonstrate that the N-terminal signal peptide and the novel conserved cysteine-rich SIGC domain at the C-terminus are critical for the adherence property and the signaling activity of this multifunctional protein. The investigation of this fungal matricellular signaling network involving Cfl1 and the master regulator of morphogenesis Znf2 provides a foundation to further elucidate intercellular communication in microbial development.

  15. Identical TCR beta-chain rearrangements in streptococcal angina and skin lesions of patients with psoriasis vulgaris.

    PubMed

    Diluvio, Laura; Vollmer, Sigrid; Besgen, Petra; Ellwart, Joachim W; Chimenti, Sergio; Prinz, Joerg C

    2006-06-01

    Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR beta-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin address in "cutaneous lymphocyte-associated Ag" (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.

  16. Transcriptionally Regulated Cell Adhesion Network Dictates Distal Tip Cell Directionality

    PubMed Central

    Wong, Ming-Ching; Kennedy, William P.; Schwarzbauer, Jean E.

    2015-01-01

    Background The mechanisms that govern directional changes in cell migration are poorly understood. The migratory paths of two distal tip cells (DTC) determine the U-shape of the C. elegans hermaphroditic gonad. The morphogenesis of this organ provides a model system to identify genes necessary for the DTCs to execute two stereotyped turns. Results Using candidate genes for RNAi knockdown in a DTC-specific strain, we identified two transcriptional regulators required for DTC turning: cbp-1, the CBP/p300 transcriptional coactivator homologue, and let-607, a CREBH transcription factor homologue. Further screening of potential target genes uncovered a network of integrin adhesion-related genes that have roles in turning and are dependent on cbp-1 and let-607 for expression. These genes include src-1/Src kinase, tln-1/talin, pat-2/α integrin and nmy-2, a nonmuscle myosin heavy chain. Conclusions Transcriptional regulation by means of cbp-1 and let-607 is crucial for determining directional changes during DTC migration. These regulators coordinate a gene network that is necessary for integrin-mediated adhesion. Overall, these results suggest that directional changes in cell migration rely on the precise gene regulation of adhesion. PMID:24811939

  17. BCOR regulates myeloid cell proliferation and differentiation.

    PubMed

    Cao, Q; Gearhart, M D; Gery, S; Shojaee, S; Yang, H; Sun, H; Lin, D-C; Bai, J-W; Mead, M; Zhao, Z; Chen, Q; Chien, W-W; Alkan, S; Alpermann, T; Haferlach, T; Müschen, M; Bardwell, V J; Koeffler, H P

    2016-05-01

    BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes. PMID:26847029

  18. Structure of a TCR-Mimic Antibody with Target Predicts Pharmacogenetics.

    PubMed

    Ataie, Niloufar; Xiang, Jingyi; Cheng, Neal; Brea, Elliott J; Lu, Wenjie; Scheinberg, David A; Liu, Cheng; Ng, Ho Leung

    2016-01-16

    Antibody therapies currently target only extracellular antigens. A strategy to recognize intracellular antigens is to target peptides presented by immune HLA receptors. ESK1 is a human, T-cell receptor (TCR)-mimic antibody that binds with subnanomolar affinity to the RMF peptide from the intracellular Wilms tumor oncoprotein WT1 in complex with HLA-A*02:01. ESK1 is therapeutically effective in mouse models of WT1(+) human cancers. TCR-based therapies have been presumed to be restricted to one HLA subtype. The mechanism for the specificity and high affinity of ESK1 is unknown. We show in a crystal structure that ESK1 Fab binds to RMF/HLA-A*02:01 in a mode different from that of TCRs. From the structure, we predict and then experimentally confirm high-affinity binding with multiple other HLA-A*02 subtypes, broadening the potential patient pool for ESK1 therapy. Using the crystal structure, we also predict potential off-target binding that we experimentally confirm. Our results demonstrate how protein structure information can contribute to personalized immunotherapy. PMID:26688548

  19. Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses

    SciTech Connect

    Fukuyama, Yoshiko; Tokuhara, Daisuke; Sekine, Shinichi; Kataoka, Kosuke; Markham, Jonathan D.; Irwin, Allyson R.; Moon, Grace H.; Tokuhara, Yuka; Fujihashi, Keiko; Davydova, Julia; Yamamoto, Masato; Gilbert, Rebekah S.; Fujihashi, Kohtaro

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer Nasal Ad-FL effectively up-regulates APC function by CD11c{sup +} DCs in mucosal tissues. Black-Right-Pointing-Pointer Nasal Ad-FL induces Notch ligand (L)-expressing CD11c{sup +} DCs. Black-Right-Pointing-Pointer Notch L-expressing DCs support the induction of Th1- and Th2-type cytokine responses. -- Abstract: Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c{sup +} dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c{sup +} DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c{sup +} DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c{sup +} DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4{sup +} T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-{gamma}, IL-2 and IL-4 producing CD4{sup +} T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c{sup +} DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.

  20. Cell shape regulates global histone acetylation in human mammaryepithelial cells

    SciTech Connect

    Le Beyec, Johanne; Xu, Ren; Lee, Sun-Young; Nelson, Celeste M.; Rizki, Aylin; Alcaraz, Jordi; Bissell, Mina J.

    2007-02-28

    Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.

  1. Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1

    PubMed Central

    Ishikawa, Eri; Kosako, Hidetaka; Yasuda, Tomoharu; Ohmuraya, Masaki; Araki, Kimi; Kurosaki, Tomohiro; Saito, Takashi; Yamasaki, Sho

    2016-01-01

    Thymic selection shapes an appropriate T cell antigen receptor (TCR) repertoire during T cell development. Here, we show that a serine/threonine kinase, protein kinase D (PKD), is crucial for thymocyte positive selection. In T cell-specific PKD-deficient (PKD2/PKD3 double-deficient) mice, the generation of CD4 single positive thymocytes is abrogated. This defect is likely caused by attenuated TCR signalling during positive selection and incomplete CD4 lineage specification in PKD-deficient thymocytes; however, TCR-proximal tyrosine phosphorylation is not affected. PKD is activated in CD4+CD8+ double positive (DP) thymocytes on stimulation with positively selecting peptides. By phosphoproteomic analysis, we identify SH2-containing protein tyrosine phosphatase-1 (SHP-1) as a direct substrate of PKD. Substitution of wild-type SHP-1 by phosphorylation-defective mutant (SHP-1S557A) impairs generation of CD4+ thymocytes. These results suggest that the PKD–SHP-1 axis positively regulates TCR signalling to promote CD4+ T cell development. PMID:27670070

  2. MicroRNAs: key regulators of stem cells.

    PubMed

    Gangaraju, Vamsi K; Lin, Haifan

    2009-02-01

    The hallmark of a stem cell is its ability to self-renew and to produce numerous differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new dimension of gene regulation in controlling stem cell fate and behaviour. PMID:19165214

  3. GATA2 regulates dendritic cell differentiation

    PubMed Central

    Onodera, Koichi; Fujiwara, Tohru; Onishi, Yasushi; Itoh-Nakadai, Ari; Okitsu, Yoko; Fukuhara, Noriko; Ishizawa, Kenichi; Shimizu, Ritsuko; Yamamoto, Masayuki

    2016-01-01

    Dendritic cells (DCs) are critical immune response regulators; however, the mechanism of DC differentiation is not fully understood. Heterozygous germ line GATA2 mutations induce GATA2-deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia/acute myeloid leukemia, and a profoundly reduced DC population, which is associated with increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. To define the role of GATA2 in DC differentiation and function, we studied Gata2 conditional knockout and haploinsufficient mice. Gata2 conditional deficiency significantly reduced the DC count, whereas Gata2 haploinsufficiency did not affect this population. GATA2 was required for the in vitro generation of DCs from Lin−Sca-1+Kit+ cells, common myeloid-restricted progenitors, and common dendritic cell precursors, but not common lymphoid-restricted progenitors or granulocyte-macrophage progenitors, suggesting that GATA2 functions in the myeloid pathway of DC differentiation. Moreover, expression profiling demonstrated reduced expression of myeloid-related genes, including mafb, and increased expression of T-lymphocyte–related genes, including Gata3 and Tcf7, in Gata2-deficient DC progenitors. In addition, GATA2 was found to bind an enhancer element 190-kb downstream region of Gata3, and a reporter assay exhibited significantly reduced luciferase activity after adding this enhancer region to the Gata3 promoter, which was recovered by GATA sequence deletion within Gata3 +190. These results suggest that GATA2 plays an important role in cell-fate specification toward the myeloid vs T-lymphocyte lineage by regulating lineage-specific transcription factors in DC progenitors, thereby contributing to DC differentiation. PMID:27259979

  4. Genome exposure and regulation in mammalian cells.

    PubMed

    Puck, T T; Webb, P; Johnson, R

    1998-09-01

    fibroblasts as revealed by in situ nick translation reveals a nuclear distribution pattern around the periphery, around the nucleoli and in punctate positions in the nuclear interior in parts of both S and G1 phases of the cell cycle. The same exposure pattern is duplicated by the pattern of DNA synthesis in S cells. It would appear that these nuclear regions represent positions of special activity. The previously proposed theory of genome regulation in mammalian cells is supported by these findings. The theory proposes that: a) gene activity requires exposure of the given locus followed by action of transcription factors on the exposed genes; b) the fiber system of the cell (cytoskeleton, nuclear fibers, and extracellular fibers) are required for normal exposure; c) active sites for gene expression and replication consist of the nuclear periphery where differentiation genes particularly are exposed; the nucleoli where at least some housekeeping genes are exposed; and possibly also punctate regions in the interior; d) noncoding sequences play a critical role in genome regulation, possibly including the transport of loci to be activated to appropriate exposure transcriptional and replicating locations. Cancer cells have lost specific differentiation gene activities, at least sometimes because of mutation of appropriate exposure genes; at least some protooncogenes and tumor suppressor genes are responsible for exposure and transport of specific differentiation gene loci to their appropriate exposure sites in the nucleus and for inducing exposure.

  5. Epigenetic regulation of hematopoietic stem cell aging

    SciTech Connect

    Beerman, Isabel

    2014-12-10

    Aging is invariably associated with alterations of the hematopoietic stem cell (HSC) compartment, including loss of functional capacity, altered clonal composition, and changes in lineage contribution. Although accumulation of DNA damage occurs during HSC aging, it is unlikely such consistent aging phenotypes could be solely attributed to changes in DNA integrity. Another mechanism by which heritable traits could contribute to the changes in the functional potential of aged HSCs is through alterations in the epigenetic landscape of adult stem cells. Indeed, recent studies on hematopoietic stem cells have suggested that altered epigenetic profiles are associated with HSC aging and play a key role in modulating the functional potential of HSCs at different stages during ontogeny. Even small changes of the epigenetic landscape can lead to robustly altered expression patterns, either directly by loss of regulatory control or through indirect, additive effects, ultimately leading to transcriptional changes of the stem cells. Potential drivers of such changes in the epigenetic landscape of aged HSCs include proliferative history, DNA damage, and deregulation of key epigenetic enzymes and complexes. This review will focus largely on the two most characterized epigenetic marks – DNA methylation and histone modifications – but will also discuss the potential role of non-coding RNAs in regulating HSC function during aging.

  6. Regulation of adipose cell development in utero.

    PubMed

    Martin, R J; Hausman, G J; Hausman, D B

    1998-12-01

    The condition of obesity is impacted by increases in fat cell number, fat cell size, or a combination of the two. It is generally believed that fat cell number is dependent on the age of onset and the degree of obesity. This review provides an update on intrauterine growth of fetal adipose tissue, the earliest period of proliferation onset, and the factors that interact to enhance or suppress development. Fetal adipose tissue development is regulated by the complex interaction of maternal, endocrine, and paracrine influences that initiate specific changes in angiogenesis, adipogenesis, and metabolism. Developmental stages and metabolic processes influenced by specific hormones and paracrine factors have been identified through examination of the offspring of obese and diabetic pregnancies, hormonal manipulation during late pregnancy in animal models, and the use of cell culture. Collectively, the results of the studies cited herein delineate the basis for imprinting or conditioning of fetal preadipocytes at the paracrine/autocrine level and a role of thyroxine, glucocorticoids, and other hormones in fetal adipose tissue development and metabolism.

  7. Regulation of polymorphonuclear cell activation by thrombopoietin.

    PubMed Central

    Brizzi, M F; Battaglia, E; Rosso, A; Strippoli, P; Montrucchio, G; Camussi, G; Pegoraro, L

    1997-01-01

    Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation. PMID:9120001

  8. Transcriptional regulation of glial cell specification.

    PubMed

    Ragone, Gianluca; Van De Bor, Véronique; Sorrentino, Sandro; Kammerer, Martial; Galy, Anne; Schenck, Annette; Bernardoni, Roberto; Miller, Alita A; Roy, Nivedita; Giangrande, Angela

    2003-03-01

    Neuronal differentiation relies on proneural factors that also integrate positional information and contribute to the specification of the neuronal type. The molecular pathway triggering glial specification is not understood yet. In Drosophila, all lateral glial precursors and glial-promoting activity have been identified, which provides us with a unique opportunity to dissect the regulatory pathways controlling glial differentiation and specification. Although glial lineages are very heterogeneous with respect to position, time of differentiation, and lineage tree, they all express and require two homologous genes, glial cell deficient/glial cell missing (glide/gcm) and glide2, that act in concert, with glide/gcm constituting the major glial-promoting factor. Here, we show that glial specification resides in glide/gcm transcriptional regulation. The glide/gcm promoter contains lineage-specific elements as well as quantitative and turmoil elements scattered throughout several kilobases. Interestingly, there is no correlation between a specific regulatory element and the type of glial lineage. Thus, the glial-promoting factor acts as a naive switch-on button that triggers gliogenesis in response to multiple pathways converging onto its promoter. Both negative and positive regulation are required to control glide/gcm expression, indicating that gliogenesis is actively repressed in some neural lineages. PMID:12618139

  9. High-resolution analysis of the human T-cell receptor repertoire

    PubMed Central

    Ruggiero, Eliana; Nicolay, Jan P.; Fronza, Raffaele; Arens, Anne; Paruzynski, Anna; Nowrouzi, Ali; Ürenden, Gökçe; Lulay, Christina; Schneider, Sven; Goerdt, Sergij; Glimm, Hanno; Krammer, Peter H.; Schmidt, Manfred; von Kalle, Christof

    2015-01-01

    Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity. Here we show that TCR ligation-anchored-magnetically captured PCR (TCR-LA-MC PCR) identifies TCR α- and β-chain diversity without sequence-associated or quantitative restrictions in healthy and diseased conditions. TCR-LA-MC PCR identifies convergent recombination events, classifies different stages of cutaneous T-cell lymphoma in vivo and demonstrates TCR reactivation after in vitro cytomegalovirus stimulation. TCR-LA-MC PCR allows ultra-deep data access to both physiological TCR diversity and mechanisms influencing clonality in all clinical settings with restricted or distorted TCR repertoires. PMID:26324409

  10. Rapid burst of H2O2 by plant growth regulators increases intracellular Ca2+ amounts and modulates CD4+ T cell activation.

    PubMed

    Ahmed, Asma; Mukherjee, Sambuddho; Deobagkar, Mukta; Naik, Tanushree; Nandi, Dipankar

    2010-11-01

    The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H(2)O(2). In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca(2+) concentrations [Ca(2+)](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNFα and IFNγ by CD4(+) T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca(2+) ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling.

  11. The phosphatase JKAP/DUSP22 inhibits t-cell receptor signalling and autoimmunity by inactivating Lck

    Technology Transfer Automated Retrieval System (TEKTRAN)

    JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knocko...

  12. T cell activation responses are differentially regulated during clinorotation and in spaceflight

    NASA Technical Reports Server (NTRS)

    Hashemi, B. B.; Penkala, J. E.; Vens, C.; Huls, H.; Cubbage, M.; Sams, C. F.

    1999-01-01

    Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell

  13. Molecular regulation of plant cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  14. Molecular regulation of plant cell wall extensibility.

    PubMed

    Cosgrove, D J

    1998-05-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized. PMID:11540640

  15. Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

    PubMed Central

    Wan, Chun-ping; Gao, Li-xin; Hou, Li-fei; Yang, Xiao-qian; He, Pei-lan; Yang, Yi-fu; Tang, Wei; Yue, Jian-min; Li, Jia; Zuo, Jian-ping

    2013-01-01

    Aim: To investigate the immunomodulating activity of astragalosides, the active compounds from a traditional tonic herb Astragalus membranaceus Bge, and to explore the molecular mechanisms underlying the actions, focusing on CD45 protein tyrosine phosphatase (CD45 PTPase), which plays a critical role in T lymphocyte activation. Methods: Primary splenocytes and T cells were prepared from mice. CD45 PTPase activity was assessed using a colorimetric assay. Cell proliferation was measured using a [3H]-thymidine incorporation assay. Cytokine proteins and mRNAs were examined with ELISA and RT-PCR, respectively. Activation markers, including CD25 and CD69, were analyzed using flow cytometry. Activation of LCK (Tyr505) was detected using Western blot analysis. Mice were injected with the immunosuppressant cyclophosphamide (CTX, 80 mg/kg), and administered astragaloside II (50 mg/kg). Results: Astragaloside I, II, III, and IV concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 μg/mL. Astragaloside II (10 and 30 nmol/L) significantly enhanced the proliferation of primary splenocytes induced by ConA, alloantigen or anti-CD3. Astragaloside II (30 nmol/L) significantly increased IL-2 and IFN-γ secretion, upregulated the mRNA levels of IFN-γ and T-bet in primary splenocytes, and promoted CD25 and CD69 expression on primary CD4+ T cells upon TCR stimulation. Furthermore, astragaloside II (100 nmol/L) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells, which could be blocked by a specific CD45 PTPase inhibitor. In CTX-induced immunosuppressed mice, oral administration of astragaloside II restored the proliferation of splenic T cells and the production of IFN-γ and IL-2. However, astragaloside II had no apparent effects on B cell proliferation. Conclusion: Astragaloside II enhances T cell activation by regulating the activity of CD45 PTPase, which may explain why Astragalus

  16. Phosphorylation Site Dynamics of Early T-cell Receptor Signaling

    PubMed Central

    Rigbolt, Kristoffer T. G.; Hu, Bin; Hlavacek, William S.; Blagoev, Blagoy

    2014-01-01

    In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein–protein interactions and phosphorylation events have been studied extensively, we lack a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites with central roles in TCR signaling. The model was used to generate predictions suggesting unexpected roles for the phosphatase PTPN6 (SHP-1) and shortcut recruitment of the actin regulator WAS. Predictions were validated experimentally. This integration of proteomics and modeling illustrates a novel, generalizable framework for solidifying quantitative understanding of a signaling network and for elucidating missing links. PMID:25147952

  17. The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

    NASA Astrophysics Data System (ADS)

    Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

    2014-02-01

    Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

  18. Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells

    PubMed Central

    O'Leary, Claire E.; Riling, Christopher R.; Spruce, Lynn A.; Ding, Hua; Kumar, Suresh; Deng, Guoping; Liu, Yuhong; Seeholzer, Steven H.; Oliver, Paula M.

    2016-01-01

    Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation. We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells. Using a three-part proteomics approach in primary T cells, we identify stabilization of Jak1 in Ndfip1/2-deficient T cells stimulated through the TCR. Jak1 degradation is aborted in activated T cells that lack Ndfips. In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells. Thus, Ndfip1/Ndfip2 regulate the cross talk between the T-cell receptor and cytokine signalling pathways to limit inappropriate T-cell responses. PMID:27088444

  19. Regulating cancer stem cells the miR way.

    PubMed

    Peter, Marcus E

    2010-01-01

    A recent study in Nature Cell Biology, Wellner et al. (2009) identifies ZEB1, a known promoter of tumor invasion, as a negative regulator of miRNA clusters that target stem cell factors. These findings provide new insight into the network of transcription factors and miRNAs that regulate cancer stem cells.

  20. Inhibition of regulated cell death by cell-penetrating peptides.

    PubMed

    Krautwald, Stefan; Dewitz, Christin; Fändrich, Fred; Kunzendorf, Ulrich

    2016-06-01

    Development of the means to efficiently and continuously renew missing and non-functional proteins in diseased cells remains a major goal in modern molecular medicine. While gene therapy has the potential to achieve this, substantial obstacles must be overcome before clinical application can be considered. A promising alternative approach is the direct delivery of non-permeant active biomolecules, such as oligonucleotides, peptides and proteins, to the affected cells with the purpose of ameliorating an advanced disease process. In addition to receptor-mediated endocytosis, cell-penetrating peptides are widely used as vectors for rapid translocation of conjugated molecules across cell membranes into intracellular compartments and the delivery of these therapeutic molecules is generally referred to as novel prospective protein therapy. As a broad coverage of the enormous amount of published data in this field is unrewarding, this review will provide a brief, focused overview of the technology and a summary of recent studies of the most commonly used protein transduction domains and their potential as therapeutic agents for the treatment of cellular damage and the prevention of regulated cell death. PMID:27048815

  1. Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes.

    PubMed

    Willinger, Tim; Staron, Matthew; Ferguson, Shawn M; De Camilli, Pietro; Flavell, Richard A

    2015-04-01

    Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell-specific deletion of dynamin 2, an essential component of endocytosis, resulted in reduced TCR signaling strength, impaired homeostatic proliferation, and the inability to undergo clonal expansion in vivo. Blocking endocytosis resulted in a failure to maintain mammalian target of rapamycin (mTOR) activity and to stably induce the transcription factor myelocytomatosis oncogene (c-Myc), which led to metabolic stress and a defect in cell growth. Our results support the concept that the TCR can continue to signal after it is internalized from the cell surface, thereby enabling sustained signaling and cell proliferation.

  2. Mesenchymal Stem Cells and Myeloid Derived Suppressor Cells: Common Traits in Immune Regulation.

    PubMed

    Vladimirovna, Irina Lyadova; Sosunova, Ekaterina; Nikolaev, Alexander; Nenasheva, Tatiana

    2016-01-01

    To protect host against immune-mediated damage, immune responses are tightly regulated. The regulation of immune responses is mediated by various populations of mature immune cells, such as T regulatory cells and B regulatory cells, but also by immature cells of different origins. In this review, we discuss regulatory properties and mechanisms whereby two distinct populations of immature cells, mesenchymal stem cells, and myeloid derived suppressor cells mediate immune regulation, focusing on their similarities, discrepancies, and potential clinical applications. PMID:27529074

  3. Mesenchymal Stem Cells and Myeloid Derived Suppressor Cells: Common Traits in Immune Regulation

    PubMed Central

    Nikolaev, Alexander

    2016-01-01

    To protect host against immune-mediated damage, immune responses are tightly regulated. The regulation of immune responses is mediated by various populations of mature immune cells, such as T regulatory cells and B regulatory cells, but also by immature cells of different origins. In this review, we discuss regulatory properties and mechanisms whereby two distinct populations of immature cells, mesenchymal stem cells, and myeloid derived suppressor cells mediate immune regulation, focusing on their similarities, discrepancies, and potential clinical applications. PMID:27529074

  4. A defect in deletion of nucleosome-specific autoimmune T cells in lupus-prone thymus: role of thymic dendritic cells.

    PubMed

    Michaels, Marissa A; Kang, Hee-Kap; Kaliyaperumal, Arunan; Satyaraj, Ebenezar; Shi, Yan; Datta, Syamal K

    2005-11-01

    To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.

  5. Impaired oxidative phosphorylation regulates necroptosis in human lung epithelial cells.

    PubMed

    Koo, Michael Jakun; Rooney, Kristen T; Choi, Mary E; Ryter, Stefan W; Choi, Augustine M K; Moon, Jong-Seok

    2015-08-28

    Cellular metabolism can impact cell life or death outcomes. While metabolic dysfunction has been linked to cell death, the mechanisms by which metabolic dysfunction regulates the cell death mode called necroptosis remain unclear. Our study demonstrates that mitochondrial oxidative phosphorylation (OXPHOS) activates programmed necrotic cell death (necroptosis) in human lung epithelial cells. Inhibition of mitochondrial respiration and ATP synthesis induced the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and necroptotic cell death. Furthermore, we demonstrate that the activation of AMP-activated protein kinase (AMPK), resulting from impaired mitochondrial OXPHOS, regulates necroptotic cell death. These results suggest that impaired mitochondrial OXPHOS contributes to necroptosis in human lung epithelial cells.

  6. Late arrival: recruiting coreceptors to the T cell receptor complex.

    PubMed

    van der Merwe, P Anton; Cordoba, Shaun-Paul

    2011-01-28

    In this issue of Immunity, Jiang et al. (2011) provide evidence that the CD8 coreceptor is recruited to the T cell receptor (TCR) complex after initial TCR triggering where it stabilizes the TCR-peptide-major histocompatibility complex interaction. PMID:21272780

  7. Identification of a Late Stage of Small Noncycling pTα−  Pre-T Cells as Immediate Precursors of T Cell Receptor α/β+  Thymocytes

    PubMed Central

    Trigueros, César; Ramiro, Almudena R.; Carrasco, Yolanda R.; de Yebenes, Virginia G.; Albar, Juan P.; Toribio, María L.

    1998-01-01

    During thymocyte development, progression from T cell receptor (TCR)β to TCRα rearrangement is mediated by a CD3-associated pre-TCR composed of the TCRβ chain paired with pre-TCRα (pTα). A major issue is how surface expression of the pre-TCR is regulated during normal thymocyte development to control transition through this checkpoint. Here, we show that developmental expression of pTα is time- and stage-specific, and is confined in vivo to a limited subset of large cycling human pre-T cells that coexpress low density CD3. This restricted expression pattern allowed the identification of a novel subset of small CD3− thymocytes lacking surface pTα, but expressing cytoplasmic TCRβ, that represent late noncycling pre-T cells in which recombination activating gene reexpression and downregulation of T early α transcription are coincident events associated with cell cycle arrest, and immediately preceding TCRα gene expression. Importantly, thymocytes at this late pre-T cell stage are shown to be functional intermediates between large pTα+ pre-T cells and TCRα/β+ thymocytes. The results support a developmental model in which pre-TCR–expressing pre-T cells are brought into cycle, rapidly downregulate surface pre-TCR, and finally become small resting pre-T cells, before the onset of TCRα gene expression. PMID:9782117

  8. Intrinsic and extrinsic mechanisms regulating satellite cell function.

    PubMed

    Dumont, Nicolas A; Wang, Yu Xin; Rudnicki, Michael A

    2015-05-01

    Muscle stem cells, termed satellite cells, are crucial for skeletal muscle growth and regeneration. In healthy adult muscle, satellite cells are quiescent but poised for activation. During muscle regeneration, activated satellite cells transiently re-enter the cell cycle to proliferate and subsequently exit the cell cycle to differentiate or self-renew. Recent studies have demonstrated that satellite cells are heterogeneous and that subpopulations of satellite stem cells are able to perform asymmetric divisions to generate myogenic progenitors or symmetric divisions to expand the satellite cell pool. Thus, a complex balance between extrinsic cues and intrinsic regulatory mechanisms is needed to tightly control satellite cell cycle progression and cell fate determination. Defects in satellite cell regulation or in their niche, as observed in degenerative conditions such as aging, can impair muscle regeneration. Here, we review recent discoveries of the intrinsic and extrinsic factors that regulate satellite cell behaviour in regenerating and degenerating muscles.

  9. Intrinsic and extrinsic mechanisms regulating satellite cell function

    PubMed Central

    Dumont, Nicolas A.; Wang, Yu Xin; Rudnicki, Michael A.

    2015-01-01

    Muscle stem cells, termed satellite cells, are crucial for skeletal muscle growth and regeneration. In healthy adult muscle, satellite cells are quiescent but poised for activation. During muscle regeneration, activated satellite cells transiently re-enter the cell cycle to proliferate and subsequently exit the cell cycle to differentiate or self-renew. Recent studies have demonstrated that satellite cells are heterogeneous and that subpopulations of satellite stem cells are able to perform asymmetric divisions to generate myogenic progenitors or symmetric divisions to expand the satellite cell pool. Thus, a complex balance between extrinsic cues and intrinsic regulatory mechanisms is needed to tightly control satellite cell cycle progression and cell fate determination. Defects in satellite cell regulation or in their niche, as observed in degenerative conditions such as aging, can impair muscle regeneration. Here, we review recent discoveries of the intrinsic and extrinsic factors that regulate satellite cell behaviour in regenerating and degenerating muscles. PMID:25922523

  10. Profiling tissue-resident T cell repertoires by RNA sequencing.

    PubMed

    Brown, Scott D; Raeburn, Lisa A; Holt, Robert A

    2015-01-01

    Deep sequencing of recombined T cell receptor (TCR) genes and transcripts has provided a view of T cell repertoire diversity at an unprecedented resolution. Beyond profiling peripheral blood, analysis of tissue-resident T cells provides further insight into immune-related diseases. We describe the extraction of TCR sequence information directly from RNA-sequencing data from 6738 tumor and 604 control tissues, with a typical yield of 1 TCR per 10 million reads. This method circumvents the need for PCR amplification of the TCR template and provides TCR information in the context of global gene expression, allowing integrated analysis of extensive RNA-sequencing data resources. PMID:26620832

  11. Patterning as a signature of human epidermal stem cell regulation

    PubMed Central

    Klein, Allon M.; Nikolaidou-Neokosmidou, Varvara; Doupé, David P.; Jones, Philip H.; Simons, Benjamin D.

    2011-01-01

    Understanding how stem cells are regulated in adult tissues is a major challenge in cell biology. In the basal layer of human epidermis, clusters of almost quiescent stem cells are interspersed with proliferating and differentiating cells. Previous studies have shown that the proliferating cells follow a pattern of balanced stochastic cell fate. This behaviour enables them to maintain homeostasis, while stem cells remain confined to their quiescent clusters. Intriguingly, these clusters reappear spontaneously in culture, suggesting that they may play a functional role in stem cell auto-regulation. We propose a model of pattern formation that explains how clustering could regulate stem cell activity in homeostatic tissue through contact inhibition and stem cell aggregation. PMID:21632613

  12. SUMOylation-mediated regulation of cell cycle progression and cancer

    PubMed Central

    Eifler, Karolin; Vertegaal, Alfred C.O.

    2016-01-01

    SUMOylation plays critical roles during cell cycle progression. Many important cell cycle regulators, including many oncogenes and tumor suppressors, are functionally regulated via SUMOylation. The dynamic SUMOylation pattern observed throughout the cell cycle is ensured via distinct spatial and temporal regulation of the SUMO machinery. Additionally, SUMOylation cooperates with other post-translational modifications to mediate cell cycle progression. Deregulation of these SUMOylation and deSUMOylation enzymes causes severe defects in cell proliferation and genome stability. Different types of cancers were recently shown to be dependent on a functioning SUMOylation system, a finding that could potentially be exploited in anti-cancer therapies. PMID:26601932

  13. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

    PubMed Central

    Chu, D H; Spits, H; Peyron, J F; Rowley, R B; Bolen, J B; Weiss, A

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling. Images PMID:8947048

  14. Co-potentiation of antigen recognition: A mechanism to boost weak T cell responses and provide immunotherapy in vivo

    PubMed Central

    Hoffmann, Michele M.; Molina-Mendiola, Carlos; Nelson, Alfreda D.; Parks, Christopher A.; Reyes, Edwin E.; Hansen, Michael J.; Rajagopalan, Govindarajan; Pease, Larry R.; Schrum, Adam G.; Gil, Diana

    2015-01-01

    Adaptive immunity is mediated by antigen receptors that can induce weak or strong immune responses depending on the nature of the antigen that is bound. In T lymphocytes, antigen recognition triggers signal transduction by clustering T cell receptor (TCR)/CD3 multiprotein complexes. In addition, it hypothesized that biophysical changes induced in TCR/CD3 that accompany receptor engagement may contribute to signal intensity. Nonclustering monovalent TCR/CD3 engagement is functionally inert despite the fact that it may induce changes in conformational arrangement or in the flexibility of receptor subunits. We report that the intrinsically inert monovalent engagement of TCR/CD3 can specifically enhance physiologic T cell responses to weak antigens in vitro and in vivo without stimulating antigen-unengaged T cells and without interrupting T cell responses to strong antigens, an effect that we term as “co-potentiation.” We identified Mono-7D6-Fab, which biophysically altered TCR/CD3 when bound and functionally enhanced immune reactivity to several weak antigens in vitro, including a gp100-derived peptide associated with melanoma. In vivo, Mono-7D6-Fab induced T cell antigen–dependent therapeutic responses against melanoma lung metastases, an effect that synergized with other anti-melanoma immunotherapies to significantly improve outcome and survival. We conclude that Mono-7D6-Fab directly co-potentiated TCR/CD3 engagement by weak antigens and that such concept can be translated into an immunotherapeutic design. The co-potentiation principle may be applicable to other receptors that could be regulated by otherwise inert compounds whose latent potency is only invoked in concert with specific physiologic ligands. PMID:26601285

  15. Regulation of Water in Plant Cells

    ERIC Educational Resources Information Center

    Kowles, Richard V.

    2010-01-01

    Cell water relationships are important topics to be included in cell biology courses. Differences exist in the control of water relationships in plant cells relative to control in animal cells. One important reason for these differences is that turgor pressure is a consideration in plant cells. Diffusion and osmosis are the underlying factors…

  16. Role of autophagy in the regulation of epithelial cell junctions.

    PubMed

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  17. Coordinated regulation of myeloid cells by tumours.

    PubMed

    Gabrilovich, Dmitry I; Ostrand-Rosenberg, Suzanne; Bronte, Vincenzo

    2012-03-22

    Myeloid cells are the most abundant nucleated haematopoietic cells in the human body and are a collection of distinct cell populations with many diverse functions. The three groups of terminally differentiated myeloid cells - macrophages, dendritic cells and granulocytes - are essential for the normal function of both the innate and adaptive immune systems. Mounting evidence indicates that the tumour microenvironment alters myeloid cells and can convert them into potent immunosuppressive cells. Here, we consider myeloid cells as an intricately connected, complex, single system and we focus on how tumours manipulate the myeloid system to evade the host immune response.

  18. Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling.

    PubMed

    Corral-Jara, Karla F; Trujillo-Ochoa, Jorge L; Realpe, Mauricio; Panduro, Arturo; Gómez-Leyva, Juan F; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia; Fierro, Nora A

    2016-01-01

    We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1. PMID:27578921

  19. Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling

    PubMed Central

    Corral-Jara, Karla F.; Gómez-Leyva, Juan F.; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia

    2016-01-01

    We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1. PMID:27578921

  20. Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling.

    PubMed

    Corral-Jara, Karla F; Trujillo-Ochoa, Jorge L; Realpe, Mauricio; Panduro, Arturo; Gómez-Leyva, Juan F; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia; Fierro, Nora A

    2016-01-01

    We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1.

  1. IL12-mediated sensitizing of T-cell receptor-dependent and -independent tumor cell killing.

    PubMed

    Braun, Matthias; Ress, Marie L; Yoo, Young-Eun; Scholz, Claus J; Eyrich, Matthias; Schlegel, Paul G; Wölfl, Matthias

    2016-07-01

    Interleukin 12 (IL12) is a key inflammatory cytokine critically influencing Th1/Tc1-T-cell responses at the time of initial antigen encounter. Therefore, it may be exploited for cancer immunotherapy. Here, we investigated how IL12, and other inflammatory cytokines, shape effector functions of human T-cells. Using a defined culture system, we followed the gradual differentiation and function of antigen-specific CD8(+) T cells from their initial activation as naïve T cells through their expansion phase as early memory cells to full differentiation as clonally expanded effector T cells. The addition of IL12 8 days after the initial priming event initiated two mechanistically separate events: First, IL12 sensitized the T-cell receptor (TCR) for antigen-specific activation, leading to an approximately 10-fold increase in peptide sensitivity and, in consequence, enhanced tumor cell killing. Secondly, IL12 enabled TCR/HLA-independent activation and cytotoxicity: this "non-specific" effect was mediated by the NK cell receptor DNAM1 (CD226) and dependent on ligand expression of the target cells. This IL12 regulated, DNAM1-mediated killing is dependent on src-kinases as well as on PTPRC (CD45) activity. Thus, besides enhancing TCR-mediated activation, we here identified for the first time a second IL12 mediated mechanism leading to activation of a receptor-dependent killing pathway via DNAM1. PMID:27622043

  2. Localization and treatment of an oxidation-sensitive defect within the TCR-coupled signalling pathway that is associated with normal and premature immunologic aging.

    PubMed

    Weber, G F; Mirza, N M; Yunis, E J; Dubey, D; Cantor, H

    1997-01-01

    The age-dependent decline in the ability of T-cells to mount a proliferative response both to mitogens and to receptor ligation is due to an age-related defect in signal transduction, since functional expression of receptors displayed by aged T-cells is not reduced. We show here that, although turnover of phosphatidylinositol is not diminished, total inositol-trisphosphate generation decreases after T-cell receptor (TCR) ligation, resulting in reduced flux of calcium. Defective inositol-trisphosphate generation may result from impaired activation of phospholipase C due to decreased tyrosine phosphorylation of this enzyme after ligation of CD3 in aged cells. Proliferation of aged T-cells, which is normally 10-30% of the level of young controls, was enhanced almost tenfold by glutathione or its precursor N-acetyl L-cysteine (NAC), reached levels of young controls and was accompanied by restoration of normal inositol-trisphosphate generation and calcium flux. These findings suggest that the T-cell antigen receptor is associated with at least two types of signal transduction modules. The first depends on synthesis and phosphorylation of phosphatidylinositol that is independent of sulphydryl groups and is not affected by senescence. The second transduction module includes tyrosine phosphorylation and activation of phospholipase C. This module is regulated by glutathione levels and is diminished in aged T-cells, that are deficient in reducing equivalents which support the PLC gamma-dependent generation of inositol-trisphosphate from phosphatidylinositol derivatives. This underlying biochemical defect also occurs earlier in strains which display premature aging due to differences in the H-2 region of MHC I.

  3. Interstitial Cells: Regulators of Smooth Muscle Function

    PubMed Central

    Sanders, Kenton M.; Ward, Sean M.; Koh, Sang Don

    2014-01-01

    Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα+ cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα+ cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues. PMID:24987007

  4. New insights in the regulation of human B cell differentiation

    PubMed Central

    Schmidlin, Heike; Diehl, Sean A.; Blom, Bianca

    2009-01-01

    B lymphocytes provide the cellular basis of the humoral immune response. All stages of this process, from B cell activation to formation of germinal centers and differentiation into memory B cells or plasma cells, are influenced by extrinsic signals and controlled by transcriptional regulation. Compared to naïve B cells, memory B cells display a distinct expression profile, which allows for their rapid secondary responses. Indisputably, many B cell malignancies result from aberrations in the circuitry controlling B cell function, particularly during the GC reaction. Here we review new insights into memory B cell subtypes, recent literature on transcription factors regulating human B cell differentiation, and further evidence for B cell lymphomagenesis emanating from errors during the GC cell reactions. PMID:19447676

  5. The diabetogenic mouse MHC class II molecule I-A[subscript g7] is endowed with a switch that modulates TCR affinity

    SciTech Connect

    Yoshida, Kenji; Corper, Adam L.; Herro, Rana; Jabri, Bana; Wilson, Ian A.; Teyton, Luc

    2011-11-16

    Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-A{sub g7}, typically have a small, uncharged amino acid residue at position 57 of their {beta} chain ({beta}57); this results in the absence of a salt bridge between {beta}57 and Arg{alpha}76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Arg{alpha}76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3{beta}. The crystal structure of one such TCR in complex with I-A{sub g7} bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 {angstrom}) electrostatic interactions existed among Arg{alpha}76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue {beta}57 of the I-Ag7 and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

  6. The diabetogenic mouse MHC class II molecule I-A[superscript g7] is endowed with a switch that modulates TCR affinity

    SciTech Connect

    Yoshida, Kenji; Corper, Adam L.; Herro, Rana; Jabri, Bana; Wilson, Ian A.; Teyton, Luc

    2010-07-22

    Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-A{sup g7}, typically have a small, uncharged amino acid residue at position 57 of their {beta} chain ({beta}57); this results in the absence of a salt bridge between {beta}57 and Arg{alpha}76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Arg{alpha}76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3{beta}. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 {angstrom}) electrostatic interactions existed among Arg{alpha}76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue {beta}57 of the I-A{sup g7} and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

  7. Mevalonate Pathway Regulates Cell Size Homeostasis and Proteostasis through Autophagy

    PubMed Central

    Miettinen, Teemu P.; Björklund, Mikael

    2015-01-01

    Summary Balance between cell growth and proliferation determines cell size homeostasis, but little is known about how metabolic pathways are involved in the maintenance of this balance. Here, we perform a screen with a library of clinically used drug molecules for their effects on cell size. We find that statins, inhibitors of the mevalonate pathway, reduce cell proliferation and increase cell size and cellular protein density in various cell types, including primary human cells. Mevalonate pathway effects on cell size and protein density are mediated through geranylgeranylation of the small GTPase RAB11, which is required for basal autophagic flux. Our results identify the mevalonate pathway as a metabolic regulator of autophagy and expose a paradox in the regulation of cell size and proteostasis, where inhibition of an anabolic pathway can cause an increase in cell size and cellular protein density. PMID:26686643

  8. Creatine kinase in cell cycle regulation and cancer.

    PubMed

    Yan, Yong-Bin

    2016-08-01

    The phosphocreatine-creatine kinase (CK) shuttle system is increasingly recognized as a fundamental mechanism for ATP homeostasis in both excitable and non-excitable cells. Many intracellular processes are ATP dependent. Cell division is a process requiring a rapid rate of energy turnover. Cell cycle regulation is also a key point to understanding the mechanisms underlying cancer progression. It has been known for about 40 years that aberrant CK levels are associated with various cancers and for over 30 years that CK is involved in mitosis regulation. However, the underlying molecular mechanisms have not been investigated sufficiently until recently. By maintaining ATP at sites of high-energy demand, CK can regulate cell cycle progression by affecting the intracellular energy status as well as by influencing signaling pathways that are essential to activate cell division and cytoskeleton reorganization. Aberrant CK levels may impair cell viability under normal or stressed conditions and induce cell death. The involvement of CK in cell cycle regulation and cellular energy metabolism makes it a potential diagnostic biomarker and therapeutic target in cancer. To understand the multiple physiological/pathological functions of CK, it is necessary to identify CK-binding partners and regulators including proteins, non-coding RNAs and participating endogenous small molecular weight chemical compounds. This review will focus on molecular mechanisms of CK in cell cycle regulation and cancer progression. It will also discuss the implications of recent mechanistic studies, the emerging problems and future challenges of the multifunctional enzyme CK. PMID:27020776

  9. [Genetic regulation of plant shoot stem cells].

    PubMed

    Al'bert, E V; Ezhova, T A

    2013-02-01

    This article describes the main features of plant stem cells and summarizes the results of studies of the genetic control of stem cell maintenance in the apical meristem of the shoot. It is demonstrated that the WUS-CLV gene system plays a key role in the maintenance of shoot apical stem cells and the formation of adventitious buds and somatic embryos. Unconventional concepts of plant stem cells are considered.

  10. Laminin regulates PDGFRβ+ cell stemness and muscle development

    PubMed Central

    Yao, Yao; Norris, Erin H.; E. Mason, Christopher; Strickland, Sidney

    2016-01-01

    Muscle-resident PDGFRβ+ cells, which include pericytes and PW1+ interstitial cells (PICs), play a dual role in muscular dystrophy. They can either undergo myogenesis to promote muscle regeneration or differentiate into adipocytes and other cells to compromise regeneration. How the differentiation and fate determination of PDGFRβ+ cells are regulated, however, remains unclear. Here, by utilizing a conditional knockout mouse line, we report that PDGFRβ+ cell-derived laminin inhibits their proliferation and adipogenesis, but is indispensable for their myogenesis. In addition, we show that laminin alone is able to partially reverse the muscle dystrophic phenotype in these mice at the molecular, structural and functional levels. Further RNAseq analysis reveals that laminin regulates PDGFRβ+ cell differentiation/fate determination via gpihbp1. These data support a critical role of laminin in the regulation of PDGFRβ+ cell stemness, identify an innovative target for future drug development and may provide an effective treatment for muscular dystrophy. PMID:27138650

  11. Regulation of antiviral T cell responses by type I interferons.

    PubMed

    Crouse, Josh; Kalinke, Ulrich; Oxenius, Annette

    2015-04-01

    Type I interferons (IFNs) are pro-inflammatory cytokines that are rapidly induced in different cell types during viral infections. The consequences of type I IFN signalling include direct antiviral activity, innate immune cell activation and regulation of adaptive immune responses. In this Review, we discuss recent conceptual advances in our understanding of indirect and direct regulation of T cell immunity by type I IFNs, which can either promote or inhibit T cell activation, proliferation, differentiation and survival. This regulation depends, to a large extent, on the timing of type I IFN exposure relative to T cell receptor signalling. Type I IFNs also provide activated T cells with resistance to natural killer cell-mediated elimination. PMID:25790790

  12. Common stemness regulators of embryonic and cancer stem cells.

    PubMed

    Hadjimichael, Christiana; Chanoumidou, Konstantina; Papadopoulou, Natalia; Arampatzi, Panagiota; Papamatheakis, Joseph; Kretsovali, Androniki

    2015-10-26

    Pluripotency of embryonic stem cells (ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal transducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors (cancer stem cells), provides a common conceptual and research framework for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies.

  13. Common stemness regulators of embryonic and cancer stem cells

    PubMed Central

    Hadjimichael, Christiana; Chanoumidou, Konstantina; Papadopoulou, Natalia; Arampatzi, Panagiota; Papamatheakis, Joseph; Kretsovali, Androniki

    2015-01-01

    Pluripotency of embryonic stem cells (ESCs) and induced pluripotent stem cells is regulated by a well characterized gene transcription circuitry. The circuitry is assembled by ESC specific transcription factors, signal transducing molecules and epigenetic regulators. Growing understanding of stem-like cells, albeit of more complex phenotypes, present in tumors (cancer stem cells), provides a common conceptual and research framework for basic and applied stem cell biology. In this review, we highlight current results on biomarkers, gene signatures, signaling pathways and epigenetic regulators that are common in embryonic and cancer stem cells. We discuss their role in determining the cell phenotype and finally, their potential use to design next generation biological and pharmaceutical approaches for regenerative medicine and cancer therapies. PMID:26516408

  14. Regulation of floral stem cell termination in Arabidopsis

    PubMed Central

    Sun, Bo; Ito, Toshiro

    2015-01-01

    In Arabidopsis, floral stem cells are maintained only at the initial stages of flower development, and they are terminated at a specific time to ensure proper development of the reproductive organs. Floral stem cell termination is a dynamic and multi-step process involving many transcription factors, chromatin remodeling factors and signaling pathways. In this review, we discuss the mechanisms involved in floral stem cell maintenance and termination, highlighting the interplay between transcriptional regulation and epigenetic machinery in the control of specific floral developmental genes. In addition, we discuss additional factors involved in floral stem cell regulation, with the goal of untangling the complexity of the floral stem cell regulatory network. PMID:25699061

  15. Autoimmunity: regulatory B cells--IL-35 and IL-21 regulate the regulators.

    PubMed

    Tedder, Thomas F; Leonard, Warren J

    2014-08-01

    IL-21 regulates the activity and number of IL-10-producing regulatory B cells (B10 cells) that modulate immune responses and limit diverse autoimmune diseases. A new study demonstrates that IL-35 has a similar function. Identifying regulatory circuits that control B10-cell function in vivo might open the door to future treatments for autoimmune diseases.

  16. Regulation of Natural Killer Cell Function by STAT3

    PubMed Central

    Cacalano, Nicholas A.

    2016-01-01

    Natural killer (NK) cells, key members of a distinct hematopoietic lineage, innate lymphoid cells, are not only critical effectors that mediate cytotoxicity toward tumor and virally infected cells but also regulate inflammation, antigen presentation, and the adaptive immune response. It has been shown that NK cells can regulate the development and activation of many other components of the immune response, such as dendritic cells, which in turn, modulate the function of NK cells in multiple synergistic feed back loops driven by cell–cell contact, and the secretion of cytokines and chemokines that control effector function and migration of cells to sites of immune activation. The signal transducer and activator of transcription (STAT)-3 is involved in driving almost all of the pathways that control NK cytolytic activity as well as the reciprocal regulatory interactions between NK cells and other components of the immune system. In the context of tumor immunology, NK cells are a first line of defense that eliminates pre-cancerous and transformed cells early in the process of carcinogenesis, through a mechanism of “immune surveillance.” Even after tumors become established, NK cells are critical components of anticancer immunity: dysfunctional NK cells are often found in the peripheral blood of cancer patients, and the lack of NK cells in the tumor microenvironment often correlates to poor prognosis. The pathways and soluble factors activated in tumor-associated NK cells, cancer cells, and regulatory myeloid cells, which determine the outcome of cancer immunity, are all critically regulated by STAT3. Using the tumor microenvironment as a paradigm, we present here an overview of the research that has revealed fundamental mechanisms through which STAT3 regulates all aspects of NK cell biology, including NK development, activation, target cell killing, and fine tuning of the innate and adaptive immune responses. PMID:27148255

  17. Regulation of Cell Adhesion Strength by Peripheral Focal Adhesion Distribution

    PubMed Central

    Elineni, Kranthi Kumar; Gallant, Nathan D.

    2011-01-01

    Cell adhesion to extracellular matrices is a tightly regulated process that involves the complex interplay between biochemical and mechanical events at the cell-adhesive interface. Previous work established the spatiotemporal contributions of adhesive components to adhesion strength and identified a nonlinear dependence on cell spreading. This study was designed to investigate the regulation of cell-adhesion strength by the size and position of focal adhesions (FA). The cell-adhesive interface was engineered to direct FA assembly to the periphery of the cell-spreading area to delineate the cell-adhesive area from the cell-spreading area. It was observed that redistributing the same adhesive area over a larger cell-spreading area significantly enhanced cell-adhesion strength, but only up to a threshold area. Moreover, the size of the peripheral FAs, which was interpreted as an adhesive patch, did not directly govern the adhesion strength. Interestingly, this is in contrast to the previously reported functional role of FAs in regulating cellular traction where sizes of the peripheral FAs play a critical role. These findings demonstrate, to our knowledge for the first time, that two spatial regimes in cell-spreading area exist that uniquely govern the structure-function role of FAs in regulating cell-adhesion strength. PMID:22208188

  18. Substrate stress relaxation regulates cell spreading

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Ovijit; Gu, Luo; Darnell, Max; Klumpers, Darinka; Bencherif, Sidi A.; Weaver, James C.; Huebsch, Nathaniel; Mooney, David J.

    2015-02-01

    Studies of cellular mechanotransduction have converged upon the idea that cells sense extracellular matrix (ECM) elasticity by gauging resistance to the traction forces they exert on the ECM. However, these studies typically utilize purely elastic materials as substrates, whereas physiological ECMs are viscoelastic, and exhibit stress relaxation, so that cellular traction forces exerted by cells remodel the ECM. Here we investigate the influence of ECM stress relaxation on cell behaviour through computational modelling and cellular experiments. Surprisingly, both our computational model and experiments find that spreading for cells cultured on soft substrates that exhibit stress relaxation is greater than cells spreading on elastic substrates of the same modulus, but similar to that of cells spreading on stiffer elastic substrates. These findings challenge the current view of how cells sense and respond to the ECM.

  19. Substrate stress relaxation regulates cell spreading

    PubMed Central

    Chaudhuri, Ovijit; Gu, Luo; Darnell, Max; Klumpers, Darinka; Bencherif, Sidi A.; Weaver, James C.; Huebsch, Nathaniel; Mooney, David J

    2015-01-01

    Studies of cellular mechanotransduction have converged upon the idea that cells sense extracellular matrix (ECM) elasticity by gauging resistance to the traction forces they exert on the ECM. However, these studies typically utilize purely elastic materials as substrates, whereas physiological ECM are viscoelastic, and exhibit stress relaxation, so that cellular traction forces exerted by cells remodel the ECM. Here we investigate the influence of ECM stress relaxation on cell behavior through computational modeling and cellular experiments. Surprisingly, both our computational model and experiments find that spreading for cells cultured on soft substrates that exhibit stress relaxation is greater than cells spreading on elastic substrates of the same modulus, but similar to that of cells spreading on stiffer elastic substrates. These findings challenge the current view of how cells sense and respond to the ECM. PMID:25695512

  20. Nanotechnology in the regulation of stem cell behavior

    NASA Astrophysics Data System (ADS)

    Wu, King-Chuen; Tseng, Ching-Li; Wu, Chi-Chang; Kao, Feng-Chen; Tu, Yuan-Kun; So, Edmund C.; Wang, Yang-Kao

    2013-10-01

    Stem cells are known for their potential to repair damaged tissues. The adhesion, growth and differentiation of stem cells are likely controlled by the surrounding microenvironment which contains both chemical and physical cues. Physical cues in the microenvironment, for example, nanotopography, were shown to play important roles in stem cell fate decisions. Thus, controlling stem cell behavior by nanoscale topography has become an important issue in stem cell biology. Nanotechnology has emerged as a new exciting field and research from this field has greatly advanced. Nanotechnology allows the manipulation of sophisticated surfaces/scaffolds which can mimic the cellular environment for regulating cellular behaviors. Thus, we summarize recent studies on nanotechnology with applications to stem cell biology, including the regulation of stem cell adhesion, growth, differentiation, tracking and imaging. Understanding the interactions of nanomaterials with stem cells may provide the knowledge to apply to cell-scaffold combinations in tissue engineering and regenerative medicine.

  1. Receptor regulation of osmolyte homeostasis in neural cells.

    PubMed

    Fisher, Stephen K; Heacock, Anne M; Keep, Richard F; Foster, Daniel J

    2010-09-15

    The capacity of cells to correct their volume in response to hyposmotic stress via the efflux of inorganic and organic osmolytes is well documented. However, the ability of cell-surface receptors, in particular G-protein-coupled receptors (GPCRs), to regulate this homeostatic mechanism has received much less attention. Mechanisms that underlie the regulation of cell volume are of particular importance to cells in the central nervous system because of the physical restrictions of the skull and the adverse impact that even small increases in cell volume can have on their function. Increases in brain volume are seen in hyponatraemia, which can arise from a variety of aetiologies and is the most frequently diagnosed electrolyte disorder in clinical practice. In this review we summarize recent evidence that the activation of GPCRs facilitates the volume-dependent efflux of osmolytes from neural cells and permits them to more efficiently respond to small, physiologically relevant, reductions in osmolarity. The characteristics of receptor-regulated osmolyte efflux, the signalling pathways involved and the physiological significance of receptor activation are discussed. In addition, we propose that GPCRs may also regulate the re-uptake of osmolytes into neural cells, but that the influx of organic and inorganic osmolytes is differentially regulated. The ability of neural cells to closely regulate osmolyte homeostasis through receptor-mediated alterations in both efflux and influx mechanisms may explain, in part at least, why the brain selectively retains its complement of inorganic osmolytes during chronic hyponatraemia, whereas its organic osmolytes are depleted. PMID:20498228

  2. Bcl-2 family proteins: master regulators of cell survival.

    PubMed

    Hatok, Jozef; Racay, Peter

    2016-08-01

    The most prominent function of proteins of the Bcl-2 family is regulation of the initiation of intrinsic (mitochondrial) pathways of apoptosis. However, recent research has revealed that in addition to regulation of mitochondrial apoptosis, proteins of the Bcl-2 family play important roles in regulating other cellular pathways with a strong impact on cell survival like autophagy, endoplasmic reticulum (ER) stress response, intracellular calcium dynamics, cell cycle progression, mitochondrial dynamics and energy metabolism. This review summarizes the recent knowledge about functions of Bcl-2 family proteins that are related to cell survival. PMID:27505095

  3. A Conserved Hydrophobic Patch on Vβ Domains Revealed by TCRβ Chain Crystal Structures: Implications for Pre-TCR Dimerization

    PubMed Central

    Zhou, Bo; Chen, Qiang; Mallis, Robert J.; Zhang, Hongmin; Liu, Jin-huan; Reinherz, Ellis L.; Wang, Jia-huai

    2011-01-01

    The αβ T cell receptor (TCR) is a multimeric complex whose β chain plays a crucial role in thymocyte development as well as antigen recognition by mature T lymphocytes. We report here crystal structures of individual β subunits, termed N15β (Vβ5.2Dβ2Jβ2.6Cβ2) and N30β (Vβ13Dβ1Jβ1.1Cβ2), derived from two αβ TCRs specific for the immunodominant vesicular stomatitis virus octapeptide (VSV-8) bound to the murine H-2Kb MHC class I molecule. The crystal packing of the N15β structure reveals a homodimer formed through two Vβ domains. The Vβ/Vβ module is topologically very similar to the Vα/Vβ module in the N15αβ heterodimer. By contrast, in the N30β structure, the Vβ domain’s external hydrophobic CFG face is covered by the neighboring molecule’s Cβ domain. In conjunction with systematic investigation of