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Sample records for antigen receptor genes

  1. Analysis of antigen receptor genes in Hodgkin's disease.

    PubMed Central

    Angel, C A; Pringle, J H; Naylor, J; West, K P; Lauder, I

    1993-01-01

    AIM--To analyse the configuration of the antigen receptor genes in Hodgkin's disease. METHODS--DNA extracted from 45 samples of Hodgkin's disease was analysed using Southern blotting and DNA hybridisation, using probes to the joining region of the immunoglobulin heavy chain gene, the constant region of kappa immunoglobulin light chain gene, and the constant region of the beta chain of the T cell receptor gene. RESULTS--A single case of nodular sclerosing disease showed clonal rearrangement of the immunoglobulin heavy and light chain genes, all other samples having germline immunoglobulin genes. The nature of the clonal population in the diseased tissue is uncertain, because the intensity of the rearranged bands did not correlate with the percentage of Reed-Sternberg cells present. The T cell receptor genes were in germline configuration in all the samples. CONCLUSIONS--Antigen receptor gene rearrangement is a rare finding in unselected cases of Hodgkin's disease. Images PMID:8388407

  2. Immunophenotypic and antigen receptor gene rearrangement analysis in T cell neoplasia.

    PubMed Central

    Knowles, D. M.

    1989-01-01

    The author reviews the immunophenotypic profiles displayed by the major clinicopathologic categories of T cell neoplasia, the immunophenotypic criteria useful in the immunodiagnosis of T cell neoplasia, and the contributions made by antigen receptor gene rearrangement analysis to the understanding of T cell neoplasia. Neoplasms belonging to distinct clinicopathologic categories of T cell neoplasia often exhibit characteristic immunophenotypic profiles. Approximately 80% of lymphoblastic lymphomas and 20% of acute lymphoblastic leukemias express phenotypes consistent with prethymic and intrathymic stages of T cell differentiation, including intranuclear terminal deoxynucleotidyl transferase. Cutaneous T cell lymphomas of mycosis fungoides type usually express pan-T cell antigens CD2, CD5, and CD3, often lack the pan-T cell antigen CD7, and usually express the mature, peripheral helper subset phenotype, CD4+ CD8-. Cutaneous T cell lymphomas of nonmycosis fungoides type and peripheral T cell lymphomas often lack one or more pan-T cell antigens and, in addition, occasionally express the anomalous CD4+ CD8+ or CD4- CD8- phenotypes. T gamma-lymphoproliferative disease is divisable into two broad categories: those cases that are CD3 antigen positive and exhibit clonal T cell receptor beta chain (TCR-beta) gene rearrangements and those cases that are CD3 antigen negative and exhibit the TCR-beta gene germline configuration. Human T cell lymphotropic virus-I (HTLV-I) associated Japanese, Carribean, and sporadic adult T cell leukemia/lymphomas usually express pan-T cell antigens, the CD4+ CD8- phenotype, and various T cell-associated activation antigens, including the interleukin-2 receptor (CD25). Immunophenotypic criteria useful in the immunodiagnosis of T cell neoplasia include, in increasing order of utility, T cell predominance, T cell subset antigen restriction, anomalous T cell subset antigen expression, and deletion of one or more pan-T cell antigens. Only in

  3. Molecular Pathways: Breaking the Epithelial Cancer Barrier for Chimeric Antigen Receptor and T-cell Receptor Gene Therapy.

    PubMed

    Hinrichs, Christian S

    2016-04-01

    Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.

  4. Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

    PubMed

    Diebold, Sandra S; Plank, Christian; Cotten, Matt; Wagner, Ernst; Zenke, Martin

    2002-11-01

    Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

  5. An evolutionarily mobile antigen receptor variable region gene: doubly rearranging NAR-TcR genes in sharks.

    PubMed

    Criscitiello, Michael F; Saltis, Mark; Flajnik, Martin F

    2006-03-28

    Distinctive Ig and T cell receptor (TcR) chains define the two major lineages of vertebrate lymphocyte yet similarly recognize antigen with a single, membrane-distal variable (V) domain. Here we describe the first antigen receptor chain that employs two V domains, which are generated by separate VDJ gene rearrangement events. These molecules have specialized "supportive" TcRdeltaV domains membrane-proximal to domains with most similarity to IgNAR V. The ancestral NAR V gene encoding this domain is hypothesized to have recombined with the TRD locus in a cartilaginous fish ancestor >200 million years ago and encodes the first V domain shown to be used in both Igs and TcRs. Furthermore, these data support the view that gamma/delta TcRs have for long used structural conformations recognizing free antigen.

  6. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism

    PubMed Central

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-01-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leukocyte antigen/killer - cell immunoglobulin - like receptor complexes are increased in people with autism. We present data, which suggests that an activating cB01/tA01 killer cell immunoglobulin - like receptor gene - content haplotype and the cognate ligand human leukocyte antigen - C1k that activates this haplotype is significantly increased in autism. This is an important observation suggesting that the interaction between two proteins encoded on different chromosomes increases natural killer cell killing in autism. PMID:27853655

  7. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism.

    PubMed

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-04-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leukocyte antigen/killer - cell immunoglobulin - like receptor complexes are increased in people with autism. We present data, which suggests that an activating cB01/tA01 killer cell immunoglobulin - like receptor gene - content haplotype and the cognate ligand human leukocyte antigen - C1k that activates this haplotype is significantly increased in autism. This is an important observation suggesting that the interaction between two proteins encoded on different chromosomes increases natural killer cell killing in autism.

  8. Antigen-receptor gene-modified T cells for treatment of glioma.

    PubMed

    Ikeda, Hiroaki; Shiku, Hiroshi

    2012-01-01

    Immunological effector cells and molecules have been shown to access intracranial tumor sites despite the existence of blood brain barrier (BBB) or immunosuppressive mechanisms associated with brain tumors. Recent progress in T-cell biology and tumor immunology made possible to develop strategies of tumor-associated antigen-specific immunotherapeutic approaches such as vaccination with defined antigens and adoptive T-cell therapy with antigen-specific T cells including gene-modified T cells for the treatment of patients with brain tumors. An array of recent reports on the trials of active and passive immunotherapy for patients with brain tumors have documented safety and some preliminary clinical efficacy, although the ultimate judgment for clinical benefits awaits rigorous evaluation in trials of later phases. Nevertheless, treatment with lymphocytes that are engineered to express tumor-specific receptor genes is a promising immunotherapy against glioma, based on the significant efficacy reported in the trials for patients with other types of malignancy. Overcoming the relative difficulty to apply immunotherapeutic approach to intracranial region, current advances in the understanding of human tumor immunology and the gene-therapy methodology will address the development of effective immunotherapy of brain tumors.

  9. Adoptive immunotherapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

    PubMed

    Fujiwara, Hiroshi

    2014-12-15

    Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI) and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL) for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as "cellular drugs". As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs), transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR) gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

  10. Recombinative events of the T cell antigen receptor delta gene in peripheral T cell lymphomas.

    PubMed Central

    Kanavaros, P; Farcet, J P; Gaulard, P; Haioun, C; Divine, M; Le Couedic, J P; Lefranc, M P; Reyes, F

    1991-01-01

    Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation. Images PMID:1991851

  11. [Novel therapy for malignant lymphoma: adoptive immuno-gene therapy using chimeric antigen receptor(CAR)-expressing T lymphocytes].

    PubMed

    Ozawa, Keiya

    2014-03-01

    Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed.

  12. Detection of circulating tumor cells using GeneScan analysis for antigen receptor gene rearrangements in canine lymphoma patients

    PubMed Central

    HIYOSHI-KANEMOTO, Saaya; GOTO-KOSHINO, Yuko; FUKUSHIMA, Kenjiro; TAKAHASHI, Masashi; KANEMOTO, Hideyuki; UCHIDA, Kazuyuki; FUJINO, Yasuhito; OHNO, Koichi; TSUJIMOTO, Hajime

    2016-01-01

    The presence of circulating tumor cells (CTCs) serves as a prognostic marker and indicator of disease relapse, as well as a means of evaluating treatment efficacy in human and canine lymphoma patients. As an extension of our previous study for the construction of clinically useful GeneScan system, we utilized the GeneScan system for detecting CTCs in canine lymphoma patients. Samples from the primary lesion and peripheral blood mononuclear cells (PBMCs) were obtained from 32 dogs with lymphoma at initial diagnosis. All samples were subjected to polymerase chain reaction (PCR) for antigen receptor gene rearrangements (PARR) followed by GeneScan analysis. Common clonal rearrangements with identical amplified fragments were detected in both the primary lesion and PBMCs in 19 of the 32 dogs (59.4%). However, the detection rate of CTCs varied among the anatomical classification of lymphoma studied. GeneScan analysis following PARR would facilitate studies on determining the clinical significance of CTCs in canine lymphoma patients. PMID:26888583

  13. In Vitro Generation of Human NK cells Expressing Chimeric Antigen Receptor through Differentiation of Gene-Modified Hematopoietic Stem Cells

    PubMed Central

    Lowe, Emily; Truscott, Laurel C.; De Oliveira, Satiro N.

    2016-01-01

    Summary NK cells represent a very promising source for adoptive cellular approaches for cancer immunotherapy, and extensive research has been conducted, including clinical trials. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer techniques is very limited. Here we describe two protocols designed to generate mature human NK cells from gene-modified hematopoietic stem cells. These protocols use chimeric antigen receptor as the transgene, but could potentially be modified for the expression any particular transgene in human NK cells. PMID:27177671

  14. The human application of gene therapy to re-program T-cell specificity using chimeric antigen receptors

    PubMed Central

    Guerrero, Alan D; Moyes, Judy S; Cooper, Laurence JN

    2014-01-01

    The adoptive transfer of T cells is a promising approach to treat cancers. Primary human T cells can be modified using viral and non-viral vectors to promote the specific targeting of cancer cells via the introduction of exogenous T-cell receptors (TCRs) or chimeric antigen receptors (CARs). This gene transfer displays the potential to increase the specificity and potency of the anticancer response while decreasing the systemic adverse effects that arise from conventional treatments that target both cancerous and healthy cells. This review highlights the generation of clinical-grade T cells expressing CARs for immunotherapy, the use of these cells to target B-cell malignancies and, particularly, the first clinical trials deploying the Sleeping Beauty gene transfer system, which engineers T cells to target CD19+ leukemia and non-Hodgkin's lymphoma. PMID:25189715

  15. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen

    PubMed Central

    Johnson, Laura A.; Morgan, Richard A.; Dudley, Mark E.; Cassard, Lydie; Yang, James C.; Hughes, Marybeth S.; Kammula, Udai S.; Royal, Richard E.; Sherry, Richard M.; Wunderlich, John R.; Lee, Chyi-Chia R.; Restifo, Nicholas P.; Schwarz, Susan L.; Cogdill, Alexandria P.; Bishop, Rachel J.; Kim, Hung; Brewer, Carmen C.; Rudy, Susan F.; VanWaes, Carter; Davis, Jeremy L.; Mathur, Aarti; Ripley, Robert T.; Nathan, Debbie A.; Laurencot, Carolyn M.

    2009-01-01

    Gene therapy of human cancer using genetically engineered lymphocytes is dependent on the identification of highly reactive T-cell receptors (TCRs) with antitumor activity. We immunized transgenic mice and also conducted high-throughput screening of human lymphocytes to generate TCRs highly reactive to melanoma/melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA− and CD45RO+ after ex vivo expansion. After infusion, the persisting cells displayed a CD45RA+ and CD45RO− phenotype. Gene-engineered cells persisted at high levels in the blood of all patients 1 month after treatment, responding patients with higher ex vivo antitumor reactivity than nonresponders. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear, and sometimes required local steroid administration to treat uveitis and hearing loss. Thus, T cells expressing highly reactive TCRs mediate cancer regression in humans and target rare cognate–antigen-containing cells throughout the body, a finding with important implications for the gene therapy of cancer. This trial was registered at www.ClinicalTrials.gov as NCI-07-C-0174 and NCI-07-C-0175. PMID:19451549

  16. T-cell receptor gene therapy targeting melanoma-associated antigen-A4 inhibits human tumor growth in non-obese diabetic/SCID/γcnull mice.

    PubMed

    Shirakura, Yoshitaka; Mizuno, Yukari; Wang, Linan; Imai, Naoko; Amaike, Chisaki; Sato, Eiichi; Ito, Mamoru; Nukaya, Ikuei; Mineno, Junichi; Takesako, Kazutoh; Ikeda, Hiroaki; Shiku, Hiroshi

    2012-01-01

    Adoptive cell therapy with lymphocytes that have been genetically engineered to express tumor-reactive T-cell receptors (TCR) is a promising approach for cancer immunotherapy. We have been exploring the development of TCR gene therapy targeting cancer/testis antigens, including melanoma-associated antigen (MAGE) family antigens, that are ideal targets for adoptive T-cell therapy. The efficacy of TCR gene therapy targeting MAGE family antigens, however, has not yet been evaluated in vivo. Here, we demonstrate the in vivo antitumor activity in immunodeficient non-obese diabetic/SCID/γc(null) (NOG) mice of human lymphocytes genetically engineered to express TCR specific for the MAGE-A4 antigen. Polyclonal T cells derived from human peripheral blood mononuclear cells were transduced with the αβ TCR genes specific for MAGE-A4, then adoptively transferred into NOG mice inoculated with MAGE-A4 expressing human tumor cell lines. The transferred T cells maintained their effector function in vivo, infiltrated into tumors, and inhibited tumor growth in an antigen-specific manner. The combination of adoptive cell therapy with antigen peptide vaccination enhanced antitumor activity, with improved multifunctionality of the transferred cells. These data suggest that TCR gene therapy with MAGE-A4-specific TCR is a promising strategy to treat patients with MAGE-A4-expressing tumors; in addition, the acquisition of multifunctionality in vivo is an important factor to predict the quality of the T-cell response during adoptive therapy with human lymphocytes.

  17. Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

    PubMed

    Fujiwara, Hiroshi

    2014-02-01

    The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

  18. Physical mapping of the human T-cell antigen receptor (TCR) {beta}-chain gene complex

    SciTech Connect

    Yashim, Y.; So, A.K.

    1994-09-01

    The genetic variation of the TCR loci and their contribution to autoimmune diseases is poorly defined, in direct contrast to the clear examples of disease association with the Class I and II alleles of the major histocompatibility complex. We have therefore started to determine the gene organization and polymorphism of the TCR {beta} locus. Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human TCR {beta}-chain gene complex. Variable gene (V{beta}) sequences for the 25 known V{beta} subfamilies were amplified by PCR and were used as probes to screen a YAC library. Five positive YACs were identified. YACs designated B3, E11 and H11 of sizes 820, 400 and 600 kbp, respectively, were analyzed for their V{beta} content by pulse-field gel electrophoresis (PFGE). YAC B3 was found to contain all 25 V{beta} subfamilies, E11 for 14 and H11 for 7. B3 was also positive for the constant region genes. Restriction enzyme mapping of B3 located V{beta} and C{beta} gene regions to four Sfi I fragments of 280, 110, 90 and 125 kbp, and was in accordance with published data. The data thus showed that YAC B3 encoded a complete and unrearranged TCR {beta}-gene locus. The map was further resolved by locating restriction sites for Sal I and Bssll II on B3. Fluorescent in situ hybridization to human metaphase chromosomes localized B3 to chromosome 7q35. However, two additional signals were obtained: one attributable to V{beta} orphon cluster on chromosome 9q21; the second to the long arm of chromosome 2. PCR amplification of a chromosome 2 somatic cell hybrid using primers for all 25 V{beta} gene families revealed the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCR {beta} locus flanked by chromosome 2 sequences. The determination of the TCR genomic organization will help extend studies of the role T-cells play in autoimmune diseases.

  19. Human Leukocyte Antigen (HLA) A*1101-Restricted Epstein-Barr Virus-Specific T-cell Receptor Gene Transfer to Target Nasopharyngeal Carcinoma.

    PubMed

    Zheng, Yong; Parsonage, Greg; Zhuang, Xiaodong; Machado, Lee R; James, Christine H; Salman, Asmaa; Searle, Peter F; Hui, Edwin P; Chan, Anthony T C; Lee, Steven P

    2015-10-01

    Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)-associated posttransplant lymphomas, and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favor outgrowth of T cells targeting viral antigens expressed within EBV(+) lymphomas, but not in NPC. Here, we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a human leukocyte antigen (HLA) A*1101-restricted TCR, which would be widely applicable because 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms, we have optimized expression of the TCR and demonstrated high-avidity antigen-specific function (proliferation, cytotoxicity, and cytokine release) in both CD8(+) and CD4(+) T cells. The engineered T cells also inhibited LMP2(+) epithelial tumor growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high-avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens.

  20. Stability of multiple antigen receptor gene rearrangements and immunophenotype in Hodgkin's disease-derived cell line L428 and variant subline L428KSA.

    PubMed

    Athan, E S; Paietta, E; Papenhausen, P R; Augenlicht, L; Wiernik, P H; Gallagher, R E

    1989-07-01

    The Hodgkin's disease (HD) derived cell line L428 and a phorbol ester-selected subline L428KSA, which have been independently passaged in tissue culture for several years, were studied for possible antigen receptor gene and immunophenotypic differences. Multiple but identical alterations of these genes were found, including: the deletion of one and rearrangement of the other immunoglobulin (Ig) heavy chain allele; the rearrangement of one kappa and one lambda light chain allele; and the rearrangement of one T cell receptor (TCR) beta allele. Restriction mapping of the Ig heavy chain locus indicated that rearrangement of the retained allele produced a JH-C gamma 4 fusion product by an isotype switch mechanism. The 14q+ chromosome [t(14q32;?)] present in both cell cultures derived either from translocation 5' (telomeric) to the rearranged JH allele or 3' (centromeric) to the deleted Ig heavy chain allele and did not involve detectable rearrangement of the c-myc, bcl 1, or bcl 2 oncogenes. No differences in the immunophenotype were found between the L428 and L428KSA cells: both expressed leukocyte activation antigens and some determinants associated with myelomonocytic cells but no lymphoid markers. It is postulated that these phenotypic characteristics derived from secondary genetic events/differentiative reprogramming which produced extinction of primary lymphoid characters, including terminal deoxynucleotidyl transferase (TdT) essential to generation of the Ig and TCR gene rearrangements, and expression of an incomplete set of myelomonocytic markers.

  1. Human Leukocyte Antigen (HLA) A*1101-restricted Epstein-Barr Virus-specific T-cell Receptor Gene Transfer to Target Nasopharyngeal Carcinoma

    PubMed Central

    Zheng, Yong; Parsonage, Greg; Zhuang, Xiaodong; Machado, Lee R; James, Christine H.; Salman, Asmaa; Searle, Peter F.; Hui, Edwin P.; Chan, Anthony T.C.; Lee, Steven P.

    2015-01-01

    Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)-associated post-transplant lymphomas and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favour outgrowth of T cells targeting viral antigens expressed within EBV+ lymphomas but not in NPC. Here we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a HLA A*1101-restricted TCR, which would be widely applicable since 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms we have optimised expression of the TCR and demonstrated high avidity antigen-specific function (proliferation, cytotoxicity, cytokine release) in both CD8+ and CD4+ T cells. The engineered T cells also inhibited LMP2+ epithelial tumour growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens. PMID:25711537

  2. Androgen receptor and prostate-specific antigen gene polymorphisms and breast cancer in African-American women.

    PubMed

    Wang, Wei; John, Esther M; Ingles, Sue Ann

    2005-12-01

    Several previous studies have found the CAG repeat polymorphism in exon 1 of the androgen receptor (AR) gene to be associated with breast cancer risk among some groups of Caucasian and Asian women. In a population-based case-control study of 488 African-American women (239 cases and 249 controls), we examined this polymorphism along with a polymorphism (-158 G/A) in an androgen-regulated gene (PSA) whose expression has been correlated with breast cancer prognosis. Overall, we did not observe any significant association between the CAG repeat polymorphism and breast cancer risk. However, among women with a first-degree family history of breast cancer, longer CAG repeats were associated with a significantly increased risk. Women carrying at least one longer allele [(CAG)n > or = 22] had a 3-fold increased risk compared to those with two shorter alleles (odds ratio, 3.18; 95% confidence interval, 1.08-9.36). There was no significant association between the PSA gene polymorphism and breast cancer risk, nor was there significant gene-gene interaction. In summary, our results further support that shorter CAG repeats (stronger AR transactivation activity) may reduce the risk of breast cancer, at least among some groups of women. Our data, however, are unable to provide evidence that PSA is the pathway through which the protective effect of androgens operates.

  3. In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia

    PubMed Central

    Minagawa, Kentaro; Jamil, Muhammad O.; AL-Obaidi, Mustafa; Pereboeva, Larisa; Salzman, Donna; Erba, Harry P.; Lamb, Lawrence S.; Bhatia, Ravi; Mineishi, Shin

    2016-01-01

    Background Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia. Methods and findings We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85–90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a

  4. Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

    PubMed Central

    Davis, Melissa B.; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M.; Ford, DeJuana; Howerth, Elizabeth W.; Monteil, Michele

    2015-01-01

    The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

  5. Detection of monoclonality in intestinal lymphoma with polymerase chain reaction for antigen receptor gene rearrangement analysis to differentiate from enteritis in dogs.

    PubMed

    Ohmura, S; Leipig, M; Schöpper, I; Hergt, F; Weber, K; Rütgen, B C; Tsujimoto, H; Hermanns, W; Hirschberger, J

    2017-03-01

    The diagnosis of canine intestinal lymphoma by morphological examination is challenging, especially when endoscopic tissue specimens are used. The utility of detection of antigen receptor gene rearrangement by polymerase chain reaction (PARR) in canine lymphoma has been well established, but its usefulness to distinguish enteritis and intestinal lymphoma remains unclear. In this retrospective study we assessed clonality of 29 primary canine intestinal lymphoma, 14 enteritis and 15 healthy control cases by PARR analysis, using formalin-fixed, paraffin-embedded full-thickness tissue specimens. We could detect monoclonal rearrangements in 22 of 29 canine intestinal lymphomas [76%; 95% confidence interval (CI) 56-90%] and polyclonal rearrangements in all of the enteritis and healthy control cases (100%; CI 88-100%). We revealed a predominance of T-cell phenotype compared to B-cell phenotype (85%; CI 65-96% and 15%; CI 4-35%, respectively). We showed that PARR analysis contributes to differentiation of canine intestinal lymphoma from enteritis and to phenotyping of lymphomas.

  6. Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors

    PubMed Central

    Moot, Robert; Raikar, Sunil S; Fleischer, Lauren; Querrey, Melissa; Tylawsky, Daniel E; Nakahara, Hirotomo; Doering, Christopher B; Spencer, H Trent

    2016-01-01

    Chimeric antigen receptors (CARs) are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs). VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig)-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition. PMID:27933313

  7. [Clonality lymphoid study through rearrangement analysis of antigen receptor].

    PubMed

    Villamizar-Rivera, Nicolás; Olaya, Natalia

    2015-01-01

    As a rule, malignant lymphoid proliferations are clonal. While most of the time the biological potential can be established through routine pathologic examination and auxiliary techniques, some cases are difficult to classify. Moreover, there are situations in which there are dominant clones whose analysis are important, such as occur in autoimmune diseases and immunodeficiency. This paper presents in an understandable way the main techniques for the study of clonality in lymphoid lesions, i.e. the analysis of rearrangements of antigen receptor genes by multiplex polymerase chain reaction (PCR) based tests.

  8. 4-1BB chimeric antigen receptors.

    PubMed

    Campana, Dario; Schwarz, Herbert; Imai, Chihaya

    2014-01-01

    In addition to T-cell receptor signals, T lymphocytes require costimulatory signals for robust activation. Among these, those mediated by 4-1BB (CD137, TNFRSF9) are critical for tumor immunity. 4-1BB is expressed in T-cell receptor-activated lymphocytes as well as natural killer cells and other hematopoietic and nonhematopoietic cells. 4-1BB ligation induces a signaling cascade that results in cytokine production, expression of antiapoptotic molecules, and enhanced immune responses. In line with the described function of 4-1BB, its addition to CD3ζ chimeric antigen receptors (CARs) increases their capacity to provoke T-cell expansion and antitumor activity. The results of preclinical studies with 4-1BB CARs have been corroborated by encouraging results from clinical trials. Advantages and disadvantages of 4-1BB CARs versus CARs bearing other costimulatory components remain to be fully elucidated. In this review, we discuss the properties of 4-1BB, the design of 4-1BB CARs, and the function of T lymphocytes and natural killer cells expressing them.

  9. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain

    PubMed Central

    1993-01-01

    CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules. PMID:8391057

  10. Selection of the lamprey VLRC antigen receptor repertoire.

    PubMed

    Holland, Stephen J; Gao, Mingming; Hirano, Masayuki; Iyer, Lakshminarayan M; Luo, Ming; Schorpp, Michael; Cooper, Max D; Aravind, L; Mariuzza, Roy A; Boehm, Thomas

    2014-10-14

    The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC(+) peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line-encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA(+) cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC(+) lymphocyte lineage.

  11. Expression of T cell antigen receptor during differentiation

    SciTech Connect

    Allison, J.P.; Lanier, L.L.; Guyden, J.; Richie, E.R.

    1986-03-01

    The authors have used flow cytometry with monoclonal antibodies, radioimmuneprecipitation with a rabbit antiserum to common epitopes of the TCR, and Northern and Southern blot analysis with cloned TCR genes to study antigen receptor (TCR) expression by normal murine and human thymocytes and by primary murine thymomas. L3T4-,Lyt2- murine thymomas corresponding to the earliest stage of thymic differentiation, were found to have rearranged TCR beta genes, and to express low levels of beta transcript, but lacked alpha gene transcript and failed to express TCR on the cell surface. L3T4+,Lyt2+ thymomas were variable, but the majority were found to contain significant levels of both alpha and beta transcripts and to express TCR at the cell surface. Similarly, alpha and beta transcripts and TCR protein were detected in sorted L3T4+,Lyt2+ murine thymocytes. Using three color fluorescence, the authors determined that app. 70% of human T4+T8+ thymocytes also expressed T3, a component of the TCR complex. These data indicate that in mouse and man expression of TCR occurs in the immature, or cortical, thymic population.

  12. The Memory Function of the B Cell Antigen Receptor.

    PubMed

    Wienands, Jürgen; Engels, Niklas

    2016-01-01

    Activated B lymphocytes preserve their antigen experience by differentiating into long-lived pools of antibody-secreting plasma cells or various types of memory B cells (MBCs). The former population constantly produces serum immunoglobulins with sufficient specificity and affinity to thwart infections with recurrent pathogens. By contrast, memory B cell populations retain their antigen receptors on the cell surface and hence need pathogen-induced differentiation steps before they can actively contribute to host defense. The terminal differentiation of MBCs into antibody-secreting plasma cells is hallmarked by the absence of the lag phase characteristic for primary antibody responses. Moreover, secondary antibody responses are predominantly driven by MBCs that bear an antigen receptor of the IgG class on their surface although IgM-positive memory populations exist as well. These fundamental principles of B cell memory were enigmatic for decades. Only recently, we have begun to understand the underlying mechanisms. This review summarizes our current understanding of how different subpopulations of MBCs are generated during primary immune responses and how their functional heterogeneity on antigen recall is controlled by different signaling capabilities of B cell antigen receptor (BCR) isotypes and by the nature of the antigen.

  13. Chimeric antigen receptor T-cell therapy for solid tumors

    PubMed Central

    Newick, Kheng; Moon, Edmund; Albelda, Steven M

    2016-01-01

    Chimeric antigen receptor (CAR) T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias). This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment. PMID:27162934

  14. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves’ Disease

    PubMed Central

    Inaba, Hidefumi; De Groot, Leslie J.; Akamizu, Takashi

    2016-01-01

    Graves’ disease (GD) is an organ-specific autoimmune disease, and thyrotropin (TSH) receptor (TSHR) is a major autoantigen in this condition. Since the extracellular domain of human TSHR (TSHR-ECD) is shed into the circulation, TSHR-ECD is a preferentially immunogenic portion of TSHR. Both genetic factors and environmental factors contribute to development of GD. Inheritance of human leukocyte antigen (HLA) genes, especially HLA-DR3, is associated with GD. TSHR-ECD protein is endocytosed into antigen-presenting cells (APCs), and processed to TSHR-ECD peptides. These peptide epitopes bind to HLA-class II molecules, and subsequently the complex of HLA-class II and TSHR-ECD epitope is presented to CD4+ T cells. The activated CD4+ T cells secrete cytokines/chemokines that stimulate B-cells to produce TSAb, and in turn hyperthyroidism occurs. Numerous studies have been done to identify T- and B-cell epitopes in TSHR-ECD, including (1) in silico, (2) in vitro, (3) in vivo, and (4) clinical experiments. Murine models of GD and HLA-transgenic mice have played a pivotal role in elucidating the immunological mechanisms. To date, linear or conformational epitopes of TSHR-ECD, as well as the molecular structure of the epitope-binding groove in HLA-DR, were reported to be related to the pathogenesis in GD. Dysfunction of central tolerance in the thymus, or in peripheral tolerance, such as regulatory T cells, could allow development of GD. Novel treatments using TSHR antagonists or mutated TSHR peptides have been reported to be effective. We review and update the role of immunogenic TSHR epitopes and HLA in GD, and offer perspectives on TSHR epitope specific treatments. PMID:27602020

  15. Engineering HIV-Specific Immunity with Chimeric Antigen Receptors.

    PubMed

    Kitchen, Scott G; Zack, Jerome A

    2016-12-01

    HIV remains a highly important public health and clinical issue despite many recent advances in attempting to develop a cure, which has remained elusive for most people infected with HIV. HIV disease can be controlled with pharmacologic therapies; however, these treatments are expensive, may have severe side effects, and are not curative. Consequently, an improved means to control or eliminate HIV replication is needed. Cytotoxic T lymphocytes (CTLs) play a critical role in controlling viral replication and are an important part in the ability of the immune response to eradicate most viral infections. There are considerable efforts to enhance CTL responses in HIV-infected individuals in hopes of providing the immune response with armaments to more effectively control viral replication. In this review, we discuss some of these efforts and focus on the development of a gene therapy-based approach to engineer hematopoietic stem cells with an HIV-1-specific chimeric antigen receptor, which seeks to provide an inexhaustible source of HIV-1-specific immune cells that are MHC unrestricted and superior to natural antiviral T cell responses. These efforts provide the basis for further development of T cell functional enhancement to target and treat chronic HIV infection in hopes of eradicating the virus from the body.

  16. Multimolecular associations of the T-cell antigen receptor.

    PubMed

    Beyers, A D; Spruyt, L L; Williams, A F

    1992-09-01

    T cells are activated when the T-cell receptor for antigen (TCR) interacts with an antigenic peptide bound to a major histocompatibility complex (MHC) molecule on the surface of another cell. It is often assumed that T-cell activation is induced by the crosslinking of TCRs. In this article, Albertus Beyers, Louise Spruyt and Alan Williams argue that this mechanism is not generally applicable. They hypothesize that the key event in T-cell activation is the formation of multimolecular complexes consisting of the TCR and several other polypeptides, including CD4 or CD8, CD2, CD5 and the associated tyrosine kinases p59(fyn) and p56(lck).

  17. Recombinant Carcinoembryonic Antigen as a Reporter Gene for Molecular Imaging

    PubMed Central

    Kenanova, Vania; Barat, Bhaswati; Olafsen, Tove; Chatziioannou, Arion; Herschman, Harvey R.; Braun, Jonathan; Wu, Anna M.

    2009-01-01

    Purpose Reporter genes can provide a way of non-invasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. Methods To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human FcγRIIb receptor. The NA3-FcγRIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Results Flow cytometry identified Jurkat clones stably expressing NA3-FcγRIIb at low, medium, and high levels. High and medium NA3-FcγRIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA 124I-labeled scFv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4h. MicroPET image quantitation showed tumor activity of 1.8(±0.2), 15.2(±1.3) and 4.6(±1.2) %ID/g at 4h, 20h and 48h, respectively. Biodistribution at 48h, demonstrated tumor uptake of 4.8(±0.8) %ID/g. Conclusion The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo. PMID:18719907

  18. Immunoglobulin heavy chain variable gene usage and (super)-antigen drive in chronic lymphocytic leukemia.

    PubMed

    Bühler, Andreas; Zenz, Thorsten; Stilgenbauer, Stephan

    2010-01-15

    Increasing evidence supports the prognostic relevance of specific immunoglobulin heavy chain variable (IGHV) genes or stereotyped B-cell receptors (BCR) in chronic lymphocytic leukemia (CLL). The clonotypic BCRs differ in their specificity and affinity toward classical antigens and/or superantigens. The BCR-triggered mechanisms are distinct but could explain in part the different clinical behavior among CLL subgroups.

  19. Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity.

    PubMed Central

    Katayama, C D; Eidelman, F J; Duncan, A; Hooshmand, F; Hedrick, S M

    1995-01-01

    The antigen receptor on T cells (TCR) has been predicted to have a structure similar to a membrane-anchored form of an immunoglobulin F(ab) fragment. Virtually all of the conserved amino acids that are important for inter- and intramolecular interactions in the VH-VL pair are also conserved in the TCR V alpha and V beta chains. A molecular model of the TCR has been constructed by homology and we have used the information from this, as well as the earlier structural predictions of others, to study the basis for specificity. Specifically, regions of a TCR cloned from an antigen-specific T cell were stitched into the corresponding framework of a second TCR. Results indicate that the substitution of amino acid sequences corresponding to the complementarity determining regions (CDRs) of immunoglobulin can convey the specificity for antigen and major histocompatibility complex molecules. These data are consistent with a role, but not an exclusive role, for CDR3 in antigen peptide recognition. Images PMID:7534228

  20. Regulator T cells: specific for antigen and/or antigen receptors?

    PubMed

    Rubin, B; de Durana, Y Diaz; Li, N; Sercarz, E E

    2003-05-01

    Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary alpha/beta T-cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoplastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR-alpha and TCR-beta chains than are necessary for surface membrane expression of TCR-alphabeta heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR-alphabeta clonotype for recognition by B-cell receptors and clonotypic TCR-alphabeta peptides for recognition by T cells?

  1. Chimaeric antigen receptor T-cell therapy for tumour immunotherapy

    PubMed Central

    Sha, Huan-huan; Wang, Dan-dan; Yan, Da-li; Hu, Yong; Yang, Su-jin; Liu, Si-wen

    2017-01-01

    Chimaeric antigen receptor (CAR) T-cell therapies, as one of the cancer immunotherapies, have heralded a new era of treating cancer. The accumulating data, especially about CAR-modified T cells against CD19 support that CAR T-cell therapy is a highly effective immune therapy for B-cell malignancies. Apart from CD19, there have been many trials of CAR T cells directed other tumour specific or associated antigens (TSAs/TAAs) in haematologic malignancies and solid tumours. This review will briefly summarize basic CAR structure, parts of reported TSAs/TAAs, results of the clinical trials of CAR T-cell therapies as well as two life-threatening side effects. Experiments in vivo or in vitro, ongoing clinical trials and the outlook for CAR T-cell therapies also be included. Our future efforts will focus on identification of more viable cancer targets and more strategies to make CAR T-cell therapy safer. PMID:28053197

  2. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.

    PubMed

    Dai, Hanren; Wang, Yao; Lu, Xuechun; Han, Weidong

    2016-07-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy.

  3. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy

    PubMed Central

    Dai, Hanren; Wang, Yao; Lu, Xuechun

    2016-01-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy. PMID:26819347

  4. Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

    PubMed

    Díaz, P; Cardenas, H; Orihuela, P A

    2016-10-01

    We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 μg/ml of red Maca plus Taxol or 2ME 5 μM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 μg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 μg/ml, but not at 80 μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells.

  5. Cloning and expression of genes encoding Haemophilus somnus antigens.

    PubMed Central

    Corbeil, L B; Chikami, G; Yarnall, M; Smith, J; Guiney, D G

    1988-01-01

    A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease. Images PMID:2843469

  6. Molecular Characterization of a Fully Human Chimeric T-Cell Antigen Receptor for Tumor-Associated Antigen EpCAM

    PubMed Central

    Shirasu, Naoto; Yamada, Hiromi; Shibaguchi, Hirotomo; Kuroki, Motomu; Kuroki, Masahide

    2012-01-01

    The transduction of T cells to express chimeric T-cell antigen receptor (CAR) is an attractive strategy for adaptive immunotherapy for cancer, because the CAR can redirect the recognition specificity of T cells to tumor-associated antigens (TAAs) on the surface of target cells, thereby avoiding the limitations of HLA restriction. However, there are considerable problems with the clinical application of CAR, mostly due to its xenogeneic components, which could be immunogenic in humans. Moreover, while extensive studies on the CARs have been performed, the detailed molecular mechanisms underlying the activation of CAR-grafted T cells remain unclear. In order to eliminate potential immunogenicity and investigate the molecular basis of the CAR-mediated T-cell activation, we constructed a novel CAR (CAR57-28ζ) specific for one of the most important TAAs, epithelial cell adhesion molecule (EpCAM), using only human-derived genes. We revealed that in Jurkat T cells, lentivirally expressed CAR57-28ζ can transmit the T-cell-activating signals sufficient to induce IL-2 production upon EpCAM stimulation. An immunofluorescent analysis clearly showed that the CAR57-28ζ induces the formation of signaling clusters containing endogenous CD3ζ at the CAR/EpCAM interaction interface. These results suggest that this CAR gene may be safely and effectively applied for adaptive T-cell immunotherapy. PMID:22547929

  7. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    PubMed

    Ataca, Pınar; Arslan, Önder

    2015-12-01

    It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified T cells. On 1 July 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.

  8. Chimeric antigen receptor engineered stem cells: a novel HIV therapy.

    PubMed

    Zhen, Anjie; Carrillo, Mayra A; Kitchen, Scott G

    2017-03-01

    Despite the success of combination antiretroviral therapy (cART) for suppressing HIV and improving patients' quality of life, HIV persists in cART-treated patients and remains an incurable disease. Financial burdens and health consequences of lifelong cART treatment call for novel HIV therapies that result in a permanent cure. Cellular immunity is central in controlling HIV replication. However, HIV adopts numerous strategies to evade immune surveillance. Engineered immunity via genetic manipulation could offer a functional cure by generating cells that have enhanced antiviral activity and are resistant to HIV infection. Recently, encouraging reports from several human clinical trials using an anti-CD19 chimeric antigen receptor (CAR) modified T-cell therapy for treating B-cell malignancies have provided valuable insights and generated remarkable enthusiasm in engineered T-cell therapy. In this review, we discuss the development of HIV-specific chimeric antigen receptors and the use of stem cell based therapies to generate lifelong anti-HIV immunity.

  9. Inclusion of Strep-Tag II in design of antigen receptors for T cell immunotherapy

    PubMed Central

    Liu, Lingfeng; Sommermeyer, Daniel; Cabanov, Alexandra; Kosasih, Paula; Hill, Tyler; Riddell, Stanley R

    2016-01-01

    The tactical introduction of Strep-tag II into synthetic antigen receptors provides engineered T cells with a marker for identification and rapid purification, and a functional element for selective antibody coated microbead-driven large-scale expansion. Such receptor designs can be applied to chimeric antigen receptors of different ligand specificities and costimulatory domains, and to T cell receptors to facilitate cGMP manufacturing of adoptive T cell therapies to treat cancer and other diseases. PMID:26900664

  10. Vγ9 and Vδ2 T cell antigen receptor genes and butyrophilin 3 (BTN3) emerged with placental mammals and are concomitantly preserved in selected species like alpaca (Vicugna pacos).

    PubMed

    Karunakaran, Mohindar M; Göbel, Thomas W; Starick, Lisa; Walter, Lutz; Herrmann, Thomas

    2014-04-01

    Human Vγ9Vδ2 T cells recognize phosphorylated products of isoprenoid metabolism (phosphoantigens) PAg with TCR comprising Vγ9JP γ-chains and Vδ2 δ-chains dependent on butyrophilin 3 (BTN3) expressed by antigen-presenting cells. They are massively activated in many infections and show anti-tumor activity and so far, they have been considered to exist only in higher primates. We performed a comprehensive analysis of databases and identified the three genes in species of both placental magnorders, but not in rodents. The common occurrence or loss of in silico translatable Vγ9, Vδ2, and BTN3 genes suggested their co-evolution based on a functional relationship. In the peripheral lymphocytes of alpaca (Vicugna pacos), characteristic Vγ9JP rearrangements and in-frame Vδ2 rearrangements were found and could be co-expressed in a TCR-negative mouse T cell hybridoma where they rescued CD3 expression and function. Finally, database sequence analysis of the extracellular domain of alpaca BTN3 revealed complete conservation of proposed PAg binding residues of human BTN3A1. In summary, we show emergence and preservation of Vγ9 and Vδ2 TCR genes with the gene of the putative antigen-presenting molecule BTN3 in placental mammals and lay the ground for analysis of alpaca as candidate for a first non-primate species to possess Vγ9Vδ2 T cells.

  11. The pharmacology of second-generation chimeric antigen receptors.

    PubMed

    van der Stegen, Sjoukje J C; Hamieh, Mohamad; Sadelain, Michel

    2015-07-01

    Second-generation chimeric antigen receptors (CARs) retarget and reprogramme T cells to augment their antitumour efficacy. The combined activating and co-stimulatory domains incorporated in these CARs critically determine the function, differentiation, metabolism and persistence of engineered T cells. CD19-targeted CARs that incorporate CD28 or 4-1BB signalling domains are the best known to date. Both have shown remarkable complete remission rates in patients with refractory B cell malignancies. Recent data indicate that CD28-based CARs direct a brisk proliferative response and boost effector functions, whereas 4-1BB-based CARs induce a more progressive T cell accumulation that may compensate for less immediate potency. These distinct kinetic features can be exploited to further develop CAR-based T cell therapies for a variety of cancers. A new field of immunopharmacology is emerging.

  12. Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis

    DTIC Science & Technology

    1988-02-02

    transcription and translation of the Bacillus megaterium protein C gene. J. Bacteriol. 158:e09-813. 9. Friedlander, A, M. 1986. Macrophages are sensitive to...of the Protective Antigen Gene of Bacillus anthracis 6. pEaltranalO opl. AMPOA’T B*u~iA S. L. Welkos, J. R. Lowe, F. Eden-McCutchan, M. Vodkin, S. M... Bacillus anthracls and the 5’ and 3’ flanking sequences were determined. Protective antigen ie one of three proteins comprising anthrax toxin. The open

  13. Cloning of the Protective Antigen Gene of Bacillus anthracis

    DTIC Science & Technology

    1983-09-01

    of the complicated precedents of duplicate toxin genes in chro- muumm mosomall and plasmid DNA of B. thuringiensis (Schnepf and Whitely, 1981; Klier...OiL V4. 34. S-W7. SW 1v 99 CwI 0193 by MT 0 009-7483/06O-002.00/0 mU"- - 1*;)-0Cloning of the Protective Antigen Gene OCT 19 MI L Sof Bacillus ...Sumnler uncertain, it is probably caused by other Bacillus antigens, 4 t which may include LF and EF. PA produced from recom- A The - "w t of a

  14. Antigen receptor-regulated exocytosis in cytotoxic T lymphocytes

    PubMed Central

    1987-01-01

    We demonstrate here that T cell receptor for antigen (TCR)-triggered exocytosis in cytotoxic T lymphocytes (CTL) is not constitutive and is regulated through crosslinking of the TCR by antigen or monoclonal anti- TCR antibodies. Morphological and biochemical data using three different biochemical markers of granules and Percoll gradient fractionation analysis are presented, suggesting that TCR-triggered exocytosis is accompanied by the loss of granules from CTL and appearance of intragranular proteins and enzymatic activities in the incubation medium. The strict requirement for crosslinking of the TCR in exocytosis triggering could be bypassed by protein kinase C activators (phorbol esters or bryostatin I and II) acting in synergy with Ca2+ ionophores. It is shown that external Ca2+ is obligatory for both the TCR-triggered and for the PMA/A23187-triggered exocytosis, since Ca2+ chelators and divalent cations that compete with Ca2+ for A23187 can inhibit exocytosis of granules. These data suggest that Ca2+ from intracellular stores is not sufficient to support exocytosis in CTL. Ca2+ channel blockers and calmodulin antagonists significantly inhibited TCR-triggered exocytosis without affecting the basal level of secretion. The described results are consistent with a model in which exocytosis of granules in CTL is triggered by the crosslinking of TCR, transmembrane protein kinase C activation, and external Ca2+ translocation through CTL plasma membrane Ca2+ channels and modulation of activity of Ca2+, calmodulin-dependent enzymes, and cytoskeletal proteins. PMID:2442289

  15. Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia

    PubMed Central

    Maude, Shannon L.; Frey, Noelle; Shaw, Pamela A.; Aplenc, Richard; Barrett, David M.; Bunin, Nancy J.; Chew, Anne; Gonzalez, Vanessa E.; Zheng, Zhaohui; Lacey, Simon F.; Mahnke, Yolanda D.; Melenhorst, Jan J.; Rheingold, Susan R.; Shen, Angela; Teachey, David T.; Levine, Bruce L.; June, Carl H.; Porter, David L.; Grupp, Stephan A.

    2014-01-01

    BACKGROUND Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor–modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease. METHODS We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×106 to 20.6×106 CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells. RESULTS A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti–interleukin-6 receptor antibody tocilizumab. CONCLUSIONS Chimeric antigen receptor–modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by

  16. 77 FR 3482 - Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen Receptors Into.../057272 and foreign equivalents thereof entitled ``Anti-MAGE-A3 T cell receptors and related materials and... Patent Application No. PCT/US2011/051537 and foreign equivalents thereof entitled ``Anti-SSX-2 T...

  17. Molecular cloning of Taenia taeniaeformis oncosphere antigen genes.

    PubMed

    Cougle, W G; Lightowlers, M W; Bogh, H O; Rickard, M D; Johnson, K S

    1991-03-01

    Infection of mice with the cestode Taenia taeniaeformis exhibits several important features common to other cestode infections, including the ability to vaccinate with crude antigen mixtures. Partial purification of the protective oncosphere antigens has been reported with a cutout from deoxycholate (DOC) acrylamide gels; this cutout was called fraction II (FII), and comprises approximately 10% of total DOC-soluble oncosphere antigen. Western blots of DOC gels probed with anti-FII antisera revealed a series of 3-5 discrete bands within the FII region. Further fractionation of the FII antigens on DOC gels was impractical due to limitations in supply of oncospheres, so a cDNA library was constructed from 150 ng of oncosphere mRNA and screened with alpha-FII antisera. Two distinct clone families were identified, oncA and oncB. Antibodies affinity-purified on either of two representative members, oncA1 and oncB1, recognised all the FII bands. Individual FII bands excised from a DOC gel resolved into an overlapping series of molecules when re-run on SDS-PAGE, indicating that each FII band consisted of several polypeptides of differing molecular weight. Immunoprecipitates resolved on SDS-PAGE revealed that alpha-FII recognised 3 major oncosphere antigens, of 62, 34 and 25 kDa; antisera against oncB precipitated both the 34- and 25-kDa antigens, whereas alpha-oncA antisera precipitated the 62-kDa antigen. We conclude that oncA and oncB encode the major antigens in the FII complex. The 62-kDa antigen encoded by oncA1 was the only common antigen precipitated by anti-FII and two other antisera raised against different protective extracts, suggesting that it may be a protective component in all three. Southern blot results indicate that oncA and oncB are distinct genes present at low copy number in the genome. Evidence is also presented suggesting that some cestode mRNAs, including oncA, may use variant polyadenylation signals.

  18. Chimeric Antigen Receptor T Cells in Hematologic Malignancies.

    PubMed

    Shank, Brandon R; Do, Bryan; Sevin, Adrienne; Chen, Sheree E; Neelapu, Sattva S; Horowitz, Sandra B

    2017-03-01

    Patients with B-cell hematologic malignancies who progress through first- or second-line chemotherapy have a poor prognosis. Early clinical trials with autologous anti-CD19 chimeric antigen receptor (CAR) T cells have demonstrated promising results for patients who have relapsed or refractory disease. Lymphodepleting conditioning regimens, including cyclophosphamide, fludarabine, pentostatin, bendamustine, interleukin-2, and total body irradiation, are often administered before the infusion of CAR T cells, allowing for greater T-cell expansion. The major toxicity associated with CAR T-cell infusions is cytokine release syndrome (CRS), a potentially life-threatening systemic inflammatory disorder. The quick onset and progression of CRS require rapid detection and intervention to reduce treatment-related mortality. Management with tocilizumab can help ameliorate the symptoms of severe CRS, allowing steroids, which diminish the expansion and persistence of CAR T cells, to be reserved for tocilizumab-refractory patients. Other toxicities of CAR T-cell therapy include neutropenia and/or febrile neutropenia, infection, tumor lysis syndrome, neurotoxicity and nausea/vomiting. A review of patients' medications is imperative to eliminate medications that may contribute to treatment-related toxicities. Studies are ongoing to help optimize patient selection, preparation, safety, and management of individuals receiving CAR T cells. Long-term follow-up will help establish the place of CAR T cells in therapy.

  19. Glycosylation of the T-cell antigen-specific receptor and its potential role in lectin-mediated cytotoxicity

    SciTech Connect

    Hubbard, S.C.; Kranz, D.M.; Longmore, G.D.; Sitkovsky, M.V.; Eisen, H.N.

    1986-03-01

    Cytotoxic T lymphocytes (CTLs) normally destroy only those cells (target cells) whose surface antigens they recognize. However, in the presence of lectins such as Con A, CTLs destroy virtually any cell, regardless of its antigens. The oligosaccharides of the T-cell antigen-specific receptor, a dimeric surface glycoprotein composed of disulfide-linked ..cap alpha.. and ..beta.. subunits, are of interest because of their potential involvement in this lectin-dependent cytotoxic activity. The authors report here that three or four asparagine-linked oligosaccharides could be enzymatically removed from each of the receptor subunits expressed by a cloned line of murine CTLs (clone 2C), consistent with the presence of glycosylation sites deduced from cDNA sequences of the ..cap alpha.. and ..beta.. genes expressed in this clone. All the N-linked glycans on the ..cap alpha.. subunit were of the complex type, but the ..beta.. subunit carried two or three endoglycosidase H-sensitive oligosaccharides. High-mannose glycans can bind tightly to Con A and, indeed, this lectin was found to bind specifically to solubilized 2C T-cell receptor. The Con A-dependent cytotoxic activity of clone 2C, but not of other CTL clones, was inhibited by a monoclonal antibody (1B2) that is specific for the T-cell receptor of clone 2C. Antibody 1B2 also inhibited clone 2C cytotoxicity mediated by phytohemagglutinin, lentil-lectin, and wheat-germ agglutinin. These results suggest that, although lectin-dependent lysis of target cells by CTLs is antigen nonspecific, the cytolytic activity can be triggered by binding of the lectin to the T-cell antigen-specific receptor.

  20. Evolutionary History of the Cancer Immunity Antigen MAGE Gene Family

    PubMed Central

    Katsura, Yukako; Satta, Yoko

    2011-01-01

    The evolutionary mode of a multi-gene family can change over time, depending on the functional differentiation and local genomic environment of family members. In this study, we demonstrate such a change in the melanoma antigen (MAGE) gene family on the mammalian X chromosome. The MAGE gene family is composed of ten subfamilies that can be categorized into two types. Type I genes are of relatively recent origin, and they encode epitopes for human leukocyte antigen (HLA) in cancer cells. Type II genes are relatively ancient and some of their products are known to be involved in apoptosis or cell proliferation. The evolutionary history of the MAGE gene family can be divided into four phases. In phase I, a single-copy state of an ancestral gene and the evolutionarily conserved mode had lasted until the emergence of eutherian mammals. In phase II, eight subfamily ancestors, with the exception for MAGE-C and MAGE-D subfamilies, were formed via retrotransposition independently. This would coincide with a transposition burst of LINE elements at the eutherian radiation. However, MAGE-C was generated by gene duplication of MAGE-A. Phase III is characterized by extensive gene duplication within each subfamily and in particular the formation of palindromes in the MAGE-A subfamily, which occurred in an ancestor of the Catarrhini. Phase IV is characterized by the decay of a palindrome in most Catarrhini, with the exception of humans. Although the palindrome is truncated by frequent deletions in apes and Old World monkeys, it is retained in humans. Here, we argue that this human-specific retention stems from negative selection acting on MAGE-A genes encoding epitopes of cancer cells, which preserves their ability to bind to highly divergent HLA molecules. These findings are interpreted with consideration of the biological factors shaping recent human MAGE-A genes. PMID:21695252

  1. Chimeric antigen receptor T-cell neuropsychiatric toxicity in acute lymphoblastic leukemia.

    PubMed

    Prudent, Vasthie; Breitbart, William S

    2017-01-04

    Chimeric antigen receptor T cells are used in the treatment of B-cell leukemias. Common chimeric antigen receptor T-cell toxicities can range from mild flu-like symptoms, such as fever and myalgia, to a more striking neuropsychiatric toxicity that can present as discrete neurological symptoms and delirium. We report here two cases of chimeric antigen receptor T-cell neuropsychiatric toxicity, one who presented as a mild delirium and aphasia that resolved without intervention, and one who presented with delirium, seizures, and respiratory insufficiency requiring intensive treatment. The current literature on the treatment and proposed mechanisms of this clinically challenging chimeric antigen receptor T-cell complication is also presented.

  2. DNA segment containing C/sub. beta. 1/, a gene for the constant region of the. beta. chain of the T-cell antigen receptor, was inserted into chromosome 6 in cells from one patients with human T-cell leukemia

    SciTech Connect

    Ino, T.; Kurosawa, Y.; Yoshida, M.C.; Hirano, M.

    1987-06-01

    DNA rearrangements that occurred in the vicinity of T-cell antigen receptor ..beta..-chain gene clusters residing on chromosome 7 were examined in human T-cell acute lymphoblastic leukemia cells. In one patient, it was observed that, for the T-cell receptor ..beta..-chain genes, a D/sub ..beta.. 1/-J/sub ..beta..2.3/ (where D is diversity and J is joining) junction was found on one chromosome, while the other chromosome kept the germ-line configuration. If this D/sub ..beta../-J/sub ..beta../ junction was formed by the customary deletion mechanism, the C/sub ..beta..1/ gene (where C is constant) located between the D/sub ..beta..1/ and J/sub BETA2.3/ loci should have disappeared from this chromosome. The C/sub ..beta..1/ gene indeed was absent from the rearranged chromosome 7, but it was found on chromosome 6 as an inserted segment. The implications of the observations are discussed.

  3. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed Central

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-01-01

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism. PMID:6896821

  4. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  5. [Application of Chimeric Antigen Receptor-Modified CAR-T/NK Cells to Treatment of Multiple Myeloma].

    PubMed

    Wang, Lei; Ou, jian-Feng; Bai, Hai

    2015-04-01

    Chimeric antigen receptor(CAR) is a synthesized transmembrane protein, which redirects the modified cells through specific or associated antigen on tumor cells. CAR-modified T/NK cells, especially CAR-T cells, are a new tool of rapidly developing of adoptive immunotherapy of tumor in recent years, they give T/NK cells the targeting cytotoxic activity and can overcome the tumor immunosuppressive microenvironment and break the state of the host immune tolerance. CAR combines the single-chain antibody to tumor-associated antigen with T/NK cells' activated motifs, giving T/NK cells' tumor targeting activity, so enhancing their cytotoxic activity and lasting the vitality by gene transduction. In this article the CAR development, comparison of CAR-T and CAR-NK cells, surface markers on MM cells and use of CAR in MM, and CAR perspectives are summarized.

  6. Changes in repeat number, sequence, and reading frame in S-antigen genes of Plasmodium falciparum.

    PubMed Central

    Saint, R B; Coppel, R L; Cowman, A F; Brown, G V; Shi, P T; Barzaga, N; Kemp, D J; Anders, R F

    1987-01-01

    The S antigens from different isolates of Plasmodium falciparum exhibit extensive size, charge, and serological diversity. We show here that the S-antigen genes behave as multiple alleles of a single locus. The size heterogeneity results from different numbers, lengths, and/or sequences of tandem repeat units encoded within the S-antigen genes. Two genes studied here encode antigenically different S antigens but nevertheless have closely related tandem repeat sequences. We show that antigenic differences can arise because repeats are translated in different reading frames. Images PMID:3313007

  7. HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells

    PubMed Central

    Zhen, Anjie; Kamata, Masakazu; Rezek, Valerie; Rick, Jonathan; Levin, Bernard; Kasparian, Saro; Chen, Irvin SY; Yang, Otto O; Zack, Jerome A; Kitchen, Scott G

    2015-01-01

    The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities. PMID:26050990

  8. HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells.

    PubMed

    Zhen, Anjie; Kamata, Masakazu; Rezek, Valerie; Rick, Jonathan; Levin, Bernard; Kasparian, Saro; Chen, Irvin Sy; Yang, Otto O; Zack, Jerome A; Kitchen, Scott G

    2015-08-01

    The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities.

  9. 20-kDa protein associated with the murine T-cell antigen receptor is phosphorylated in response to activation by antigen or concanavalin A

    SciTech Connect

    Samelson, L.E.; Harford, J.; Schwartz, R.H.; Klausner, R.D.

    1985-04-01

    Antigen or concanavalin A activation of a murine T-cell hybrid specific for pigeon cytochrome resulted in phosphorylation of a 20-kDa protein that was specifically coprecipitated by a monoclonal antibody binding the T-cell antigen receptor. There was no evidence for phosphorylation of the antigen receptor itself. The phosphorylation of the 20-kDa polypeptide was dependent on the concentration of antigen or lectin used to activate the T-cell hybrid and reached a maximum 40 min after the addition of antigen. The 20-kDa protein was also radioiodinated with a hydrophobic photoactivatable labeling reagent. The amount of iodinated 20-kDa protein immunoprecipitable with the anti-receptor antibody did not increase with T-cell activation, indicating that the phosphorylation occurred on a molecule that was constitutively associated with the antigen receptor. Concanavalin A also induced phosphorylation of a 20-kDa polypeptide in a second antigen-specific major histocompatibility complex-restricted T-cell hybrid. Again, the phosphorylated polypeptide was precipitated only by a monoclonal antibody specific for the antigen receptor on this hybrid. Thus, the antigen or concanavalin A-induced activation of T-cell hybrids results in the rapid phosphorylation of a 20-kDa protein that is associated with the T-cell receptor.

  10. Gene frequencies of human neutrophil antigens in the Tunisian blood donors and Berbers.

    PubMed

    Abid, S; Zili, M; Bouzid, L; Kibech, R; Foudhaili, N; Joudi, K; Ren Regaya, Z; Abdennaji, B; Mrad, R; Boukef, K

    2001-08-01

    Human neutrophil antigens play an important role in provoking immune neutropenia and transfusion-reactions. The aim of this study was to determine granulocyte-specific antigens on the neutrophil Fc gamma receptor IIIb (Fc gamma RIIIb, CD16b), namely, the HNA-1a(NA1) and HNA-1b(NA2) antigens and their gene frequencies in Tunisian blood donors and Berbers. One hundred and ninety-nine unrelated healthy Tunisian blood donors and Berbers were typed for HNA-1a and HNA-1b(NA1 and NA2), using polymerase chain reaction with sequence-specific primers (PCR-SSP). In 24 granulocyte samples, the HNA-1a and HNA-1b phenotypes was additionally determined by the granulocyte immunofluorescence test (GIFT) and correlated with the genotyping results. A subsequent analysis of the genotyping study showed that, the HNA-1a and HNA-1b gene frequencies observed, were 0.342 and 0.658 for Berbers, and 0.311 and 0.668 for blood donors, respectively. In the genotyping study conducted, it was determined that the HNA-1a and HNA-1b gene frequencies observed in Tunisian blood donors and Berbers are similar to those previously reported in other white populations.

  11. Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

    PubMed Central

    Zhao, Bo; Mar, Jessica C.; Maruo, Seiji; Lee, Sungwook; Gewurz, Benjamin E.; Johannsen, Eric; Holton, Kristina; Rubio, Renee; Takada, Kenzo; Quackenbush, John; Kieff, Elliott

    2011-01-01

    EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen–dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine–cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10−4. PMID:21173222

  12. Chimeric antigen receptors for the adoptive T cell therapy of hematologic malignancies.

    PubMed

    Davila, Marco L; Bouhassira, Diana C G; Park, Jae H; Curran, Kevin J; Smith, Eric L; Pegram, Hollie J; Brentjens, Renier

    2014-04-01

    The genetic modification of autologous T cells with chimeric antigen receptors (CARs) represents a breakthrough for gene engineering as a cancer therapy for hematologic malignancies. By targeting the CD19 antigen, we have demonstrated robust and rapid anti-leukemia activity in patients with heavily pre-treated and chemotherapy-refractory B cell acute lymphoblastic leukemia (B-ALL). We demonstrated rapid induction of deep molecular remissions in adults, which has been recently confirmed in a case report involving a child with B-ALL. In contrast to the results when treating B-ALL, outcomes have been more modest in patients with chronic lymphocytic leukemia (CLL) or other non-hodgkin's lymphoma (NHL). We review the clinical trial experience targeting B-ALL and CLL and speculate on the possible reasons for the different outcomes and propose potential optimization to CAR T cell therapy when targeting CLL or other indolent NHL. Lastly, we discuss the pre-clinical development and potential for clinical translation for using CAR T cells against multiple myeloma and acute myeloid leukemia. We highlight the potential risks and benefits by targeting these poor outcome hematologic malignancies.

  13. Interleukin 18 secretion and its effect in improving Chimeric Antigen Receptors efficiency

    NASA Astrophysics Data System (ADS)

    Kim, Jae-Kun

    Clinical trials have shown that chimeric antigen receptor T cells modified to target cancer cells expressing a surface antigen found on immature B-cells. The purpose of this experiment is to take a pro-inflammatory cytokine, and analyze its effect in improving the efficiency of the T cells. IL-18 has been previously shown to recruit T cells to the tumor site and improve their secretion of cytotoxic cytokines. A human model of the proposed armored T cell has been created and has shown success in combating cancer cells in vitro. The next step is to design and produce a murine model to test in vivo in immunocompetent mice. This research project aimed to create two models: one utilizing 2A peptides and another utilizing IRES elements as a multicistronic vector. Both models would require the insertion of the desired genes into SFG backbones. IRES, a DNA element which acts as a binding site for the transcriptional machinery to recognize which part of the DNA to transcribe, commonly found in bicistronic vectors, is large with 500-600 base pairs, and has a lower transgene expression rate. P2A is smaller, only consisting of about 20 amino acids, and typically has a higher transgene expression rate, which may or may not result in higher effectiveness of the model. I would like to thank Dr. Renier Brentjens for being a mentor who cared about giving his interns as much educational value as possible.

  14. Optimizing T-cell receptor gene therapy for hematologic malignancies

    PubMed Central

    Morris, Emma C.

    2016-01-01

    Recent advances in genetic engineering have enabled the delivery of clinical trials using patient T cells redirected to recognize tumor-associated antigens. The most dramatic results have been seen with T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19, a differentiation antigen expressed in B cells and B lineage malignancies. We propose that antigen expression in nonmalignant cells may contribute to the efficacy of T-cell therapy by maintaining effector function and promoting memory. Although CAR recognition is limited to cell surface structures, T-cell receptors (TCRs) can recognize intracellular proteins. This not only expands the range of tumor-associated self-antigens that are amenable for T-cell therapy, but also allows TCR targeting of the cancer mutagenome. We will highlight biological bottlenecks that potentially limit mutation-specific T-cell therapy and may require high-avidity TCRs that are capable of activating effector function when the concentrations of mutant peptides are low. Unexpectedly, modified TCRs with artificially high affinities function poorly in response to low concentration of cognate peptide but pose an increased safety risk as they may respond optimally to cross-reactive peptides. Recent gene-editing tools, such as transcription activator–like effector nucleases and clustered regularly interspaced short palindromic repeats, provide a platform to delete endogenous TCR and HLA genes, which removes alloreactivity and decreases immunogenicity of third-party T cells. This represents an important step toward generic off-the-shelf T-cell products that may be used in the future for the treatment of large numbers of patients. PMID:27207802

  15. Novel antigen design for the generation of antibodies to G-protein-coupled receptors.

    PubMed

    Larsson, K; Hofström, C; Lindskog, C; Hansson, M; Angelidou, P; Hökfelt, T; Uhlén, M; Wernérus, H; Gräslund, T; Hober, S

    2011-07-29

    Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

  16. Genomic organization of the human T-cell antigen-receptor alpha/delta locus.

    PubMed

    Satyanarayana, K; Hata, S; Devlin, P; Roncarolo, M G; De Vries, J E; Spits, H; Strominger, J L; Krangel, M S

    1988-11-01

    Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.

  17. Minicircle-Based Engineering of Chimeric Antigen Receptor (CAR) T Cells.

    PubMed

    Hudecek, Michael; Gogishvili, Tea; Monjezi, Razieh; Wegner, Julia; Shankar, Ram; Kruesemann, Christa; Miskey, Csaba; Ivics, Zoltán; Schmeer, Marco; Schleef, Martin

    2016-01-01

    Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.

  18. Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

    PubMed Central

    Heath, D G; Cleary, P P

    1989-01-01

    The partial nucleotide sequence for an Fc-receptor gene from an M-type 76 group A streptococcus was determined. DNA sequence analysis revealed considerable sequence similarity between the Fc-receptor and M-protein genes in their proposed promoter regions, signal sequences, and 3' termini. Additional analysis indicated that the deduced Fc-receptor protein contains a proline-rich region and membrane anchor region highly similar to that of M protein. In view of these results, we postulated that Fc-receptor and M-protein genes of group A streptococci are the products of gene duplication from a common ancestral gene. It is proposed that DNA sequence similarity between these two genes may allow for extragenic homologous recombination as a means of generating antigenic diversity in these two surface proteins. PMID:2660147

  19. Evolution of an expanded mannose receptor gene family.

    PubMed

    Staines, Karen; Hunt, Lawrence G; Young, John R; Butter, Colin

    2014-01-01

    Sequences of peptides from a protein specifically immunoprecipitated by an antibody, KUL01, that recognises chicken macrophages, identified a homologue of the mammalian mannose receptor, MRC1, which we called MRC1L-B. Inspection of the genomic environment of the chicken gene revealed an array of five paralogous genes, MRC1L-A to MRC1L-E, located between conserved flanking genes found either side of the single MRC1 gene in mammals. Transcripts of all five genes were detected in RNA from a macrophage cell line and other RNAs, whose sequences allowed the precise definition of spliced exons, confirming or correcting existing bioinformatic annotation. The confirmed gene structures were used to locate orthologues of all five genes in the genomes of two other avian species and of the painted turtle, all with intact coding sequences. The lizard genome had only three genes, one orthologue of MRC1L-A and two orthologues of the MRC1L-B antigen gene resulting from a recent duplication. The Xenopus genome, like that of most mammals, had only a single MRC1-like gene at the corresponding locus. MRC1L-A and MRC1L-B genes had similar cytoplasmic regions that may be indicative of similar subcellular migration and functions. Cytoplasmic regions of the other three genes were very divergent, possibly indicating the evolution of a new functional repertoire for this family of molecules, which might include novel interactions with pathogens.

  20. Lysophosphatidic Acid (LPA) Receptor 5 Inhibits B Cell Antigen Receptor Signaling and Antibody Response1

    PubMed Central

    Shotts, Kristin; Donovan, Erin E.; Strauch, Pamela; Pujanauski, Lindsey M.; Victorino, Francisco; Al-Shami, Amin; Fujiwara, Yuko; Tigyi, Gabor; Oravecz, Tamas; Pelanda, Roberta; Torres, Raul M.

    2014-01-01

    Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically and whose levels are elevated in certain pathological settings such as cancer and infections. Here, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13 – Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-trisphosphate receptor activity. We further show that LPA5 also limits antigen-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced antibody responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity. PMID:24890721

  1. Regulation of cancer germline antigen gene expression: implications for cancer immunotherapy.

    PubMed

    Akers, Stacey N; Odunsi, Kunle; Karpf, Adam R

    2010-05-01

    Cancer germline (CG; also known as cancer-testis) antigen genes are normally expressed in germ cells and trophoblast tissues and are aberrantly expressed in a variety of human malignancies. CG antigen genes have high clinical relevance as they encode a class of immunogenic and highly selective tumor antigens. CG antigen-directed immunotherapy is undergoing clinical evaluation for the treatment of a number of solid tumor malignancies and has been demonstrated to be safe, provoke immune responses and be of therapeutic benefit. Achieving an improved understanding of the mechanisms of CG antigen gene regulation will facilitate the continued development of targeted therapeutic approaches against tumors expressing these antigens. Substantial evidence suggests epigenetic mechanisms, particularly DNA methylation, as a primary regulator of CG antigen gene expression in normal and cancer cells as well as in stem cells. The roles of sequence-specific transcription factors and signal transduction pathways in controlling CG antigen gene expression are less clear but are emerging. A combinatorial therapeutic approach involving epigenetic modulatory drugs and CG antigen immunotherapy is suggested based on these data and is being actively pursued. In this article, we review the mechanisms of CG antigen gene regulation and discuss the implications of these mechanisms for the development of cancer immunotherapy approaches targeting CG antigens.

  2. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

    PubMed Central

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  3. Evidence for horizontal gene transfer of two antigenically distinct O antigens in Bordetella bronchiseptica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antigenic variation is one mechanism pathogens use to avoid immune-mediated competition between closely related strains. Here, we show that two Bordetella bronchiseptica strains, RB50 and 1289, express two antigenically distinct O-antigen serotypes (O1 and O2 respectively). When 18 additional B. b...

  4. Chimeric antigen receptor (CAR)-directed adoptive immunotherapy: a new era in targeted cancer therapy.

    PubMed

    Chen, Yamei; Liu, Delong

    2014-01-01

    As a result of the recent advances in molecular immunology, virology, genetics, and cell processing, chimeric antigen receptor (CAR)-directed cancer therapy has finally arrived for clinical application. CAR-directed adoptive immunotherapy represents a novel form of gene therapy, cellular therapy, and immunotherapy, a combination of three in one. Early phase clinical trial was reported in patients with refractory chronic lymphoid leukemia with 17p deletion. Accompanying the cytokine storm and tumor lysis syndrome was the shocking disappearance of the leukemia cells refractory to chemotherapy and monoclonal antibodies. CAR therapy was reproduced in both children and adults with refractory acute lymphoid leukemia. The CAR technology is being explored for solid tumor therapy, such as glioma. Close to 30 clinical trials are underway in the related fields (www.clinicaltrials.gov). Further improvement in gene targeting, cell expansion, delivery constructs (such as using Sleeping Beauty or Piggyback transposons) will undoubtedly enhance clinical utility. It is foreseeable that CAR-engineered T cell therapy will bring targeted cancer therapy into a new era.

  5. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  6. A 55-kilodalton immunodominant antigen of Porphyromonas gingivalis W50 has arisen via horizontal gene transfer.

    PubMed

    Hanley, S A; Aduse-Opoku, J; Curtis, M A

    1999-03-01

    A 55-kDa outer membrane protein of Porphyromonas gingivalis W50 is a significant target of the serum immunoglobulin G antibody response of periodontal disease patients and hence may play an important role in host-bacterium interactions in periodontal disease. The gene encoding the 55-kDa antigen (ragB, for receptor antigen B) was isolated on a 9.5-kb partial Sau3AI fragment of P. gingivalis W50 chromosomal DNA in pUC18 by immunoscreening with a monoclonal antibody to this antigen. The 1.6-kb open reading frame (ORF) encoding RagB was located via subcloning and nested-deletion analysis. Sequence analysis demonstrated the presence of an upstream 3.1-kb ORF (ragA) which is cotranscribed with ragB. A number of genetic characteristics suggest that the ragAB locus was acquired by a horizontal gene transfer event. These include a significantly reduced G+C content relative to that of the P. gingivalis chromosome (42 versus 48%) and the presence of mobility elements flanking this locus in P. gingivalis W50. Furthermore, Southern blotting and PCR analyses showed a restricted distribution of this locus in laboratory and clinical isolates of this bacterium. The association of ragAB+ P. gingivalis with clinical status was examined by PCR analysis of subgingival samples. ragAB+ was not detected in P. gingivalis-positive shallow pockets from periodontal disease patients but was present in 36% of the P. gingivalis-positive samples from deep pockets. These data suggest that the ragAB locus was acquired by certain P. gingivalis strains via horizontal gene transfer and that the acquisition of this locus may facilitate the survival of these strains at sites of periodontal destruction.

  7. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  8. Human epidermal Langerhans cells cointernalize by receptor-mediated endocytosis "nonclassical" major histocompatibility complex class I molecules (T6 antigens) and class II molecules (HLA-DR antigens).

    PubMed Central

    Hanau, D; Fabre, M; Schmitt, D A; Garaud, J C; Pauly, G; Tongio, M M; Mayer, S; Cazenave, J P

    1987-01-01

    HLA-DR and T6 surface antigens are expressed only by Langerhans cells and indeterminate cells in normal human epidermis. We have previously demonstrated that T6 antigens are internalized in Langerhans cells and indeterminate cells by receptor-mediated endocytosis. This process is induced by the binding of BL6, a monoclonal antibody directed against T6 antigens. In the present study, using a monoclonal antibody directed against HLA-DR antigens, on human epidermal cells in suspension, we show that the surface HLA-DR antigens are also internalized by receptor-mediated endocytosis in Langerhans and indeterminate cells. Moreover, using immunogold double labeling, we demonstrate that T6 and HLA-DR antigens are internalized through common coated regions of the membrane of Langerhans or indeterminate cells. The receptor-mediated endocytosis that is induced involves coated pits and vesicles, receptosomes, lysosomes, and also, in Langerhans cells, the Birbeck granules. Thus, T6 antigens, which are considered to be "unusual" or "nonclassical" major histocompatibility complex class I molecules, and the major histocompatibility complex class II molecules, HLA-DR, are internalized in Langerhans and indeterminate cells through common receptor-mediated endocytosis organelles. Images PMID:3106979

  9. T Cell Receptors that Recognize the Tyrosinase Tumor Antigen | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute, Surgery Branch, Tumor Immunology Section, is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize T Cells Attacking Cancer: T Cell Receptors that Recognize the Tyrosinase Tumor Antigen

  10. ɣδ T cell receptor ligands and modes of antigen recognition

    PubMed Central

    Champagne, Eric

    2011-01-01

    T lymphocytes expressing the γδ-type of T cell receptors for antigens contribute to all aspects of immune responses, including defenses against viruses, bacteria, parasites and tumors, allergy and autoimmunity. Multiple subsets have been individualized in humans as well as in mice and they appear to recognize in a TCR-dependent manner antigens as diverse as small non-peptidic molecules, soluble or membrane-anchored polypeptides and molecules related to MHC antigens on cell surfaces, implying diverse modes of antigen recognition. We review here the γδ TCR ligands which have been identified along the years and their characteristics, with emphasis on a few systems which have been extensively studied such as human γδ T cells responding to phosphoantigens or murine γδ T cells activated by allogeneic MHC antigens. We discuss a speculative model of antigen recognition involving simultaneous TCR recognition of MHC-like and non-MHC ligands which could fit with most available data and shares many similarities with the classical model of MHC-restricted antigen recognition for peptides or lipids by T cells subsets with αβ-type TCRs. PMID:21298486

  11. Polymeric structure and host Toll-like receptor 4 dictate immunogenicity of NY-ESO-1 antigen in vivo.

    PubMed

    Liu, Yanan; Tian, Xiaoli; Leitner, Wolfgang W; Aldridge, Michael E; Zheng, Junying; Yu, Zhiya; Restifo, Nicholas P; Weiss, Richard; Scheiblhofer, Sandra; Xie, Chong; Sun, Ren; Cheng, Genhong; Zeng, Gang

    2011-10-28

    In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T cell-dependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-to-Ser) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern.

  12. T-cell intracellular antigens function as tumor suppressor genes.

    PubMed

    Sánchez-Jiménez, C; Ludeña, M D; Izquierdo, J M

    2015-03-05

    Knockdown of T-cell intracellular antigens TIA1 and TIAR in transformed cells triggers cell proliferation and tumor growth. Using a tetracycline-inducible system, we report here that an increased expression of TIA1 or TIAR in 293 cells results in reduced rates of cell proliferation. Ectopic expression of these proteins abolish endogenous TIA1 and TIAR levels via the regulation of splicing of their pre-mRNAs, and partially represses global translation in a phospho-eukaryotic initiation factor 2 alpha-dependent manner. This is accompanied by cell cycle arrest at G1/S and cell death through caspase-dependent apoptosis and autophagy. Genome-wide profiling illustrates a selective upregulation of p53 signaling pathway-related genes. Nude mice injected with doxycycline-inducible cells expressing TIA1 or TIAR retard, or even inhibit, growth of xenotumors. Remarkably, low expressions of TIA1 and TIAR correlate with poor prognosis in patients with lung squamous cell carcinoma. These findings strongly support the concept that TIA proteins act as tumor suppressor genes.

  13. Atypical natural killer T-cell receptor recognition of CD1d–lipid antigens

    PubMed Central

    Le Nours, Jérôme; Praveena, T.; Pellicci, Daniel G.; Gherardin, Nicholas A.; Ross, Fiona J.; Lim, Ricky T.; Besra, Gurdyal S.; Keshipeddy, Santosh; Richardson, Stewart K.; Howell, Amy R.; Gras, Stephanie; Godfrey, Dale I.; Rossjohn, Jamie; Uldrich, Adam P.

    2016-01-01

    Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d–α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1+ type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7–8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A′-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d–α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition. PMID:26875526

  14. Plasmodium falciparum complicated malaria: Modulation and connectivity between exportome and variant surface antigen gene families.

    PubMed

    Subudhi, Amit Kumar; Boopathi, P A; Pandey, Isha; Kohli, Ramandeep; Karwa, Rohan; Middha, Sheetal; Acharya, Jyoti; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2015-05-01

    In temperate and sub-tropical regions of Asia and Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from adult patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variant surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison between Plasmodium falciparum complicated and uncomplicated isolates) have been observed. The exportome has been analyzed using similar parameters. Gene ontology term based functional enrichment of differentially regulated genes identified, up-regulated genes statistically enriched (P<0.05) to critical biological processes like generation of precursor metabolite and energy, chromosome organization and electron transport chain. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group B and C genes whose proteins are predicted to interact with CD36 receptor in the host, the up-regulation of domain cassette 13 (DC13) containing var group A, as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of mostly var A group genes have been seen. A protein-protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variant surface antigens (VSA) and the exportome. The up

  15. Molecular and genetic analyses of the putative Proteus O antigen gene locus.

    PubMed

    Wang, Quan; Torzewska, Agnieszka; Ruan, Xiaojuan; Wang, Xiaoting; Rozalski, Antoni; Shao, Zhujun; Guo, Xi; Zhou, Haijian; Feng, Lu; Wang, Lei

    2010-08-01

    Proteus species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The Proteus O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of Proteus isolates. Even though most Proteus O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative Proteus O antigen locus and demonstrated this region's high degree of heterogeneity by comparing sequences of 40 Proteus isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative Proteus O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally, Proteus-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68 Proteus O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative Proteus O antigen locus, and we describe a novel molecular classification method for the identification of different Proteus serogroups.

  16. Antigen receptor-induced B lymphocyte apoptosis mediated via a protease of the caspase family.

    PubMed

    Andjelic, S; Liou, H C

    1998-02-01

    An extensive body of data, in a variety of systems, denoted the caspase family of proteases as a key player in the execution of programmed cell death. This family consists of cysteine proteases that cleave after asparagine-containing motifs. It is well established that the caspases are essential for the apoptosis mediated by Fas (CD95) and TNF receptor p55, molecules that contain the "death domain" in the cytoplasmic tail. However, little is known about the mechanisms underlying the antigen receptor-mediated cell death in B lymphocytes, a process instrumental in negative selection of potentially autoreactive B cells. Here, we investigated the involvement of caspases in cell death triggered via the antigen receptor in B lymphocytes (BCR) by using specific inhibitors. Initially, we used a well-established cell line, CH31, which is a model of B cell tolerance, to demonstrate that these proteases indeed participate in the BCR-induced apoptotic pathway. Next, we confirmed the physiological relevance of the caspase-mediated cell death pathway in splenic B cell populations isolated ex vivo that were induced to undergo apoptosis by extensive cross-linking of their BCR. Most interestingly, our data demonstrated that caspases regulate not only the nuclear DNA fragmentation, but also the surface membrane phosphatidylserine translocation as well as the degradation of a specific nuclear substrate. Taken together, this report supports the hypothesis that regulation of the caspase family is crucial in controlling the life/death decision in B lymphocytes mediated by the antigen receptor signal transduction.

  17. Proteinase 3, Wegener's autoantigen: from gene to antigen.

    PubMed

    van der Geld, Y M; Limburg, P C; Kallenberg, C G

    2001-02-01

    Proteinase 3 (PR3) is one of four serine protease homologues in the azurophilic granules of neutrophils and granules of monocytes. It is of importance that anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis (WG) are mainly directed against PR3 only. Furthermore, PR3 is overexpressed in a variety of acute and chronic myeloid leukemia cells. Cytotoxic T lymphocytes specific for a PR3-derived peptide have been shown to specifically lyse leukemia cells that overexpress PR3. This review will focus on PR3 and the characteristics of PR3 that might implicate this particular antigen in the pathogenesis of WG and as target for immunotherapy in myeloid leukemias. We will discuss the genetic localization and gene regulation of PR3, the processing, storage, and expression of the PR3 protein, and the physiological functions of PR3, and compare this with the three other neutrophil-derived serine proteases: human leukocyte elastase, cathepsin G, and azurocidin. Three main differences are described between PR3 and the other serine proteases. This makes PR3 a very intriguing protein with a large array of physiological functions, some of which may play a role in ANCA-associated vasculitidis and myeloid leukemia.

  18. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

    NASA Astrophysics Data System (ADS)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

    1986-08-01

    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  19. The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features.

    PubMed

    Wan, Zhengpeng; Liu, Wanli

    2012-07-01

    B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell's encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.

  20. A Human Minor Histocompatibility Antigen Resulting from Differential Expression due to a Gene Deletion

    PubMed Central

    Murata, Makoto; Warren, Edus H.; Riddell, Stanley R.

    2003-01-01

    Minor histocompatibility antigens (minor H antigens) are targets of graft-versus-host disease and graft-versus-leukemia responses after allogeneic human leukocyte antigen identical hematopoietic stem cell transplantation. Only a few human minor H antigens have been molecularly characterized and in all cases, amino acid differences between homologous donor and recipient proteins due to nucleotide polymorphisms in the respective genes were responsible for immunogenicity. Here, we have used cDNA expression cloning to identify a novel human minor H antigen encoded by UGT2B17, an autosomal gene in the multigene UDP-glycosyltransferase 2 family that is selectively expressed in liver, intestine, and antigen-presenting cells. In contrast to previously defined human minor H antigens, UGT2B17 is immunogenic because of differential expression of the protein in donor and recipient cells as a consequence of a homozygous gene deletion in the donor. Deletion of individual members of large gene families is a common form of genetic variation in the population and our results provide the first evidence that differential protein expression as a consequence of gene deletion is a mechanism for generating minor H antigens in humans. PMID:12743171

  1. T-cell receptor-CD4 physical association in a murine T-cell hybridoma: induction by antigen receptor ligation.

    PubMed Central

    Mittler, R S; Goldman, S J; Spitalny, G L; Burakoff, S J

    1989-01-01

    By employing flow cytometric analysis and fluorescence resonance energy transfer (FRET), we examined the physical relationship between the T-cell receptor-CD3 complex (Ti-CD3) and the CD4 molecule on helper T cells. Through the use of an L3T4-negative murine T-cell hybridoma infectant expressing the human CD4 gene and having antigen specificity for HLA-DR, we show that binding of the Ti-CD3 complex with an anti-CD3 monoclonal antibody induces its redistribution proximal to cell-surface CD4. FRET efficiency was 9.4% on cells labeled with rhodaminated anti-CD3 and fluoresceinated anti-CD4. FRET was found to be temperature dependent, since similarly treated cells held at 4 degrees C displayed a FRET efficiency of less than 1%. Energy transfer was evident within 3 min after warming cells to 37 degrees C. Energy transfer was not detected between Ti-CD3 and the abundantly expressed leukocyte common antigen (CD45). Of greater significance was our observation that hybridomas infected with a truncated CD4 gene lacking the cytoplasmic domain failed to transfer energy despite the fact that CD4 was expressed on the cell surface at levels equivalent to or greater than the wild type. These studies suggest that after crosslinking of the Ti-CD3 on CD4+ T cells, a physical association occurs between the antigen receptor complex and CD4 and that the association is dependent upon the presence of the cytoplasmic domain of CD4. PMID:2530583

  2. Evaluation of the Antigen-Experienced B-Cell Receptor Repertoire in Healthy Children and Adults

    PubMed Central

    IJspeert, Hanna; van Schouwenburg, Pauline A.; van Zessen, David; Pico-Knijnenburg, Ingrid; Driessen, Gertjan J.; Stubbs, Andrew P.; van der Burg, Mirjam

    2016-01-01

    Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1–CDR3 sequence, but different subclasses. Together, these data suggest that

  3. The mouse B-cell antigen receptor: definition and assembly of the core receptor of the five immunoglobulin isotypes.

    PubMed

    Neuberger, M S; Patel, K J; Dariavach, P; Nelms, K; Peaker, C J; Williams, G T

    1993-04-01

    We have shown that the core antigen receptor of all five isotypes is composed of immunoglobulin in association with a common heterodimeric alpha/beta sheath. The stoichiometry of the association is unknown although preliminary evidence points to it being an IgH2L2 [alpha/beta]2 association. Studies with chimaeric molecules indicate that much of the immunoglobulin-sheath interaction must occur through the carboxyterminal end of the molecule with particular importance being given to the linker-transmembrane region. The glycosylation of the alpha chain differs according to the isotype with which it is associated. There are two sites for N-linked glycosylation on the alpha chain (Asn-30 and Asn-40); both sites are used. Mutation of Asn-30 alone decreases but does not abolish surface expression of the antigen receptor complex. Mutation of both sites prevents expression of the surface IgM[alpha/beta] complex but not of a surface IgD[alpha/beta] complex. Moreover, the pattern of alpha glycosylation is considerably affected by changes in the linker region between C mu 4 and the transmembrane, giving further support to the importance of this region in immunoglobulin-sheath interaction. Unlike IgM, IgD and IgG2b do not require alpha/beta for transport to the cell surface and can be expressed on the surface without either sheath or glycosyl phosphatidylinositol anchor. This finding may reflect that the IgD transmembrane region is significantly less hydrophobic than that of IgM; however, it should be noted that is not clear whether naked IgD exists in vivo. In fact, we have found that the alpha/beta sheath is necessary in order to facilitate efficient internalization and presentation of antigen by membrane immunoglobulin. The sheath presumably also plays a major role in potentiating transmembrane signalling. However, mutant receptors that do not associate with the alpha/beta sheath are nevertheless able to trigger phosphorylation of cellular proteins on tyrosine residues

  4. Expression of CD3-associated antigen-binding receptors on suppressor T cells.

    PubMed Central

    Kuchroo, V K; Steele, J K; Billings, P R; Selvaraj, P; Dorf, M E

    1988-01-01

    Three suppressor T (Ts)-cell hybridomas specific for 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were selected for surface expression of cluster determinant 3 (CD3) by using antibody (anti-CD3) or antigen (NP-bovine serum albumin) panning procedures followed by cloning at limiting dilution. The CD3-selected Ts hybridomas showed a 1-2 logarithmic enrichment in suppressor activity when compared to the parent lines; they also specifically bound NP-coupled sheep red blood cells in rosette assays. This antigen-binding ability could be down-modulated by anti-CD3 antibody. Similarly, surface expression of CD3 was specifically down-modulated by preincubation of these hybridomas with antigen. Anti-CD3 monoclonal antibody under reducing conditions coprecipitated a broad band of 38-50 kDa associated with two CD3 (25 and 16 kDa) bands. T-cell receptor, anti-alpha-specific monoclonal antibody also immunoprecipitated a broad band in the 41 to 49-kDa region. The combined results suggest that, like helper and cytotoxic T lymphocytes, Ts cells also bear antigen-specific receptors associated with CD3 molecules. Images PMID:2973609

  5. GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs.

    PubMed

    Eicke, Nicola; Günthert, Andreas R; Viereck, Volker; Siebold, Doreen; Béhé, Martin; Becker, Tamara; Emons, Günter; Gründker, Carsten

    2005-10-01

    We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor

  6. Prospects and limitations of T cell receptor gene therapy.

    PubMed

    Jorritsma, Annelies; Schotte, Remko; Coccoris, Miriam; de Witte, Moniek A; Schumacher, Ton N M

    2011-08-01

    Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining molecule in T cell function, adoptive transfer of TCR genes into patient T cells may be used as an alternative approach for the transfer of tumor-specific T cell immunity. On theoretical grounds, TCR gene therapy has two substantial advantages over conventional cellular transfer. First, it circumvents the demanding process of in vitro generation of large numbers of specific immune cells. Second, it allows the use of a set of particularly effective TCR genes in large patient groups. Conversely, TCR gene therapy may be associated with a number of specific problems that are not confronted during classical cellular therapy. Here we review our current understanding of the potential and possible problems of TCR gene therapy, as based on in vitro experiments, mouse model systems and phase I clinical trials. Furthermore, we discuss the prospects of widespread clinical application of this gene therapy approach for the treatment of human cancer.

  7. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.

    PubMed

    MacDonald, Katherine G; Hoeppli, Romy E; Huang, Qing; Gillies, Jana; Luciani, Dan S; Orban, Paul C; Broady, Raewyn; Levings, Megan K

    2016-04-01

    Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

  8. Chimeric antigen receptor T cells: a novel therapy for solid tumors.

    PubMed

    Yu, Shengnan; Li, Anping; Liu, Qian; Li, Tengfei; Yuan, Xun; Han, Xinwei; Wu, Kongming

    2017-03-29

    The chimeric antigen receptor T (CAR-T) cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs) expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII) was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2), and mesothelin (MSLN), as well as the challenges for CAR-T cell therapy.

  9. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor.

    PubMed

    Streltsov, V A; Varghese, J N; Carmichael, J A; Irving, R A; Hudson, P J; Nuttall, S D

    2004-08-24

    The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-A structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the "bottom" of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain/variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended beta-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes.

  10. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire

    PubMed Central

    1991-01-01

    We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all. PMID:1836010

  11. Novel Aeromonas hydrophila PPD134/91 Genes Involved in O-Antigen and Capsule Biosynthesis

    PubMed Central

    Zhang, Y. L.; Arakawa, E.; Leung, K. Y.

    2002-01-01

    The sequences of the O-antigen and capsule gene clusters of the virulent Aeromonas hydrophila strain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11 A. hydrophila strains of different serotypes. A. hydrophila PPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster of A. hydrophila PPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster of Escherichia coli and other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulent A. hydrophila strain PPD35/85 to survive in naïve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion of A. hydrophila PPD134/91 to carp epithelial cells. PMID:11953367

  12. Requirement for caspase-8 in NF-kappaB activation by antigen receptor.

    PubMed

    Su, Helen; Bidère, Nicolas; Zheng, Lixin; Cubre, Alan; Sakai, Keiko; Dale, Janet; Salmena, Leonardo; Hakem, Razqallah; Straus, Stephen; Lenardo, Michael

    2005-03-04

    Caspase-8, a proapoptotic protease, has an essential role in lymphocyte activation and protective immunity. We show that caspase-8 deficiency (CED) in humans and mice specifically abolishes activation of the transcription factor nuclear factor kappaB (NF-kappaB) after stimulation through antigen receptors, Fc receptors, or Toll-like receptor 4 in T, B, and natural killer cells. Caspase-8 also causes the alphabeta complex of the inhibitor of NF-kappaB kinase (IKK) to associate with the upstream Bcl10-MALT1 (mucosa-associated lymphatic tissue) adapter complex. Recruitment of the IKKalpha, beta complex, its activation, and the nuclear translocation of NF-kappaB require enzyme activity of full-length caspase-8. These findings thus explain the paradoxical association of defective apoptosis and combined immunodeficiency in human CED.

  13. The androgen receptor gene mutations database.

    PubMed

    Patterson, M N; Hughes, I A; Gottlieb, B; Pinsky, L

    1994-09-01

    The androgen receptor gene mutations database is a comprehensive listing of mutations published in journals and meetings proceedings. The majority of mutations are point mutations identified in patients with androgen insensitivity syndrome. Information is included regarding the phenotype, the nature and location of the mutations, as well as the effects of the mutations on the androgen binding activity of the receptor. The current version of the database contains 149 entries, of which 114 are unique mutations. The database is available from EMBL (NetServ@EMBL-Heidelberg.DE) or as a Macintosh Filemaker file (mc33001@musica.mcgill.ca).

  14. Immunoreceptor tyrosine-based activation motif (ITAM), a unique module linking antigen and Fc receptors to their signaling cascades.

    PubMed

    Isakov, N

    1997-01-01

    Signal transduction by the T cell and B cell antigen receptors and by receptors for a variety of immunoglobulins' Fc region is strictly dependent on a receptor subunit cytoplasmic module termed immunoreceptor tyrosine-based activation motif (ITAM). This module exists in one or more copies in each of the receptor-associated signal-transducing molecules and it possesses two repeats of the consensus sequence Tyr-X-X-Leu/Ile spaced by six to eight amino acids. Receptor engagement is followed by a rapid and transient phosphorylation of tyrosine residues within their ITAMs, thereby creating temporary binding sites for Src homology 2 (SH2)-containing signaling molecules operating downstream of the activated receptor. The purpose of this review is to discuss recent findings on the functional role of ITAMs in antigen and Fc receptor-mediated signal transduction, with a particular emphasis on kinases operating upstream and downstream of the ITAMs.

  15. Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination

    PubMed Central

    Qi, Qian; Cavanagh, Mary M.; Le Saux, Sabine; NamKoong, Hong; Kim, Chulwoo; Turgano, Emerson; Liu, Yi; Wang, Chen; Mackey, Sally; Swan, Gary E.; Dekker, Cornelia L.; Olshen, Richard A.; Boyd, Scott D.; Weyand, Cornelia M.; Tian, Lu; Goronzy, Jörg J.

    2016-01-01

    Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood is able to escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. While all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells, but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important read-out to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection. PMID:27030598

  16. Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

    PubMed Central

    Tabarin, Thibault; Yamamoto, Yui; Ma, Yuanqing; Nicovich, Philip R.; Bridgeman, John S.; Cohnen, André; Benzing, Carola; Gao, Yijun; Crowther, Michael D.; Tungatt, Katie; Dolton, Garry; Sewell, Andrew K.; Price, David A.; Acuto, Oreste; Parton, Robert G.; Gooding, J. Justin; Rossy, Jérémie; Rossjohn, Jamie; Gaus, Katharina

    2016-01-01

    Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination. PMID:27573839

  17. Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering.

    PubMed

    Molnár, Eszter; Swamy, Mahima; Holzer, Martin; Beck-García, Katharina; Worch, Remigiusz; Thiele, Christoph; Guigas, Gernot; Boye, Kristian; Luescher, Immanuel F; Schwille, Petra; Schubert, Rolf; Schamel, Wolfgang W A

    2012-12-14

    The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRβ chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRβ chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

  18. Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

    PubMed

    Pageon, Sophie V; Tabarin, Thibault; Yamamoto, Yui; Ma, Yuanqing; Bridgeman, John S; Cohnen, André; Benzing, Carola; Gao, Yijun; Crowther, Michael D; Tungatt, Katie; Dolton, Garry; Sewell, Andrew K; Price, David A; Acuto, Oreste; Parton, Robert G; Gooding, J Justin; Rossy, Jérémie; Rossjohn, Jamie; Gaus, Katharina

    2016-09-13

    Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

  19. Characterization of thymus-derived lymphocytes expressing Ti alpha-beta CD3 gamma delta epsilon zeta-zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta- eta antigen receptor isoforms: analysis by gene transfection

    PubMed Central

    1990-01-01

    To characterize the function of the CD3 eta subunit of the T cell receptor (TCR), we have used cDNAs encoding CD3 zeta, CD3 eta, or both to reconstitute a variant of a cytochrome c-specific, I-Ek-restricted murine T cell hybridoma, termed MA5.8, which lacks CD3 zeta and CD3 eta proteins. We provide direct evidence that assembly and surface expression of TCRs can be mediated by either of these subunits separately or together. However, the level of TCR expression on zeta transfectants is up to one order of magnitude greater than that on eta transfectants, implying that CD3 eta is weakly associated with the pentameric Ti alpha-beta CD3 gamma delta epsilon complex and/or inefficient at salvaging the incomplete TCR from lysosomal degradation. As a component of the TCR, the CD3 eta subunit preferentially forms a heterodimer with CD3 zeta, but is also able to form a CD3 eta-eta homodimer. Crosslinking of Ti alpha-beta CD3 gamma delta epsilon zeta- zeta, Ti alpha-beta CD3 gamma delta epsilon eta-eta, or Ti alpha-beta CD3 gamma delta epsilon zeta-zeta/zeta-eta TCR isotypes with anti-CD3 epsilon monoclonal antibody or a cytochrome c peptide epitope on I-Ek antigen-presenting cells mediates signal transduction resulting in reversible cell-cycle arrest of transfected clones. Given the potential for diversity of signals generated by these functional TCR isotypes and the expression of the CD3 eta gene product in the thymus, CD3 eta is likely to play a role in selection and/or activation of thymocytes during development. PMID:2145389

  20. The B-cell antigen receptor integrates adaptive and innate immune signals

    PubMed Central

    Otipoby, Kevin L.; Waisman, Ari; Derudder, Emmanuel; Srinivasan, Lakshmi; Franklin, Andrew; Rajewsky, Klaus

    2015-01-01

    B cells respond to antigens by engagement of their B-cell antigen receptor (BCR) and of coreceptors through which signals from helper T cells or pathogen-associated molecular patterns are delivered. We show that the proliferative response of B cells to the latter stimuli is controlled by BCR-dependent activation of phosphoinositidyl 3-kinase (PI-3K) signaling. Glycogen synthase kinase 3β and Foxo1 are two PI-3K-regulated targets that play important roles, but to different extents, depending on the specific mitogen. These results suggest a model for integrating signals from the innate and the adaptive immune systems in the control of the B-cell immune response. PMID:26371314

  1. Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

    PubMed Central

    Cartellieri, Marc; Bachmann, Michael; Feldmann, Anja; Bippes, Claudia; Stamova, Slava; Wehner, Rebekka; Temme, Achim; Schmitz, Marc

    2010-01-01

    CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs). First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells. PMID:20467460

  2. Intracerebral delivery of a third generation EGFRvIII-specific chimeric antigen receptor is efficacious against human glioma.

    PubMed

    Choi, Bryan D; Suryadevara, Carter M; Gedeon, Patrick C; Herndon, James E; Sanchez-Perez, Luis; Bigner, Darell D; Sampson, John H

    2014-01-01

    Chimeric antigen receptors (CAR)-transduced T cells hold great promise in the treatment of malignant disease. Here, we demonstrate that intracerebral injection with a human, epidermal growth factor receptor variant III (EGFRvIII)-specific, third generation CAR successfully treats glioma in mice. Importantly, these results endorse clinical translation of this CAR in patients with EGFRvIII-expressing brain tumors.

  3. Receptor-mediated endocytosis of carcinoembryonic antigen by rat liver Kupffer cells.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1985-01-01

    In vivo, carcinoembryonic antigen (CEA) is removed from the circulation by the liver Kupffer cells. Immunologically identifiable CEA is transferred from these macrophages to the hepatocytes, where degradation is completed. Circulatory clearance of CEA is specific, rapid [t1/2 = 3.7 +/- 0.9 (S.D.) min], and saturable. In vitro, Kupffer cells take up CEA by a saturable process which is time/temperature dependent and colchicine sensitive. Isolated Kupffer cells endocytose CEA with an apparent Km of 6 X 10(-8) M. There are approximately 16,000 CEA binding sites per cell. Nonspecific cross-reacting antigen (NCA), a glycoprotein structurally similar to CEA, is recognized with lower affinity by the same receptor. Endocytosis is independent of the nonreducing terminal sugars on the molecule: CEA modified by Smith degradation inhibits Kupffer cell recognition of native CEA. Since performic acid oxidized CEA also inhibits endocytosis, receptor binding is similarly independent of intact protein conformation. Isolated Kupffer cells have mannose and/or N-acetyl glucosamine receptor activity but do not internalize CEA by that mechanism. Galactose-terminated glycoproteins impede CEA and NCA clearance in vivo but not Kupffer cell endocytosis in vitro. Radiolabeled CEA released from isolated Kupffer cells following endocytosis shows no apparent molecular weight change. However, the released CEA contains species with higher isoelectric points, suggesting that perhaps the removal of sialic acid and the resulting exposure of galactose residues mediate the subsequent transfer to the hepatocyte.

  4. The paracaspase MALT1 cleaves the LUBAC subunit HOIL1 during antigen receptor signaling.

    PubMed

    Douanne, Tiphaine; Gavard, Julie; Bidère, Nicolas

    2016-05-01

    Antigen-receptor-mediated activation of lymphocytes relies on a signalosome comprising CARMA1 (also known as CARD11), BCL10 and MALT1 (the CBM complex). The CBM activates nuclear factor κB (NF-κB) transcription factors by recruiting the 'linear ubiquitin assembly complex' (LUBAC), and unleashes MALT1 paracaspase activity. Although MALT1 enzyme shapes NF-κB signaling, lymphocyte activation and contributes to lymphoma growth, the identity of its substrates continues to be elucidated. Here, we report that the LUBAC subunit HOIL1 (also known as RBCK1) is cleaved by MALT1 following antigen receptor engagement. HOIL1 is also constitutively processed in the 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), which exhibits aberrant MALT1 activity. We further show that the overexpression of MALT1-insensitive HOIL1 mitigates T-cell-receptor-mediated NF-κB activation and subsequent cytokine production in lymphocytes. Thus, our results unveil HOIL1 as a negative regulator of lymphocyte activation cleaved by MALT1. This cleavage could therefore constitute an appealing therapeutic target for modulating immune responses.

  5. Positive and negative regulation of antigen receptor signaling by the Shc family of protein adapters.

    PubMed

    Finetti, Francesca; Savino, Maria Teresa; Baldari, Cosima T

    2009-11-01

    The Shc adapter family includes four members that are expressed as multiple isoforms and participate in signaling by a variety of cell-surface receptors. The biological relevance of Shc proteins as well as their variegated function, which relies on their highly conserved modular structure, is underscored by the distinct and dramatic phenotypic alterations resulting from deletion of individual Shc isoforms both in the mouse and in two model organisms, Drosophila melanogaster and Caenorhabditis elegans. The p52 isoform of ShcA couples antigen and cytokine receptors to Ras activation in both lymphoid and myeloid cells. However, the recognition of the spectrum of activities of p52ShcA in the immune system has been steadily expanding in recent years to other fundamental processes both at the cell and organism levels. Two other Shc family members, p66ShcA and p52ShcC/Rai, have been identified recently in T and B lymphocytes, where they antagonize survival and attenuate antigen receptor signaling. These developments reveal an unexpected and complex interplay of multiple Shc proteins in lymphocytes.

  6. Social regulation of cortisol receptor gene expression

    PubMed Central

    Korzan, Wayne J.; Grone, Brian P.; Fernald, Russell D.

    2014-01-01

    In many social species, individuals influence the reproductive capacity of conspecifics. In a well-studied African cichlid fish species, Astatotilapia burtoni, males are either dominant (D) and reproductively competent or non-dominant (ND) and reproductively suppressed as evidenced by reduced gonadotropin releasing hormone (GnRH1) release, regressed gonads, lower levels of androgens and elevated levels of cortisol. Here, we asked whether androgen and cortisol levels might regulate this reproductive suppression. Astatotilapia burtoni has four glucocorticoid receptors (GR1a, GR1b, GR2 and MR), encoded by three genes, and two androgen receptors (ARα and ARβ), encoded by two genes. We previously showed that ARα and ARβ are expressed in GnRH1 neurons in the preoptic area (POA), which regulates reproduction, and that the mRNA levels of these receptors are regulated by social status. Here, we show that GR1, GR2 and MR mRNAs are also expressed in GnRH1 neurons in the POA, revealing potential mechanisms for both androgens and cortisol to influence reproductive capacity. We measured AR, MR and GR mRNA expression levels in a microdissected region of the POA containing GnRH1 neurons, comparing D and ND males. Using quantitative PCR (qPCR), we found D males had higher mRNA levels of ARα, MR, total GR1a and GR2 in the POA compared with ND males. In contrast, ND males had significantly higher levels of GR1b mRNA, a receptor subtype with a reduced transcriptional response to cortisol. Through this novel regulation of receptor type, neurons in the POA of an ND male will be less affected by the higher levels of cortisol typical of low status, suggesting GR receptor type change as a potential adaptive mechanism to mediate high cortisol levels during social suppression. PMID:25013108

  7. The T cell antigen receptor: the Swiss army knife of the immune system

    PubMed Central

    Attaf, M; Legut, M; Cole, D K; Sewell, A K

    2015-01-01

    The mammalian T cell receptor (TCR) orchestrates immunity by responding to many billions of different ligands that it has never encountered before and cannot adapt to at the protein sequence level. This remarkable receptor exists in two main heterodimeric isoforms: αβ TCR and γδ TCR. The αβ TCR is expressed on the majority of peripheral T cells. Most αβ T cells recognize peptides, derived from degraded proteins, presented at the cell surface in molecular cradles called major histocompatibility complex (MHC) molecules. Recent reports have described other αβ T cell subsets. These ‘unconventional’ T cells bear TCRs that are capable of recognizing lipid ligands presented in the context of the MHC-like CD1 protein family or bacterial metabolites bound to the MHC-related protein 1 (MR1). γδ T cells constitute a minority of the T cell pool in human blood, but can represent up to half of total T cells in tissues such as the gut and skin. The identity of the preferred ligands for γδ T cells remains obscure, but it is now known that this receptor can also functionally engage CD1-lipid, or immunoglobulin (Ig) superfamily proteins called butyrophilins in the presence of pyrophosphate intermediates of bacterial lipid biosynthesis. Interactions between TCRs and these ligands allow the host to discriminate between self and non-self and co-ordinate an attack on the latter. Here, we describe how cells of the T lymphocyte lineage and their antigen receptors are generated and discuss the various modes of antigen recognition by these extraordinarily versatile receptors. PMID:25753381

  8. Nanostructured materials detect epidermal growth factor receptor, neuron specific enolase and carcinoembryonic antigen

    NASA Astrophysics Data System (ADS)

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Badulescu, Marius

    2015-09-01

    New nanostructured materials based on thin films of Cu and Ni deposited on textile material (veil), as well as gold nanostructured microspheres were used for the design of new stochastic sensors. The stochastic sensors were able to detect simultaneously a panel of biomarkers comprising epidermal growth factor receptor, neuron specific enolase, and carcinoembryonic antigen from whole blood samples with high reliabilities - recovery tests higher than 97.00%, with a RSD (%) lower than 0.1%. The stochastic sensors had shown high sensitivities and low determination levels for the detection of the proposed panel of biomarkers making early detection of lung cancer possible by fast screening of whole blood.

  9. Molecular characterization of swine leukocyte antigen (SLA) class II genes in outbred pig populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly polymorphic swine leukocyte antigen (SLA) genes are one of the most important determinants in swine immune, disease and vaccine responses. Thus, understanding how SLA gene polymorphism affects immunity, especially in outbred pig populations with a diverse genetic background, requires accu...

  10. Phylogenetic analysis of the swine leukocyte antigen - 2 gene for Korean native pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to investigate genetic distances of the SLA-2 gene, to characterize SLA-2 alleles, and to provide basic genetic information of Korean pigs. The swine leukocyte antigen - 2 (SLA-2) gene in the MHC classical region was cloned with spleen tissues from Korean native pigs ...

  11. 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.

  12. Direct Delivery of Antigens to Dendritic Cells via Antibodies Specific for Endocytic Receptors as a Promising Strategy for Future Therapies

    PubMed Central

    Lehmann, Christian H. K.; Heger, Lukas; Heidkamp, Gordon F.; Baranska, Anna; Lühr, Jennifer J.; Hoffmann, Alana; Dudziak, Diana

    2016-01-01

    Dendritic cells (DCs) are the most potent professional antigen presenting cells and are therefore indispensable for the control of immunity. The technique of antibody mediated antigen targeting to DC subsets has been the basis of intense research for more than a decade. Many murine studies have utilized this approach of antigen delivery to various kinds of endocytic receptors of DCs both in vitro and in vivo. Today, it is widely accepted that different DC subsets are important for the induction of select immune responses. Nevertheless, many questions still remain to be answered, such as the actual influence of the targeted receptor on the initiation of the immune response to the delivered antigen. Further efforts to better understand the induction of antigen-specific immune responses will support the transfer of this knowledge into novel treatment strategies for human diseases. In this review, we will discuss the state-of-the-art aspects of the basic principles of antibody mediated antigen targeting approaches. A table will also provide a broad overview of the latest studies using antigen targeting including addressed DC subset, targeted receptors, outcome, and applied coupling techniques. PMID:27043640

  13. Coupling of HIV-1 Antigen to the Selective Autophagy Receptor SQSTM1/p62 Promotes T-Cell-Mediated Immunity

    PubMed Central

    Andersen, Aram Nikolai; Landsverk, Ole Jørgen; Simonsen, Anne; Bogen, Bjarne; Corthay, Alexandre; Øynebråten, Inger

    2016-01-01

    Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4+ and CD8+ T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8+ T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans. PMID:27242780

  14. Linking the T cell receptor to the single cell transcriptome in antigen-specific human T cells.

    PubMed

    Eltahla, Auda A; Rizzetto, Simone; Pirozyan, Mehdi R; Betz-Stablein, Brigid D; Venturi, Vanessa; Kedzierska, Katherine; Lloyd, Andrew R; Bull, Rowena A; Luciani, Fabio

    2016-07-01

    Heterogeneity of T cells is a hallmark of a successful adaptive immune response, harnessing the vast diversity of antigen-specific T cells into a coordinated evolution of effector and memory outcomes. The T cell receptor (TCR) repertoire is highly diverse to account for the highly heterogeneous antigenic world. During the response to a virus multiple individual clones of antigen specific CD8+ (Ag-specific) T cells can be identified against a single epitope and multiple epitopes are recognised. Advances in single-cell technologies have provided the potential to study Ag-specific T cell heterogeneity at both surface phenotype and transcriptome levels, thereby allowing investigation of the diversity within the same apparent sub-population. We propose a new method (VDJPuzzle) to reconstruct the native TCRαβ from single cell RNA-seq data of Ag-specific T cells and then to link these with the gene expression profile of individual cells. We applied this method using rare Ag-specific T cells isolated from peripheral blood of a subject who cleared hepatitis C virus infection. We successfully reconstructed productive TCRαβ in 56 of a total of 63 cells (89%), with double α and double β in 18, and 7% respectively, and double TCRαβ in 2 cells. The method was validated via standard single cell PCR sequencing of the TCR. We demonstrate that single-cell transcriptome analysis can successfully distinguish Ag-specific T cell populations sorted directly from resting memory cells in peripheral blood and sorted after ex vivo stimulation. This approach allows a detailed analysis of the TCR diversity and its relationship with the transcriptional profile of different clones.

  15. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    PubMed

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2013-01-01

    Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR(+) T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+) tumor targets. This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  16. T-Cell Receptor Gene Therapy of Established Tumors in a Murine Melanoma Model

    PubMed Central

    Abad, John D.; Wrzensinski, Claudia; Overwijk, Willem; De Witte, Moniek A.; Jorritsma, Annelies; Hsu, Gary; Gattinoni, Luca; Cohen, Cyrille J.; Paulos, Chrystal M.; Palmer, Douglas C.; Haanen, John B. A. G.; Schumacher, Ton N. M.; Rosenberg, Steven A.; Restifo, Nicholas P.; Morgan, Richard A.

    2008-01-01

    Summary Adoptive cell transfer therapy using tumor-infiltrating lymphocytes for patients with metastatic melanoma has demonstrated significant objective response rates. One major limitation of these current therapies is the frequent inability to isolate tumor-reactive lymphocytes for treatment. Genetic engineering of peripheral blood lymphocytes with retroviral vectors encoding tumor antigen-specific T-cell receptors (TCRs) bypasses this restriction. To evaluate the efficacy of TCR gene therapy, a murine treatment model was developed. A retroviral vector was constructed encoding the pmel-1 TCR genes targeting the B16 melanoma antigen, gp100. Transduction of C57BL/6 lymphocytes resulted in efficient pmel-1 TCR expression. Lymphocytes transduced with this retrovirus specifically recognized gp100-pulsed target cells as measured by interferon-γ secretion assays. Upon transfer into B16 tumor-bearing mice, the genetically engineered lymphocytes significantly slowed tumor development. The effectiveness of tumor treatment was directly correlated with the number of TCR-engineered T cells administered. These results demonstrated that TCR gene therapy targeting a native tumor antigen significantly delayed the growth of established tumors. When C57BL/6 lymphocytes were added to antigen-reactive pmel-1 T cells, a reduction in the ability of pmel-1 T cell to treat B16 melanomas was seen, suggesting that untransduced cells may be deleterious to TCR gene therapy. This model may be a powerful tool for evaluating future TCR gene transfer-based strategies. PMID:18157006

  17. T-cell receptor gene therapy of established tumors in a murine melanoma model.

    PubMed

    Abad, John D; Wrzensinski, Claudia; Overwijk, Willem; De Witte, Moniek A; Jorritsma, Annelies; Hsu, Cary; Gattinoni, Luca; Cohen, Cyrille J; Paulos, Chrystal M; Palmer, Douglas C; Haanen, John B A G; Schumacher, Ton N M; Rosenberg, Steven A; Restifo, Nicholas P; Morgan, Richard A

    2008-01-01

    Adoptive cell transfer therapy using tumor-infiltrating lymphocytes for patients with metastatic melanoma has demonstrated significant objective response rates. One major limitation of these current therapies is the frequent inability to isolate tumor-reactive lymphocytes for treatment. Genetic engineering of peripheral blood lymphocytes with retroviral vectors encoding tumor antigen-specific T-cell receptors (TCRs) bypasses this restriction. To evaluate the efficacy of TCR gene therapy, a murine treatment model was developed. A retroviral vector was constructed encoding the pmel-1 TCR genes targeting the B16 melanoma antigen, gp100. Transduction of C57BL/6 lymphocytes resulted in efficient pmel-1 TCR expression. Lymphocytes transduced with this retrovirus specifically recognized gp100-pulsed target cells as measured by interferon-gamma secretion assays. Upon transfer into B16 tumor-bearing mice, the genetically engineered lymphocytes significantly slowed tumor development. The effectiveness of tumor treatment was directly correlated with the number of TCR-engineered T cells administered. These results demonstrated that TCR gene therapy targeting a native tumor antigen significantly delayed the growth of established tumors. When C57BL/6 lymphocytes were added to antigen-reactive pmel-1 T cells, a reduction in the ability of pmel-1 T cell to treat B16 melanomas was seen, suggesting that untransduced cells may be deleterious to TCR gene therapy. This model may be a powerful tool for evaluating future TCR gene transfer-based strategies.

  18. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction.

    PubMed

    Nika, Konstantina; Soldani, Cristiana; Salek, Mogjiborahman; Paster, Wolfgang; Gray, Adrian; Etzensperger, Ruth; Fugger, Lars; Polzella, Paolo; Cerundolo, Vincenzo; Dushek, Omer; Höfer, Thomas; Viola, Antonella; Acuto, Oreste

    2010-06-25

    T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to approximately 40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-zeta phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.

  19. Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells

    PubMed Central

    Maher, John

    2012-01-01

    Chimeric antigen receptor- (CAR-) based immunotherapy has been under development for almost 25 years, over which period it has progressed from a new but cumbersome technology to an emerging therapeutic modality for malignant disease. The approach involves the genetic engineering of fusion receptors (CARs) that couple the HLA-independent binding of cell surface target molecules to the delivery of a tailored activating signal to host immune cells. Engineered CARs are delivered most commonly to peripheral blood T cells using a range of vector systems, most commonly integrating viral vectors. Preclinical refinement of this approach has proceeded over several years to the point that clinical testing is now being undertaken at several centres, using increasingly sophisticated and therapeutically successful genetic payloads. This paper considers several aspects of the pre-clinical and clinical development of CAR-based immunotherapy and how this technology is acquiring an increasing niche in the treatment of both solid and haematological malignancies. PMID:23304553

  20. [DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors].

    PubMed

    Cardozo, Daniela Maira; Guelsin, Gláucia Andréia; Clementino, Samaia Laface; Melo, Fabiano Cavalcante de; Braga, Marco Antônio; Souza, Cleonice de; Moliterno, Ricardo Alberto; Visentainer, Jeane Eliete Laguila

    2009-01-01

    The objective of this study was to standardize a method for extracting high-quality DNA from samples of coagulated blood. Forty-eight samples of human coagulated blood were used for DNA extraction by means of the EZ-DNA commercial kit (Biological Industries, Beit Haemek, Israel), the Neoscience column kit (One Lambda Inc., San Diego, CA, USA) and a modified salting-out method. Only the salting-out method was able to extract high concentrations of DNA (mean, 180 ng/(1/4)microl), which were measured using the Qubit fluorescence detector (Invitrogen, USA). This method enabled amplification of HLA (human leukocyte antigen) genes using the Luminex PCR-SSO (polymerase chain reaction - sequence-specific oligonucleotide) technology, which demands good quality DNA, and amplification of KIR (killer-cell immunoglobulin-like receptor) genes using an in-house PCR-SSP (polymerase chain reaction - sequence-specific primer) technique, which demands a specific concentration of DNA (10 ng/(1/4)microl). We concluded that the modified salting-out technique was very efficient, simple and fast for DNA extraction from human coagulated blood samples, with the aim of genotyping the HLA and KIR genes.

  1. Improved efficiency in amplification of Escherichia coli o-antigen gene clusters using genome-wide sequence comparison

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: In many bacteria including E. coli, genes encoding O-antigens are clustered in the chromosome, with a 39-bp JUMPstart sequence and gnd gene located upstream and downstream of the cluster, respectively. For determining the DNA sequence of the E. coli O-antigen gene cluster, one set of P...

  2. The Evolution of Mammalian Olfactory Receptor Genes

    PubMed Central

    Issel-Tarver, L.; Rine, J.

    1997-01-01

    We performed a comparative study of four subfamilies of olfactory receptor genes first identified in the dog to assess changes in the gene family during mammalian evolution, and to begin linking the dog genetic map to that of humans. The human subfamilies were localized to chromosomes 7, 11, and 19. The two subfamilies that were tightly linked in the dog genome were also tightly linked in the human genome. The four subfamilies were compared in human (primate), horse (perissodactyl), and a variety of artiodactyls and carnivores. Some changes in gene number were detected, but overall subfamily size appeared to have been established before the divergence of these mammals 60-100 million years ago. PMID:9017400

  3. Multilocus Sequence Analysis of Housekeeping Genes and Antigenic Determinant Genes in Bordetella pertussis Strains Isolated in Korea

    PubMed Central

    Jung, Sang-Oun; Moon, Yu Mi; Kim, So-Hyeon; Sung, Hwa Young; Kwon, Seung-Jik; Kang, Yeon Ho; Yu, Jae Yon

    2011-01-01

    Objectives To confirm genotype diversities of clinical isolates of Bordetella pertussis and to evaluate the risk of pertussis outbreak in Korea. Methods Seven housekeeping genes and 10 antigenic determinant genes from clinical B. pertussis isolates were analyzed by Multilocus sequence typing (MLST). Results More variant pattern was observed in antigenic determinant genes. Especially, PtxS1 gene was the most variant gene; five genotypes were observed from eight global genotypes. In the bacterial type, the number of observed sequence types in the isolates was seven and the most frequent form was type 1 (79.6%). This major sequence type also showed a time-dependent transition pattern. Older isolates (1968 and 1975) showed type 1 and 6 in housekeeping genes and antigenic determinant genes, respectively. However, these were changed to type 2 and 1 in isolates 1999–2008. This transition was mainly attributed to genotype change of PtxS1 and Fim3 gene; the tendency of genotype change was to avoid vaccine-derived genotype. In addition, there was second transition in 2009. In this period, only the sequence type of antigenic determinant genes was changed to type 2. Based Upon Related Sequence Types (BURST) analysis confirmed that there were two clonal complexes (ACCI and ACCII) in the Korean isolates. Moreover, the recently increased sequence type was revealed as AST2 derived from AST 3 in ACCI. Conclusions Genotype changes in Korean distributing strains are still progressing and there was a specific driving force in antigenic determinant genes. Therefore continuous surveillance of genotype change of the distributing strains should be performed to confirm interrelationship of genotype change with vaccine immunity. PMID:24159461

  4. Identifying Individual T Cell Receptors of Optimal Avidity for Tumor Antigens

    PubMed Central

    Hebeisen, Michael; Allard, Mathilde; Gannon, Philippe O.; Schmidt, Julien; Speiser, Daniel E.; Rufer, Nathalie

    2015-01-01

    Cytotoxic T cells recognize, via their T cell receptors (TCRs), small antigenic peptides presented by the major histocompatibility complex (pMHC) on the surface of professional antigen-presenting cells and infected or malignant cells. The efficiency of T cell triggering critically depends on TCR binding to cognate pMHC, i.e., the TCR–pMHC structural avidity. The binding and kinetic attributes of this interaction are key parameters for protective T cell-mediated immunity, with stronger TCR–pMHC interactions conferring superior T cell activation and responsiveness than weaker ones. However, high-avidity TCRs are not always available, particularly among self/tumor antigen-specific T cells, most of which are eliminated by central and peripheral deletion mechanisms. Consequently, systematic assessment of T cell avidity can greatly help distinguishing protective from non-protective T cells. Here, we review novel strategies to assess TCR–pMHC interaction kinetics, enabling the identification of the functionally most-relevant T cells. We also discuss the significance of these technologies in determining which cells within a naturally occurring polyclonal tumor-specific T cell response would offer the best clinical benefit for use in adoptive therapies, with or without T cell engineering. PMID:26635796

  5. Design and Development of Therapies using Chimeric Antigen Receptor-Expressing T cells

    PubMed Central

    Dotti, Gianpietro; Gottschalk, Stephen; Savoldo, Barbara; Brenner, Malcolm K

    2013-01-01

    Summary Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking and effector functions of a T-cell. This article describes how the past two decades have seen a crescendo of research which has now begun to translate these potential benefits into effective treatments for patients with cancer. We describe the basic design of CARs, describe how antigenic targets are selected, and the initial clinical experience with CART cells. Our review then describes our own and other investigators’ work aimed at improving the function of CARs and reviews the clinical studies in hematological and solid malignancies that are beginning to exploit these approaches. Finally, we show the value of adding additional engineering features to CAR-T cells, irrespective of their target, to render them better suited to function in the tumor environment, and discuss how the safety of these heavily modified cells may be maintained. PMID:24329793

  6. Nanoclusters of the resting T cell antigen receptor (TCR) localize to non-raft domains.

    PubMed

    Beck-García, Katharina; Beck-García, Esmeralda; Bohler, Sheila; Zorzin, Carina; Sezgin, Erdinc; Levental, Ilya; Alarcón, Balbino; Schamel, Wolfgang W A

    2015-04-01

    In the last decade an increasing number of plasma membrane (PM) proteins have been shown to be non-randomly distributed but instead forming submicron-sized oligomers called nanoclusters. Nanoclusters exist independently of the ligand-bound state of the receptors and their existence implies a high degree of lateral organisation of the PM and its proteins. The mechanisms that drive receptor nanoclustering are largely unknown. One well-defined example of a transmembrane receptor that forms nanoclusters is the T cell antigen receptor (TCR), a multisubunit protein complex whose nanoclustering influences its activity. Membrane lipids, namely cholesterol and sphingomyelin, have been shown to contribute to TCR nanoclustering. However, the identity of the membrane microdomain in which the TCR resides remains controversial. Using a GFP-labeled TCR we show here that the resting TCR localized in the disordered domain of giant PM vesicles (GPMVs) and PM spheres (PMSs) and that single and nanoclustered TCRs are found in the high-density fractions in sucrose gradients. Both findings are indicative of non-raft localization. We discuss possible mechanisms of TCR nanoclustering in T cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

  7. Engineering AAV receptor footprints for gene therapy.

    PubMed

    Madigan, Victoria J; Asokan, Aravind

    2016-06-01

    Adeno-associated viruses (AAV) are currently at the forefront of human gene therapy clinical trials as recombinant vectors. Significant progress has been made in elucidating the structure, biology and tropisms of different naturally occurring AAV isolates in the past decade. In particular, a spectrum of AAV capsid interactions with host receptors have been identified and characterized. These studies have enabled a better understanding of key determinants of AAV cell recognition and entry in different hosts. This knowledge is now being applied toward engineering new, lab-derived AAV capsids with favorable transduction profiles. The current review conveys a structural perspective of capsid-glycan interactions and provides a roadmap for generating synthetic strains by engineering AAV receptor footprints.

  8. A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

    PubMed Central

    Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

    2014-01-01

    Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

  9. Structure and gene cluster of the O-antigen of Escherichia coli O133.

    PubMed

    Shashkov, Alexander S; Zhang, Yuanyuan; Sun, Qiangzheng; Guo, Xi; Senchenkova, Sof'ya N; Perepelov, Andrei V; Knirel, Yuriy A

    2016-07-22

    The O-specific polysaccharide (O-antigen) of Escherichia coli O133 was obtained by mild acid hydrolysis of the lipopolysaccharide of E. coli O133. The structure of the hexasaccharide repeating unit of the polysaccharide was elucidated by (1)H and (13)C NMR spectroscopy, including a two-dimensional (1)H-(1)H ROESY experiment: Functions of genes in the O-antigen gene cluster were putatively identified by comparison with sequences in the available databases and, particularly, an encoded predicted multifunctional glycosyltransferase was assigned to three α-l-rhamnosidic linkages.

  10. Antibodies Raised Against Chlamydial Lipopolysaccharide Antigens Reveal Convergence in Germline Gene Usage and Differential Epitope Recognition

    PubMed Central

    Brooks, Cory L; Müller-Loennies, Sven; Borisova, Svetlana N.; Brade, Lore; Kosma, Paul; Hirama, Tomoko; MacKenzie, C. Roger; Brade, Helmut; Evans, Stephen V

    2011-01-01

    In order to explore monoclonal antibody recognition carbohydrate antigens, several structures from two monoclonal antibodies directed against carbohydrate epitopes derived from chlamydial LPS have been solved to high resolution. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen. S54-10 uses an unrelated CDR H3 to recognize its antigen in a fashion analogous to S45-18; however, S73-2 recognizes the same antigen as S45-18 and S54-10 in a wholly unrelated manner. Together, these antibody-antigen structures provide snapshots into how the immune system uses the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes, and how unrelated CDR H3 sequences can result in convergent binding of clinically-relevant bacterial antigens. PMID:20000757

  11. New advances in leukaemia immunotherapy by the use of Chimeric Artificial Antigen Receptors (CARs): state of the art and perspectives for the near future.

    PubMed

    Biagi, Ettore; Marin, Virna; Attianese, Greta Maria Paola Giordano; Pizzitola, Irene; Tettamanti, Sarah; Cribioli, Elisabetta; Biondi, Andrea

    2011-09-22

    Leukaemia immunotherapy represents a fascinating and promising field of translational research, particularly as an integrative approach of bone marrow transplantation. Adoptive immunotherapy by the use of donor-derived expanded leukaemia-specific T cells has showed some kind of clinical response, but the major advance is nowadays represented by gene manipulation of donor immune cells, so that they acquire strict specificity towards the tumour target and potent lytic activity, followed by significant proliferation, increased survival and possibly anti-tumour memory state. This is achieved by gene insertion of Chimeric T-cell Antigen Receptors (CARs), which are artificial molecules containing antibody-derived fragments (to bind the specific target), joined with potent signalling T-Cell Receptor (TCR)-derived domains that activate the manipulated cells. This review will discuss the main application of this approach particularly focusing on the paediatric setting, raising advantages and disadvantages and discussing relevant perspectives of use in the nearest future.

  12. Treatment of solid tumors with chimeric antigen receptor-engineered T cells: current status and future prospects.

    PubMed

    Di, Shengmeng; Li, Zonghai

    2016-04-01

    Chimeric antigen receptors (CARs) are artificial recombinant receptors that generally combine the antigen-recognition domain of a monoclonal antibody with T cell activation domains. Recent years have seen great success in clinical trials employing CD19-specific CAR-T cell therapy for B cell leukemia. Nevertheless, solid tumors remain a major challenge for CAR-T cell therapy. This review summarizes the preclinical and clinical studies on the treatment of solid tumors with CAR-T cells. The major hurdles for the success of CAR-T and the novel strategies to address these hurdles have also been described and discussed.

  13. Characterization of am404, an amber mutation in the simian virus 40 T antigen gene.

    PubMed Central

    Rawlins, D R; Collis, P; Muzyczka, N

    1983-01-01

    We analyzed the biological activity of an amber mutation, am404, at map position 0.27 in the T antigen gene of simian virus 40. Immunoprecipitation of extracts from am404-infected cells demonstrated the presence of an amber protein fragment (am T antigen) of the expected molecular weight (67,000). Differential immunoprecipitation with monoclonal antibody demonstrated that am T antigen was missing the carboxy-terminal antigenic determinants. The amber mutant was shown to be defective for most of the functions associated with wild-type T antigen. The mutant did not replicate autonomously, but this defect could be complemented by a helper virus (D. R. Rawlins and N. Muzyczka, J. Virol. 36:611-616, 1980). The mutant failed to transform nonpermissive rodent cells and did not relieve the host range restriction of adenovirus 2 in monkey cells. However, stimulation of host cell DNA, whose functional region domain has been mapped within that portion of the protein synthesized by the mutant, could be demonstrated in am404-infected cells. A number of unexpected observations were made. First, the am T antigen was produced in unusually large amounts in a simian virus 40-transformed monkey cell line (COS-1), but overproduction was not seen in nontransformed monkey cells regardless of whether or not a helper virus was present. This feature of the mutant was presumably the result of the inability of am T antigen to autoregulate, the level of wild-type T antigen in COS-1 cells, and the unusually short half-life of am T antigen in vivo. Pulse-chase experiments indicated that am T antigen had an intracellular half-life of approximately 10 min. In addition, although the am T antigen retained the major phosphorylation site found in simian virus 40 T antigen, it was not phosphorylated. Thus, phosphorylation of simian virus 40 T antigen is not required for the stimulation of host cell DNA synthesis. Finally, fusion of am404-infected monkey cells with Escherichia coli protoplasts

  14. The Androgen Receptor Gene Mutations Database.

    PubMed

    Gottlieb, B; Lehvaslaiho, H; Beitel, L K; Lumbroso, R; Pinsky, L; Trifiro, M

    1998-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 272 to 309 in the past year. We have expanded the database: (i) by giving each entry an accession number; (ii) by adding information on the length of polymorphic polyglutamine (polyGln) and polyglycine (polyGly) tracts in exon 1; (iii) by adding information on large gene deletions; (iv) by providing a direct link with a completely searchable database (courtesy EMBL-European Bioinformatics Institute). The addition of the exon 1 polymorphisms is discussed in light of their possible relevance as markers for predisposition to prostate or breast cancer. The database is also available on the internet (http://www.mcgill. ca/androgendb/ ), from EMBL-European Bioinformatics Institute (ftp. ebi.ac.uk/pub/databases/androgen ), or as a Macintosh FilemakerPro or Word file (MC33@musica.mcgill.ca).

  15. Aberrant Cosmc genes result in Tn antigen expression in human colorectal carcinoma cell line HT-29

    PubMed Central

    Yu, Xiaofeng; Du, Zhenzhen; Sun, Xuhong; Shi, Chuanqin; Zhang, Huaixiang; Hu, Tao

    2015-01-01

    The Tn antigen, which arises from mutation in the Cosmc gene is one of the most common tumor associated carbohydrate antigens. Cosmc resides in X24 encoded by a single gene and functions as a specific molecular chaperone for T-synthase. While the Tn antigen cannot be detected in normal cells, Cosmc mutations inactivate T-synthase and consequently result in Tn antigen expression within certain cancers. In addition to this Cosmc mutation-induced expression, the Tn antigen is also expressed in such cell lines as Jurkat T, LSC and LS174T. Whether the Cosmc mutation is present in the colon cancer cell line HT-29 is still unclear. Here, we isolate HT-29-Tn+ cells from HT-29 cells derived from a female colon cancer patient. These HT-29-Tn+ cells show a loss of the Cosmc gene coding sequence (CDS) leading to an absence of T-synthase activity and Tn antigen expression. Additionally, almost no methylation of Cosmc CpG islands was detected in HT-29-Tn+ as well as in HT-29-Tn- and Tn- tumor cells from male patients. In contrast, the methylation frequency of CpG island of Cosmc in normal female cells was ~50%. Only one active allele of Cosmc existed in HT-29-Tn+ and HT-29-Tn- cells as based upon detection of SNP sites. These results indicate that Tn antigens expression and T-synthase inactivity in HT-29-Tn+ cells can be related to the absence of CDS in Cosmc active alleles, while an inactive allele deletion of Cosmc in HT-29 cells has no influence on Cosmc function. PMID:26045765

  16. Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators

    PubMed Central

    Schnabel, Renate B.; Baumert, Jens; Barbalic, Maja; Dupuis, Josée; Ellinor, Patrick T.; Durda, Peter; Dehghan, Abbas; Bis, Joshua C.; Illig, Thomas; Morrison, Alanna C.; Jenny, Nancy S.; Keaney, John F.; Gieger, Christian; Tilley, Cathy; Yamamoto, Jennifer F.; Khuseyinova, Natalie; Heiss, Gerardo; Doyle, Margaret; Blankenberg, Stefan; Herder, Christian; Walston, Jeremy D.; Zhu, Yanyan; Vasan, Ramachandran S.; Klopp, Norman; Boerwinkle, Eric; Larson, Martin G.; Psaty, Bruce M.; Peters, Annette; Ballantyne, Christie M.; Witteman, Jacqueline C. M.

    2010-01-01

    To identify the genetic basis of circulating concentrations of monocyte chemoattractant protein-1 (MCP-1), we conducted genome-wide association analyses for MCP-1 in 3 independent cohorts (n = 9598). The strongest association was for serum MCP-1 with a nonsynonymous polymorphism, rs12075 (Asp42Gly) in DARC, the gene for Duffy antigen receptor for chemokines, a known vascular reservoir of proinflammatory cytokines (minor allele frequency, 45.6%; P < 1.0 * 10−323). This association was supported by family-based genetic linkage at a locus encompassing the DARC gene (genome-wide P = 8.0 * 10−13). Asp42Gly accounted for approximately 20% of the variability in serum MCP-1 concentrations and also was associated with serum concentrations of interleukin-8 and RANTES. While exploring a lack of association between this polymorphism and EDTA plasma MCP-1 concentrations (P = .82), we determined that both clotting and exogenous heparan sulfate (unfractionated heparin) released substantial amounts of MCP-1 from Darc. Quantitative immunoflow cytometry failed to identify meaningful Asp42Gly-associated differences in Darc expression, suggesting that a functional change is responsible for the differential cytokine binding. We conclude that Asp42Gly is a major regulator of erythrocyte Darc-mediated cytokine binding and thereby the circulating concentrations of several proinflammatory cytokines. We have also identified for the first time 2 mechanisms for the release of reservoir chemokines with possible clinical implications. PMID:20040767

  17. Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform

    PubMed Central

    Magnani, Chiara F.; Turazzi, Nice; Benedicenti, Fabrizio; Calabria, Andrea; Tenderini, Erika; Tettamanti, Sarah; Attianese, Greta M.P. Giordano; Cooper, Laurence J.N.; Aiuti, Alessandro; Montini, Eugenio; Biondi, Andrea; Biagi, Ettore

    2016-01-01

    Chimeric antigen receptor (CAR)-modified T-cell adoptive immunotherapy is a remarkable therapeutic option proven effective in the treatment of hematological malignancies. In order to optimize cell manufacturing, we sought to develop a novel clinical-grade protocol to obtain CAR-modified cytokine-induced killer cells (CIKs) using the Sleeping Beauty (SB) transposon system. Administration of irradiated PBMCs overcame cell death of stimulating cells induced by non-viral transfection, enabling robust gene transfer together with efficient T-cell expansion. Upon single stimulation, we reached an average of 60% expression of CD123- and CD19- specific 3rd generation CARs (CD28/OX40/TCRzeta). Furthermore, modified cells displayed persistence of cell subsets with memory phenotype, specific and effective lytic activity against leukemic cell lines and primary blasts, cytokine secretion, and proliferation. Adoptive transfer of CD123.CAR or CD19.CAR lymphocytes led to a significant anti-tumor response against acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) disseminated diseases in NSG mice. Notably, we found no evidence of integration enrichment near cancer genes and transposase expression at the end of the differentiation. Taken all together, our findings describe a novel donor-derived non-viral CAR approach that may widen the repertoire of available methods for T cell-based immunotherapy. PMID:27323395

  18. Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform.

    PubMed

    Magnani, Chiara F; Turazzi, Nice; Benedicenti, Fabrizio; Calabria, Andrea; Tenderini, Erika; Tettamanti, Sarah; Giordano Attianese, Greta M P; Cooper, Laurence J N; Aiuti, Alessandro; Montini, Eugenio; Biondi, Andrea; Biagi, Ettore

    2016-08-09

    Chimeric antigen receptor (CAR)-modified T-cell adoptive immunotherapy is a remarkable therapeutic option proven effective in the treatment of hematological malignancies. In order to optimize cell manufacturing, we sought to develop a novel clinical-grade protocol to obtain CAR-modified cytokine-induced killer cells (CIKs) using the Sleeping Beauty (SB) transposon system. Administration of irradiated PBMCs overcame cell death of stimulating cells induced by non-viral transfection, enabling robust gene transfer together with efficient T-cell expansion. Upon single stimulation, we reached an average of 60% expression of CD123- and CD19- specific 3rd generation CARs (CD28/OX40/TCRzeta). Furthermore, modified cells displayed persistence of cell subsets with memory phenotype, specific and effective lytic activity against leukemic cell lines and primary blasts, cytokine secretion, and proliferation. Adoptive transfer of CD123.CAR or CD19.CAR lymphocytes led to a significant anti-tumor response against acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) disseminated diseases in NSG mice. Notably, we found no evidence of integration enrichment near cancer genes and transposase expression at the end of the differentiation. Taken all together, our findings describe a novel donor-derived non-viral CAR approach that may widen the repertoire of available methods for T cell-based immunotherapy.

  19. Trypanosome Surface Antigen Genes: Analysis Using Recombinant DNA.

    DTIC Science & Technology

    1984-06-15

    glycoprotein ( VSG ) genes. umerous syringe passaged and cyclically transmitted, frequently expressed VATs have been isolated, monoclonal antibodies prepared...to their VSGs , and he expressed VSG genes have been cloned. We have shown that many diverse tocks express VSG epitopes related to the early IsTst...epitopes. The VSG ene organization in the genome and sequence organization has been haracterized. We have confirmed sequence homology at the 3’ terminus

  20. Identification of a family of muscarinic acetylcholine receptor genes

    SciTech Connect

    Bonner, T.I.; Buckley, N.J.; Young, A.C.; Brann, M.R.

    1987-07-31

    Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.

  1. Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.

    PubMed

    Bendle, Gavin M; Linnemann, Carsten; Hooijkaas, Anna I; Bies, Laura; de Witte, Moniek A; Jorritsma, Annelies; Kaiser, Andrew D M; Pouw, Nadine; Debets, Reno; Kieback, Elisa; Uckert, Wolfgang; Song, Ji-Ying; Haanen, John B A G; Schumacher, Ton N M

    2010-05-01

    The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy-induced autoimmune pathology.

  2. Development of a T cell receptor targeting an HLA-A*0201 restricted epitope from the cancer-testis antigen SSX2 for adoptive immunotherapy of cancer.

    PubMed

    Abate-Daga, Daniel; Speiser, Daniel E; Chinnasamy, Nachimuthu; Zheng, Zhili; Xu, Hui; Feldman, Steven A; Rosenberg, Steven A; Morgan, Richard A

    2014-01-01

    The clinical success of adoptive immunotherapy of cancer relies on the selection of target antigens that are highly expressed in tumor cells but absent in essential normal tissues. A group of genes that encode the cancer/testis or cancer germline antigens have been proposed as ideal targets for immunotherapy due to their high expression in multiple cancer types and their restricted expression in immunoprivileged normal tissues. In the present work we report the isolation and characterization of human T cell receptors (TCRs) with specificity for synovial sarcoma X breakpoint 2 (SSX2), a cancer/testis antigen expressed in melanoma, prostate cancer, lymphoma, multiple myeloma and pancreatic cancer, among other tumors. We isolated seven HLA-A2 restricted T cell receptors from natural T cell clones derived from tumor-infiltrated lymph nodes of two SSX2-seropositive melanoma patients, and selected four TCRs for cloning into retroviral vectors. Peripheral blood lymphocytes (PBL) transduced with three of four SSX2 TCRs showed SSX241-49 (KASEKIFYV) peptide specific reactivity, tumor cell recognition and tetramer binding. One of these, TCR-5, exhibited tetramer binding in both CD4 and CD8 cells and was selected for further studies. Antigen-specific and HLA-A*0201-restricted interferon-γ release, cell lysis and lymphocyte proliferation was observed following culture of TCR engineered human PBL with relevant tumor cell lines. Codon optimization was found to increase TCR-5 expression in transduced T cells, and this construct has been selected for development of clinical grade viral vector producing cells. The tumor-specific pattern of expression of SSX2, along with the potent and selective activity of TCR-5, makes this TCR an attractive candidate for potential TCR gene therapy to treat multiple cancer histologies.

  3. MOLECULAR CHARACTERIZATION OF SWINE LEUKOCYTE ANTIGEN (SLA) CLASS I GENES IN OUTBRED PIG POPULATIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly polymorphic swine leukocyte antigen (SLA) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to t...

  4. No Evidence of Human Leukocyte Antigen Gene Association With Rheumatic Fever Among Children in Samoa.

    PubMed

    Erdem, Guliz; Seifried, Steven E

    2015-03-01

    Human leukocyte antigens (HLAs) have been implicated in rheumatic fever pathogenesis. This pilot whole genome association study compares genotypes of Samoan children with rheumatic fever to unaffected siblings and unrelated healthy controls. No risk-related genotypes were associated with HLA genes. Thirteen Regions of Interest were identified as candidates for further study.

  5. M-Type Phospholipase A2 Receptor as Target Antigen in Idiopathic Membranous Nephropathy

    PubMed Central

    Beck, Laurence H.; Bonegio, Ramon G.B.; Lambeau, Gérard; Beck, David M.; Powell, David W.; Cummins, Timothy D.; Klein, Jon B.; Salant, David J.

    2009-01-01

    BACKGROUND Idiopathic membranous nephropathy, a common form of the nephrotic syndrome, is an antibody-mediated autoimmune glomerular disease. Serologic diagnosis has been elusive because the target antigen is unknown. METHODS We performed Western blotting of protein extracts from normal human glomeruli with serum samples from patients with idiopathic or secondary membranous nephropathy or other proteinuric or autoimmune diseases and from normal controls. We used mass spectrometry to analyze the reactive protein bands and confirmed the identity and location of the target antigen with a monospecific antibody. RESULTS Serum samples from 26 of 37 patients (70%) with idiopathic but not secondary membranous nephropathy specifically identified a 185-kD glycoprotein in non-reduced glomerular extract. Mass spectrometry of the reactive protein band detected the M-type phospholipase A2 receptor (PLA2R). Reactive serum specimens recognized recombinant PLA2R and bound the same 185-kD glomerular protein as did the monospecific anti-PLA2R antibody. Anti-PLA2R autoantibodies in serum samples from patients with membranous nephropathy were mainly IgG4, the predominant immunoglobulin subclass in glomerular deposits. PLA2R was expressed in podocytes in normal human glomeruli and colocalized with IgG4 in immune deposits in glomeruli of patients with membranous nephropathy. IgG eluted from such deposits in patients with idiopathic membranous nephropathy, but not in those with lupus membranous or IgA nephropathy, recognized PLA2R. CONCLUSIONS A majority of patients with idiopathic membranous nephropathy have antibodies against a conformation-dependent epitope in PLA2R. PLA2R is present in normal podocytes and in immune deposits in patients with idiopathic membranous nephropathy, indicating that PLA2R is a major antigen in this disease. PMID:19571279

  6. Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

    PubMed Central

    Kovalenko, Oleg V.; Olland, Andrea; Piché-Nicholas, Nicole; Godbole, Adarsh; King, Daniel; Svenson, Kristine; Calabro, Valerie; Müller, Mischa R.; Barelle, Caroline J.; Somers, William; Gill, Davinder S.; Mosyak, Lidia; Tchistiakova, Lioudmila

    2013-01-01

    The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs. PMID:23632026

  7. Atypical antigen recognition mode of a shark immunoglobulin new antigen receptor (IgNAR) variable domain characterized by humanization and structural analysis.

    PubMed

    Kovalenko, Oleg V; Olland, Andrea; Piché-Nicholas, Nicole; Godbole, Adarsh; King, Daniel; Svenson, Kristine; Calabro, Valerie; Müller, Mischa R; Barelle, Caroline J; Somers, William; Gill, Davinder S; Mosyak, Lidia; Tchistiakova, Lioudmila

    2013-06-14

    The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.

  8. Chimeric Antigen Receptor T Cells against CD19 for Multiple Myeloma

    PubMed Central

    Garfall, Alfred L.; Maus, Marcela V.; Hwang, Wei-Ting; Lacey, Simon F.; Mahnke, Yolanda D.; Melenhorst, J. Joseph; Zheng, Zhaohui; Vogl, Dan T.; Cohen, Adam D.; Weiss, Brendan M.; Dengel, Karen; Kerr, Naseem D.S.; Bagg, Adam; Levine, Bruce L.; June, Carl H.; Stadtmauer, Edward A.

    2015-01-01

    SUMMARY A patient with refractory multiple myeloma received an infusion of CTL019 cells, a cellular therapy consisting of autologous T cells transduced with an anti-CD19 chimeric antigen receptor, after myeloablative chemotherapy (melphalan, 140 mg per square meter of body-surface area) and autologous stem-cell transplantation. Four years earlier, autologous transplantation with a higher melphalan dose (200 mg per square meter) had induced only a partial, transient response. Autologous transplantation followed by treatment with CTL019 cells led to a complete response with no evidence of progression and no measurable serum or urine monoclonal protein at the most recent evaluation, 12 months after treatment. This response was achieved despite the absence of CD19 expression in 99.95% of the patient’s neoplastic plasma cells. (Funded by Novartis and others; ClinicalTrials.gov number, NCT02135406.) PMID:26352815

  9. Improving Therapy of Chronic Lymphocytic Leukemia (CLL) with Chimeric Antigen Receptor (CAR) T Cells

    PubMed Central

    Fraietta, Joseph A.; Schwab, Robert D.; Maus, Marcela V.

    2016-01-01

    Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically-engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and re-directing the immune system against cancer. This review will briefly summarize T cell therapies in development for CLL disease. We discuss the role of T cell function and phenotype, T cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. PMID:27040708

  10. Preclinical targeting of aggressive T-cell malignancies using anti-CD5 chimeric antigen receptor.

    PubMed

    Chen, K H; Wada, M; Pinz, K G; Liu, H; Lin, K-W; Jares, A; Firor, A E; Shuai, X; Salman, H; Golightly, M; Lan, F; Senzel, L; Leung, E L; Jiang, X; Ma, Y

    2017-02-10

    The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.Leukemia advance online publication, 10 February 2017; doi:10.1038/leu.2017.8.

  11. ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells

    PubMed Central

    Chen, Xi; Madar, Aviv; Carpenito, Carmine; McGettigan, Shannon E.; Frigault, Matthew J.; Lee, Jihyun; Posey, Avery D.; Scholler, John; Scholler, Nathalie; Bonneau, Richard

    2014-01-01

    With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies. PMID:24986688

  12. Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

    PubMed Central

    Papadouli, I; Potamianos, S; Hadjidakis, I; Bairaktari, E; Tsikaris, V; Sakarellos, C; Cung, M T; Marraud, M; Tzartos, S J

    1990-01-01

    The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible. PMID:1695844

  13. ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells.

    PubMed

    Guedan, Sonia; Chen, Xi; Madar, Aviv; Carpenito, Carmine; McGettigan, Shannon E; Frigault, Matthew J; Lee, Jihyun; Posey, Avery D; Scholler, John; Scholler, Nathalie; Bonneau, Richard; June, Carl H

    2014-08-14

    With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies.

  14. A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

    PubMed Central

    Cartellieri, Marc; Koristka, Stefanie; Arndt, Claudia; Feldmann, Anja; Stamova, Slava; von Bonin, Malte; Töpfer, Katrin; Krüger, Thomas; Geib, Mathias; Michalk, Irene; Temme, Achim; Bornhäuser, Martin; Lindemann, Dirk; Ehninger, Gerhard; Bachmann, Michael P.

    2014-01-01

    Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes. PMID:24699869

  15. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.

    PubMed

    Bonifaz, Laura C; Bonnyay, David P; Charalambous, Anna; Darguste, Dara I; Fujii, Shin-Ichiro; Soares, Helena; Brimnes, Marie K; Moltedo, Bruno; Moran, Thomas M; Steinman, Ralph M

    2004-03-15

    The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic alpha-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the alphaDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell-mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

  16. In Vivo Targeting of Antigens to Maturing Dendritic Cells via the DEC-205 Receptor Improves T Cell Vaccination

    PubMed Central

    Bonifaz, Laura C.; Bonnyay, David P.; Charalambous, Anna; Darguste, Dara I.; Fujii, Shin-Ichiro; Soares, Helena; Brimnes, Marie K.; Moltedo, Bruno; Moran, Thomas M.; Steinman, Ralph M.

    2004-01-01

    The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic α-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the αDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell–mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models. PMID:15024047

  17. Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

    PubMed Central

    Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

    2015-01-01

    Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

  18. Regulatory Features for Odorant Receptor Genes in the Mouse Genome

    PubMed Central

    Degl’Innocenti, Andrea; D’Errico, Anna

    2017-01-01

    The odorant receptor genes, seven transmembrane receptor genes constituting the vastest mammalian gene multifamily, are expressed monogenically and monoallelicaly in each sensory neuron in the olfactory epithelium. This characteristic, often referred to as the one neuron–one receptor rule, is driven by mostly uncharacterized molecular dynamics, generally named odorant receptor gene choice. Much attention has been paid by the scientific community to the identification of sequences regulating the expression of odorant receptor genes within their loci, where related genes are usually arranged in genomic clusters. A number of studies identified transcription factor binding sites on odorant receptor promoter sequences. Similar binding sites were also found on a number of enhancers that regulate in cis their transcription, but have been proposed to form interchromosomal networks. Odorant receptor gene choice seems to occur via the local removal of strongly repressive epigenetic markings, put in place during the maturation of the sensory neuron on each odorant receptor locus. Here we review the fast-changing state of art for the study of regulatory features for odorant receptor genes. PMID:28270833

  19. Regulatory Features for Odorant Receptor Genes in the Mouse Genome.

    PubMed

    Degl'Innocenti, Andrea; D'Errico, Anna

    2017-01-01

    The odorant receptor genes, seven transmembrane receptor genes constituting the vastest mammalian gene multifamily, are expressed monogenically and monoallelicaly in each sensory neuron in the olfactory epithelium. This characteristic, often referred to as the one neuron-one receptor rule, is driven by mostly uncharacterized molecular dynamics, generally named odorant receptor gene choice. Much attention has been paid by the scientific community to the identification of sequences regulating the expression of odorant receptor genes within their loci, where related genes are usually arranged in genomic clusters. A number of studies identified transcription factor binding sites on odorant receptor promoter sequences. Similar binding sites were also found on a number of enhancers that regulate in cis their transcription, but have been proposed to form interchromosomal networks. Odorant receptor gene choice seems to occur via the local removal of strongly repressive epigenetic markings, put in place during the maturation of the sensory neuron on each odorant receptor locus. Here we review the fast-changing state of art for the study of regulatory features for odorant receptor genes.

  20. Genetic variability in swine leukocyte antigen class II and Toll-like receptors affects immune responses to vaccination for bacterial infections in pigs.

    PubMed

    Shinkai, H; Arakawa, A; Tanaka-Matsuda, M; Ide-Okumura, H; Terada, K; Chikyu, M; Kawarasaki, T; Ando, A; Uenishi, H

    2012-12-01

    The genes encoding swine leukocyte antigen (SLA) and Toll-like receptor (TLR) are highly polymorphic in pig populations, and likely have influences on infection and the effects of vaccination. We explored the associations of different genotypes of SLA class II and of the genes TLR1, TLR4, TLR5, and TLR6 with antibody responses after vaccination against Erysipelothrix rhusiopathiae (ER) and Actinobacillus pleuropneumoniae (APP) serotypes 1, 2, and 5 in 191 Duroc pigs maintained under specific pathogen-free conditions. We demonstrated close relationships between SLA class II and ER antibody response and between TLR genes other than TLR4 and APP antibody responses. Pigs with specific haplotypes in SLA class II or TLR5 showed decreased antibody response to ER vaccination or increased responses to APP2 and APP5 vaccination, respectively. It might be possible to breed for responsiveness to vaccination and to implement new vaccine development strategies unaffected by genetic backgrounds of pigs.

  1. Increased sensitivity of antigen-experienced T cells through the enrichment of oligomeric T cell receptor complexes.

    PubMed

    Kumar, Rashmi; Ferez, María; Swamy, Mahima; Arechaga, Ignacio; Rejas, María Teresa; Valpuesta, Jose M; Schamel, Wolfgang W A; Alarcon, Balbino; van Santen, Hisse M

    2011-09-23

    Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an "avidity maturation" mechanism underlies T cell antigenic memory.

  2. Location of Receptor-Binding Region of Protective Antigen from Bacillus anthracis

    DTIC Science & Technology

    1991-08-07

    internalization by receptor- mediated endocytosis (2). Molecular cloning of the PA and EF genes into the F. -Qjj vector pBR322 plasmid was accomplished...Leppla, S. H. and Schmidt, J. J. (1988) Gene 69, 287-300. 12. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning , A Laboratory Manual...from SDS-PAGE gels except for pBKPPAA65, which was calculated from plasmid DNA sequence data. 7[ . 7 Figure 1. Molecular Cloning of ]. anthracis pq

  3. Neutralization of reovirus: the gene responsible for the neutralization antigen

    PubMed Central

    1977-01-01

    The S1 genome segment of reovirus is linked to type specificity as determined by neutralization antibody. This gene segment codes for a minor outer capsid polypeptide (sigma1). Therefore, sigma1 is the peptide responsible for induction of neutralization antibody and confers type specificity. This biologic property of reovirus was defined using hybrid recombinants clones between reovirus types 1 and 3 and 2 and 3. PMID:925604

  4. Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

    PubMed Central

    McConnell, Sean C.; Hernandez, Kyle M.; Wcisel, Dustin J.; Kettleborough, Ross N.; Stemple, Derek L.; Andrade, Jorge; de Jong, Jill L. O.

    2016-01-01

    Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e. We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates. PMID:27493218

  5. Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse

    PubMed Central

    Reversat, Anne; Yuseff, Maria-Isabel; Lankar, Danielle; Malbec, Odile; Obino, Dorian; Maurin, Mathieu; Penmatcha, Naga Venkata Gayathri; Amoroso, Alejandro; Sengmanivong, Lucie; Gundersen, Gregg G.; Mellman, Ira; Darchen, François; Desnos, Claire; Pierobon, Paolo; Lennon-Duménil, Ana-Maria

    2015-01-01

    B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR–antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR–antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells. PMID:25631815

  6. Increased expression of TLR-2, COX-2, and SOD-2 genes in the peripheral blood leukocytes of opisthorchiasis patients induced by Opisthorchis viverrini antigen.

    PubMed

    Yongvanit, Puangrat; Thanan, Raynoo; Pinlaor, Somchai; Sithithaworn, Paiboon; Loilome, Watcharin; Namwat, Nisana; Techasen, Anchalee; Dechakhamphu, Somkid

    2012-05-01

    Re-infection with liver fluke, Opisthorchis viverrini, increases proinflammatory molecules involved in inflammation-mediated disease and carcinogenesis in an animal model. To clarify whether these genes respond to parasite antigen in peripheral blood leukocytes (PBL) of opisthorchiasis patients, we examined the transcriptional level of oxidant-generating (toll-like receptor 2 (TLR-2), nuclear factor-kappa B (NF-KB), and cyclooxygenase 2 (COX-2)), anti-oxidant-generating (manganese superoxide dismutase 2 (SOD-2) and catalase (CAT)), proinflammatory cytokine (interleukin (IL)-1β), and anti-inflammatory cytokine (IL-10), in PBL exposed to parasite antigen in O. viverrini-infected patients compared with healthy individuals in an in vitro experiment. After O. viverrini antigen-treated PBL, quantitative RT-PCR analysis revealed that increased expression of cytokines and oxidant-generating genes in PBL was similar between O. viverrini-infected and healthy groups. Interestingly, compared with healthy subjects, increase of TLR-2, COX-2, and SOD-2 and decreased CAT mRNA expression levels were observed in O. viverrini-infected group. The results indicate that O. viverrini antigen induces upregulation of TLR-2, COX-2, and SOD-2 and downregulation of CAT genes in opisthorchiasis patients, suggesting that imbalance of oxidant/anti-oxidant transcripts during re-infection may be involved in the inflammatory-driven carcinogenesis. These molecules may be used as the chemopreventive target for intervention of opisthorchiasis patients in an endemic area.

  7. The androgen receptor gene mutations database.

    PubMed

    Gottlieb, B; Trifiro, M; Lumbroso, R; Vasiliou, D M; Pinsky, L

    1996-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. We have added (if available) data on the androgen binding phenotype of the mutant AR, the clinical phenotype of the affected persons, the family history and whether the pathogenicity of a mutation has been proven. Exonic mutations are now listed in 5'-->3' sequence regardless of type and single base pair changes are presented in codon context. Splice site and intronic mutations are listed separately. The database has allowed us to substantiate and amplify the observation of mutational hot spots within exons encoding the AR androgen binding domain. The database is available from EML (ftp://www.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker file (MC33@musica.mcgill.ca).

  8. The androgen receptor gene mutations database.

    PubMed

    Gottlieb, B; Trifiro, M; Lumbroso, R; Pinsky, L

    1997-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 212 to 272. We have expanded the database: (i) by adding a large amount of new data on somatic mutations in prostatic cancer tissue; (ii) by defining a new constitutional phenotype, mild androgen insensitivity (MAI); (iii) by placing additional relevant information on an internet site (http://www.mcgill.ca/androgendb/ ). The database has allowed us to examine the contribution of CpG sites to the multiplicity of reports of the same mutation in different families. The database is also available from EMBL (ftp.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker Pro or Word file (MC33@musica,mcgill.ca)

  9. Actin restricts FcεRI diffusion and facilitates antigen-induced receptor immobilisation

    PubMed Central

    Andrews, Nicholas L.; Lidke, Keith A.; Pfeiffer, Janet R.; Burns, Alan R.; Wilson, Bridget S.; Oliver, Janet M.; Lidke, Diane S.

    2010-01-01

    The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcεRI motion and GFP-tagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganisation of actin structures occurs over seconds, making the location and dimensions of actin-defined domains time dependent. Multiple FcεRI often maintain extended close proximity without detectable correlated motion, suggesting that they are co-confined within membrane domains. FcεRI signalling is activated by cross-linking with multivalent antigen. We show that receptors become immobilised within seconds of cross-linking. Disruption of the actin cytoskeleton results in delayed immobilisation kinetics and increased diffusion of cross-linked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their long-range mobility, sequestration, and response to ligand binding. PMID:18641640

  10. Constitutively Active Lck Kinase in T Cells Drives Antigen Receptor Signal Transduction

    PubMed Central

    Nika, Konstantina; Soldani, Cristiana; Salek, Mogjiborahman; Paster, Wolfgang; Gray, Adrian; Etzensperger, Ruth; Fugger, Lars; Polzella, Paolo; Cerundolo, Vincenzo; Dushek, Omer; Höfer, Thomas; Viola, Antonella; Acuto, Oreste

    2010-01-01

    Summary T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to ∼40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-ζ phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors. PMID:20541955

  11. Chimeric antigen receptor T cell therapy in AML: How close are we?

    PubMed

    Gill, Saar

    2016-12-01

    The majority of patients presenting with acute myeloid leukemia (AML) initially respond to chemotherapy but post-remission therapy is required to consolidate this response and achieve long-term disease-free survival. The most effective form of post-remission therapy relies on T cell immunotherapy in the form of allogeneic hematopoietic cell transplantation (HCT). However, patients with active disease cannot usually expect to be cured with HCT. This inherent dichotomy implies that traditional T cell-based immunotherapy in the form of allogeneic HCT stops being efficacious somewhere between the measurable residual disease (MRD) and the morphologically obvious range. This is in part because the full power of T cells must be restrained in order to avoid lethal graft-versus-host disease (GVHD) and partly because only a sub-population of donor T cells are expected to be able to recognize AML cells via their T cell receptor. Chimeric antigen receptor (CAR) T cell therapy, most advanced in the treatment of patients with B-cell malignancies, may circumvent some of these limitations. However, major challenges remain to be overcome before CAR T cell therapy can be safely applied to AML.

  12. Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

    PubMed

    Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent; Sant'angelo, Derek; Sadelain, Michel

    2009-01-01

    MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.

  13. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses.

    PubMed

    Fedorov, Victor D; Themeli, Maria; Sadelain, Michel

    2013-12-11

    T cell therapies have demonstrated long-term efficacy and curative potential for the treatment of some cancers. However, their use is limited by damage to bystander tissues, as seen in graft-versus-host disease after donor lymphocyte infusion, or "on-target, off-tumor" toxicities incurred in some engineered T cell therapies. Nonspecific immunosuppression and irreversible T cell elimination are currently the only means to control such deleterious responses, but at the cost of abrogating therapeutic benefits or causing secondary complications. On the basis of the physiological paradigm of immune inhibitory receptors, we designed antigen-specific inhibitory chimeric antigen receptors (iCARs) to preemptively constrain T cell responses. We demonstrate that CTLA-4- or PD-1-based iCARs can selectively limit cytokine secretion, cytotoxicity, and proliferation induced through the endogenous T cell receptor or an activating chimeric receptor. The initial effect of the iCAR is temporary, thus enabling T cells to function upon a subsequent encounter with the antigen recognized by their activating receptor. iCARs thus provide a dynamic, self-regulating safety switch to prevent, rather than treat, the consequences of inadequate T cell specificity.

  14. Utility of polymerase chain reaction for analysis of antigen receptor rearrangement in staging and predicting prognosis in dogs with lymphoma.

    PubMed

    Lana, Susan E; Jackson, Tracey L; Burnett, Robert C; Morley, Paul S; Avery, Anne C

    2006-01-01

    In lymphoid neoplasia, molecular assays to confirm clonality rely on the fact that lymphoid cells normally contain DNA regions with unique sequences, resulting from recombination of the V, D, and J genes. The purpose of this study was to determine the utility of polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) for molecular staging and predicting prognosis in canine lymphoma. We hypothesized that the PARR assay would offer a sensitive method for detecting neoplastic cells in blood, and that the presence of such cells would be a negative prognostic finding compared with dogs with no detectable circulating tumor cells. Eighty-six patients with histologically or cytologically confirmed lymphoma were studied from initial diagnosis until death or euthanasia. All patients had PARR assays of a representative tumor-infiltrated lymph node and peripheral whole blood. Sixty-two patients had clonal rearrangements detected in the lymph node and were able to be staged by PARR. Seventeen patients (27%) had no detectable tumor in their blood and 45 (73%) were blood positive. Our findings showed that (1) PARR correlated with clinical stage in that the PARR assay was more likely to detect tumor cells in blood in stage 5 lymphomas, (2) PARR was more sensitive for detecting circulating tumor cells than visual assessment of blood or bone marrow because 80% of stage 3 lymphomas were blood-PARR-positive, and (3) PCR stage was not prognostic for disease-free interval (DFI) or survival.

  15. Human cord blood T-cell receptor alpha beta cell responses to protein antigens of Paracoccidioides brasiliensis yeast forms.

    PubMed Central

    Munk, M E; Kaufmann, S H

    1995-01-01

    Paracoccidioides brasiliensis causes a chronic granulomatous mycosis, prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated the response of naive cord blood T cells to P. brasiliensis lysates. Our results show: (1) P. brasiliensis stimulates T-cell expansion, interleukin-2 (IL-2) production and differentiation into cytotoxic T cells; (2) T-cell stimulation depends on P. brasiliensis processing and major histocompatibility complex (MHC) class II expression; (3) the responsive T-cell population expresses alpha beta T-cell receptors (TCR) with different V beta gene products, CD4 and CD45RO; (4) the P. brasiliensis components involved in T-cell expansion primarily reside in a high molecular weight (100,000 MW) and a low molecular weight (< 1000 MW) protein fraction. These results indicate that protein antigens of P. brasiliensis stimulate cord blood CD4 alpha beta T cells, independent from in vivo presensitization, and thus question direct correlation of positive in vitro responses with protective immunity in vivo. PMID:7890308

  16. Induction of Human Blood Group A Antigen Expression on Mouse Cells, Using Lentiviral Gene Transduction

    PubMed Central

    Fan, Xiaohu; Lang, Haili; Zhou, Xianpei; Zhang, Li; Yin, Rong; Maciejko, Jessica; Giannitsos, Vasiliki; Motyka, Bruce; Medin, Jeffrey A.; Platt, Jeffrey L.

    2010-01-01

    Abstract The ABO histo-blood group system is the most important antigen system in transplantation medicine, yet no small animal model of the ABO system exists. To determine the feasibility of developing a murine model, we previously subcloned the human α-1,2-fucosyltransferase (H-transferase, EC 2.4.1.69) cDNA and the human α-1,3-N-acetylgalactosaminyltransferase (A-transferase, EC 2.4.1.40) cDNA into lentiviral vectors to study their ability to induce human histo-blood group A antigen expression on mouse cells. Herein we investigated the optimal conditions for human A and H antigen expression in murine cells. We determined that transduction of a bicistronic lentiviral vector (LvEF1-AH-trs) resulted in the expression of A antigen in a mouse endothelial cell line. We also studied the in vivo utility of this vector to induce human A antigen expression in mouse liver. After intrahepatic injection of LvEF1-AH-trs, A antigen expression was observed on hepatocytes as detected by immunohistochemistry and real-time RT-PCR. In human group A erythrocyte-sensitized mice, A antigen expression in the liver was associated with tissue damage, and deposition of antibody and complement. These results suggest that this gene transfer strategy can be used to simulate the human ABO blood group system in a murine model. This model will facilitate progress in the development of interventions for ABO-incompatible transplantation and transfusion scenarios, which are difficult to develop in clinical or large animal settings. PMID:20163247

  17. A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor.

    PubMed

    Urbanska, Katarzyna; Lanitis, Evripidis; Poussin, Mathilde; Lynn, Rachel C; Gavin, Brian P; Kelderman, Sander; Yu, Jason; Scholler, Nathalie; Powell, Daniel J

    2012-04-01

    Adoptive immunotherapies composed of T cells engineered to express a chimeric antigen receptor (CAR) offer an attractive strategy for treatment of human cancer. However, CARs have a fixed antigen specificity such that only one tumor-associated antigen (TAA) can be targeted, limiting the efficacy that can be achieved because of heterogeneous TAA expression. For this reason, a more generalized and effective application of CAR therapy would benefit from the capability to produce large panels of CARs against many known TAAs. In this study, we show a novel strategy to extend the recognition specificity potential of a bioengineered lymphocyte population, allowing flexible approaches to redirect T cells against various TAAs. Our strategy employs a biotin-binding immune receptor (BBIR) composed of an extracellular-modified avidin linked to an intracellular T-cell signaling domain. BBIR T cells recognized and bound exclusively to cancer cells pretargeted with specific biotinylated molecules. The versatility afforded by BBIRs permitted sequential or simultaneous targeting of a combination of distinct antigens. Together, our findings show that a platform of universal T-cell specificity can significantly extend conventional CAR approaches, permitting the tailored generation of T cells of unlimited antigen specificity for improving the effectiveness of adoptive T-cell immunotherapies for cancer.

  18. Structure and expression of a mouse major histocompatibility antigen gene, H-2Ld.

    PubMed Central

    Evans, G A; Margulies, D H; Camerini-Otero, R D; Ozato, K; Seidman, J G

    1982-01-01

    A genomic clone encoding H-2Ld, a mouse major transplantation antigen, has been identified and the structure of the H-2Ld gene has been partially determined. We isolated 35 genomic clones from a BALB/c (H-2d) genomic library by hybridization to mouse or human probes. One of these clones encodes H-2Ld as determined by two criteria. First, the gene encodes a protein that is identical at the 76 known amino acid positions for H-2Ld. Second, when introduced into L cells by DNA-mediated gene transfer, a new H-2 antigen is expressed that is recognized by anti-H-2Ld monoclonal antibodies. The sequence of the H-2Ld protein predicted by the DNA sequences shows more than 80% homology to known H-2 antigens. H-2L-like sequences are found in mutant H-2Kb molecules, suggesting that gene conversion or reciprocal recombination may play a role in the development of H-2 polymorphism. PMID:6952248

  19. Mucosal Immunization with Iron Receptor Antigens Protects against Urinary Tract Infection

    PubMed Central

    Smith, Sara N.; Mobley, Harry L. T.

    2009-01-01

    Uncomplicated infections of the urinary tract, caused by uropathogenic Escherichia coli, are among the most common diseases requiring medical intervention. A preventive vaccine to reduce the morbidity and fiscal burden these infections have upon the healthcare system would be beneficial. Here, we describe the results of a large-scale selection process that incorporates bioinformatic, genomic, transcriptomic, and proteomic screens to identify six vaccine candidates from the 5379 predicted proteins encoded by uropathogenic E. coli strain CFT073. The vaccine candidates, ChuA, Hma, Iha, IreA, IroN, and IutA, all belong to a functional class of molecules that is involved in iron acquisition, a process critical for pathogenesis in all microbes. Intranasal immunization of CBA/J mice with these outer membrane iron receptors elicited a systemic and mucosal immune response that included the production of antigen-specific IgM, IgG, and IgA antibodies. The cellular response to vaccination was characterized by the induction and secretion of IFN-γ and IL-17. Of the six potential vaccine candidates, IreA, Hma, and IutA provided significant protection from experimental infection. In immunized animals, class-switching from IgM to IgG and production of antigen-specific IgA in the urine represent immunological correlates of protection from E. coli bladder colonization. These findings are an important first step toward the development of a subunit vaccine to prevent urinary tract infections and demonstrate how targeting an entire class of molecules that are collectively required for pathogenesis may represent a fundamental strategy to combat infections. PMID:19806177

  20. Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

    PubMed

    Mock, Ulrike; Nickolay, Lauren; Philip, Brian; Cheung, Gordon Weng-Kit; Zhan, Hong; Johnston, Ian C D; Kaiser, Andrew D; Peggs, Karl; Pule, Martin; Thrasher, Adrian J; Qasim, Waseem

    2016-08-01

    Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

  1. Contrasting Population Structures of the Genes Encoding Ten Leading Vaccine-Candidate Antigens of the Human Malaria Parasite, Plasmodium falciparum

    PubMed Central

    Barry, Alyssa E.; Schultz, Lee; Buckee, Caroline O.; Reeder, John C.

    2009-01-01

    The extensive diversity of Plasmodium falciparum antigens is a major obstacle to a broadly effective malaria vaccine but population genetics has rarely been used to guide vaccine design. We have completed a meta-population genetic analysis of the genes encoding ten leading P. falciparum vaccine antigens, including the pre-erythrocytic antigens csp, trap, lsa1 and glurp; the merozoite antigens eba175, ama1, msp's 1, 3 and 4, and the gametocyte antigen pfs48/45. A total of 4553 antigen sequences were assembled from published data and we estimated the range and distribution of diversity worldwide using traditional population genetics, Bayesian clustering and network analysis. Although a large number of distinct haplotypes were identified for each antigen, they were organized into a limited number of discrete subgroups. While the non-merozoite antigens showed geographically variable levels of diversity and geographic restriction of specific subgroups, the merozoite antigens had high levels of diversity globally, and a worldwide distribution of each subgroup. This shows that the diversity of the non-merozoite antigens is organized by physical or other location-specific barriers to gene flow and that of merozoite antigens by features intrinsic to all populations, one important possibility being the immune response of the human host. We also show that current malaria vaccine formulations are based upon low prevalence haplotypes from a single subgroup and thus may represent only a small proportion of the global parasite population. This study demonstrates significant contrasts in the population structure of P. falciparum vaccine candidates that are consistent with the merozoite antigens being under stronger balancing selection than non-merozoite antigens and suggesting that unique approaches to vaccine design will be required. The results of this study also provide a realistic framework for the diversity of these antigens to be incorporated into the design of next

  2. Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production.

    PubMed

    Weijtens, M E; Hart, E H; Bolhuis, R L

    2000-01-01

    Genetically engineered expression of tumor-specific single chain antibody chimeric receptors (ch-Rec) on human T lymphocytes endow these cells with the parental monoclonal antibody (mAb) dictated tumor specificity and may be useful for clinical immuno-genetherapy. Therefore it was of importance to assess how the densities of tumor-specific receptors and tumor associated antigens (TAA), respectively, affect primary human T lymphocyte functions in relation to target cell susceptibilities to lysis. We therefore studied the functional balance between ch-Rec densities on human T lymphocytes and TAA on tumor cells. The gene construct encoding a ch-Rec derived from (1) a renal carcinoma cell (RCC) specific mouse mAb (G250), and (2) the human signal transducing Fc(epsilon)RI gamma-chain was used. To obtain ch-RecHIGH-POS and ch-RecLOW-POS T lymphocytes, two distinct retroviral vectors were used to introduce the gene constructs into primary human T lymphocytes. Levels of ch-Rec-redirected T lymphocyte mediated tumor cell lysis, as well as lymphokine production were determined using RCC lines as target/stimulator cells, which express either no or increasing densities of the TAA. A functional and dynamic balance between ch-Rec densities on cytotoxic T lymphocytes (CTLs) on the one hand and TAA densities on RCCs on the other, was found. In short, ch-RecHIGH-POS CTLs are triggered by TAAHIGH-POS as well as TAALOW-POS RCCs to lyse tumor cells and produce (IFN-gamma and TNF-alpha) lymphokine. In contrast, ch-RecLOW-POS T lymphocytes are only triggered for cytolysis and lymphokine production by relatively TAAHIGH-POS RCCs. In conclusion, (1) the activation of T lymphocyte responses is co-determined by the expression levels of the ch-Rec on T lymphocytes and the TAA on tumor cells and (2) at relatively high T lymphocyte ch-Rec expression levels the CTLs lyse tumor cells with a wide range of TAA densities. Gene Therapy (2000) 7, 35-42.

  3. Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells.

    PubMed

    Ahuja, Jaya; Kampani, Karan; Datta, Suman; Wigdahl, Brian; Flaig, Katherine E; Jain, Pooja

    2006-02-01

    Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.

  4. [Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen].

    PubMed

    Rukavtsova, E B; Gaiazova, A R; Chebotareva, E N; Bur'ianova, Ia I

    2009-08-01

    The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01-0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.

  5. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  6. 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.

  7. MAPkinase: a second site of G-protein regulation of B-cell activation via the antigen receptors.

    PubMed Central

    Deehan, M R; Klaus, G G; Holman, M J; Harnett, W; Harnett, M M

    1998-01-01

    Ligation of the antigen receptors on B cells transduces transmembrane signals leading to the induction of DNA synthesis. We now show that a pertussis toxin-sensitive heterotrimeric G-protein(s) of the Gi class plays a key role in the regulation of surface immunoglobulin (sIg)-mediated DNA synthesis in B cells. This site of G-protein regulation is distinct from that we have previously reported to govern the coupling of the antigen receptors on B cells to the phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate. We have, moreover, identified a candidate target for this new G-protein regulation by showing that mitogen-activating protein kinase (MAPkinase) activity, which plays a key role in the transduction of sIg-mediated proliferative signals in B cells, is abrogated by pre-exposure to pertussis toxin that covalently modifies and inactivates heterotrimeric G-proteins of the Gi class. Furthermore, our data suggest that this pertussis toxin-sensitive G-protein couples the antigen receptors to MAPkinase activation, at least in part, by regulating sIg-coupling to Lyn, Syk and perhaps Blk and Fyn activity, results consistent with studies in other systems which show that classical G-protein-coupled receptors recruit such protein tyrosine kinases to tranduce MAPkinase activation. Interestingly, however, this G-protein plays no apparent role in the control of up-regulation of major histocompatibility complex class II expression on B cells, suggesting that such G-protein-regulated-tyrosine kinase and MAPkinase activation is not required for the induction of this biological response following antigen receptor ligation. Images Figure 5 PMID:9824472

  8. Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product.

    PubMed Central

    Gassmann, G S; Jacobs, E; Deutzmann, R; Göbel, U B

    1991-01-01

    The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis. Images PMID:1704884

  9. Cloning of Plasmodium yoelii Genes Expressing Three Different Sporozoite-Specific Antigens

    DTIC Science & Technology

    1989-01-01

    advances in the development of a recombinant vaccine against Plasmodium falcipatum sporozoites, there is still an urgent need for a reliable rodent model to...carry out complex vaccine protocols difficult to perform in primates or man. The purpose of this research was to clone the P. yoelii genes coding for...sporozoite antigens that have potential as vaccine candidates in a rodent model. Results our positive clones (B10, 885, B143 and 8155) were identified

  10. Fcγ receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3+ dendritic cells.

    PubMed

    Flinsenberg, Thijs W H; Compeer, Ewoud B; Koning, Dan; Klein, Mark; Amelung, Femke J; van Baarle, Debbie; Boelens, Jaap Jan; Boes, Marianne

    2012-12-20

    The reactivation of human cytomegalovirus (HCMV) poses a serious health threat to immune compromised individuals. As a treatment strategy, dendritic cell (DC) vaccination trials are ongoing. Recent work suggests that BDCA-3(+) (CD141(+)) subset DCs may be particularly effective in DC vaccination trials. BDCA-3(+) DCs had however been mostly characterized for their ability to cross-present antigen from necrotic cells. We here describe our study of human BDCA-3(+) DCs in elicitation of HCMV-specific CD8(+) T-cell clones. We show that Fcgamma-receptor (FcγR) antigen targeting facilitates antigen cross-presentation in several DC subsets, including BDCA-3(+) DCs. FcγR antigen targeting stimulates antigen uptake by BDCA-1(+) rather than BDCA-3(+) DCs. Conversely, BDCA-3(+) DCs and not BDCA-1(+) DCs show improved cross-presentation by FcγR targeting, as measured by induced release of IFNγ and TNF by antigen-specific CD8(+) T cells. FcγR-facilitated cross-presentation requires antigen processing in both an acidic endosomal compartment and by the proteasome, and did not induce substantial DC maturation. FcγRII is the most abundantly expressed FcγR on both BDCA-1(+) and BDCA-3(+) DCs. Furthermore we show that BDCA-3(+) DCs express relatively more stimulatory FcγRIIa than inhibitory FcγRIIb in comparison with BDCA-1(+) DCs. These studies support the exploration of FcγR antigen targeting to BDCA-3(+) DCs for human vaccination purposes.

  11. Regulation of cytochrome P450 (CYP) genes by nuclear receptors.

    PubMed Central

    Honkakoski, P; Negishi, M

    2000-01-01

    Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID:10749660

  12. The induction of antigen-specific CTL by in situ Ad-REIC gene therapy.

    PubMed

    Ariyoshi, Y; Watanabe, M; Eikawa, S; Yamazaki, C; Sadahira, T; Hirata, T; Araki, M; Ebara, S; Nasu, Y; Udono, H; Kumon, H

    2016-05-01

    An adenovirus vector carrying the human Reduced Expression in Immortalized Cell (REIC)/Dkk-3 gene (Ad-REIC) mediates simultaneous induction of cancer-selective apoptosis and augmentation of anticancer immunity. In our preclinical and clinical studies, in situ Ad-REIC gene therapy showed remarkable direct and indirect antitumor effects to realize therapeutic cancer vaccines. We herein aimed to confirm the induction of tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) by Ad-REIC. Using an ovalbumin (OVA), a tumor-associated antigen, expressing E.G7 tumor-bearing mouse model, we investigated the induction and expansion of OVA-specific CTLs responsible for indirect, systemic effects of Ad-REIC. The intratumoral administration of Ad-REIC mediated clear antitumor effects with the accumulation of OVA-specific CTLs in the tumor tissues and spleen. The CD86-positive dendritic cells (DCs) were upregulated in the tumor draining lymph nodes of Ad-REIC-treated mice. In a dual tumor-bearing mouse model in the left and right back, Ad-REIC injection in one side significantly suppressed the tumor growth on both sides and significant infiltration of OVA-specific CTLs into non-injected tumor was also detected. Consequently, in situ Ad-REIC gene therapy is expected to realize a new-generation cancer vaccine via anticancer immune activation with DC and tumor antigen-specific CTL expansion.

  13. A Toll-like receptor 2 agonist-fused antigen enhanced antitumor immunity by increasing antigen presentation and the CD8 memory T cells population

    PubMed Central

    Wu, Chiao-Chieh; Liu, Shih-Jen; Chen, Hsin-Wei; Shen, Kuan-Yin; Leng, Chih-Hsiang

    2016-01-01

    The induction of long-lived effector CD8+ T cells is key to the development of efficient cancer vaccines. In this study, we demonstrated that a Toll-like receptor 2 (TLR2) agonist-fused antigen increased antigen presentation via TLR2 signaling and induced effector memory-like CD8+ T cells against cancer after immunization. The N-terminus of ovalbumin (OVA) was biologically fused with a bacterial lipid moiety TLR2 agonist to produce a recombinant lipidated ovalbumin (rlipo-OVA). We demonstrated that rlipo-OVA activated bone marrow-derived dendritic cells (BM-DCs) maturation and increased antigen presentation by major histocompatibility complex (MHC) class I via TLR2. After immunization, rlipo-OVA skewed the immune response towards T helper (Th) 1 and induced OVA-specific cytotoxic T lymphocyte (CTL) responses. Moreover, immunization with rlipo-OVA induced higher numbers of effector memory (CD44+CD62L−) CD8+ T cells compared with recombinant ovalbumin (rOVA) alone or rOVA mixed with the TLR2 agonist Pam3CSK4. Accordingly, the CD27+CD43+ effector memory CD8+ T cells expressed high levels of the long-lived CD127 marker. The administration of rlipo-OVA could inhibit tumor growth, but the anti-tumor effects were lost after the depletion of CD8 or CD127 cells in vivo. These findings suggested that the TLR2 agonist-fused antigen induced long-lived memory CD8+ T cells for efficient cancer therapy. PMID:27127171

  14. Transient Cannabinoid Receptor 2 Blockade during Immunization Heightens Intensity and Breadth of Antigen-specific Antibody Responses in Young and Aged mice

    PubMed Central

    Dotsey, Emmanuel; Ushach, Irina; Pone, Egest; Nakajima, Rie; Jasinskas, Algis; Argueta, Donovan A.; Dillon, Andrea; DiPatrizio, Nicholas; Davies, Huw; Zlotnik, Albert; Crompton, Peter D.; Felgner, Philip L.

    2017-01-01

    The hallmark of vaccines is their ability to prevent the spread of infectious pathogens and thereby serve as invaluable public health tool. Despite their medical relevance, there is a gap in our understanding of the physiological factors that mediate innate and adaptive immune response to vaccines. The endocannabinoid (eCB) system is a critical modulator of homeostasis in vertebrates. Our results indicate that macrophages and dendritic cells produce the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG) upon antigen activation. We have also established that 2-AG levels are upregulated in the serum and in the lymph node of mice during vaccination. We hypothesized that the intrinsic release of eCBs from immune cells during activation by pathogenic antigens mitigate inflammation, but also suppress overall innate and adaptive immune response. Here we demonstrate, for the first time, that transient administration of the cannabinoid receptor 2 antagonist AM630 (10 mg/kg) or inverse agonist JTE907 (3 mg/kg) during immunization heightens the intensity and breadth of antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues. PMID:28209996

  15. Natural killer cells, killer immunoglobulin-like receptors and human leucocyte antigen class I in disease

    PubMed Central

    Boyton, R J; Altmann, D M

    2007-01-01

    Natural killer cells constitute a potent, rapid part of the innate immune response to infection or transformation, and also generate a link to priming of adaptive immunity. Their function can encompass direct cytotoxicity as well as the release of cytokines and chemokines. In humans, a major component of natural killer (NK) cell target recognition depends mainly on the surveillance of human leucocyte antigen (HLA) class I molecules by killer immunoglobulin-like receptors (KIR). Different KIR can transmit inhibitory or activatory signals to the cell, and effector function is considered to result from the balance of these contributing signals. The regulation of NK cell responses depends on a number of variables: KIR genotype, HLA genotype, heterozygosity versus homozygosity for these, whether there is cognate recognition between the HLA and KIR products carried by an individual, clonal variation between individual NK cells in KIR expression, and the specific modulation of HLA expression by infection, transformation or peptide binding. Different HLA/KIR genotypes can impart different thresholds of activation to the NK cell repertoire and such genotypic variation has been found to confer altered risk in a number of diseases including human immunodeficiency virus (HIV) susceptibility and progression, hepatitis C virus clearance, idiopathic bronchiectasis, autoimmunity and cancer. PMID:17521317

  16. Suboptimal B-cell antigen receptor signaling activity in vivo elicits germinal center counterselection mechanisms.

    PubMed

    Königsberger, Sebastian; Weis, Vanessa; Prodöhl, Jan; Stehling, Martin; Hobeika, Elias; Reth, Michael; Kiefer, Friedemann

    2015-02-01

    Syk and Zap-70 constitute a closely related nonreceptor protein tyrosine kinase family, of which both members are functionally indispensable for conferring their respective antigen receptors with enzymatic activity. In this study, we analyze the impact of altering BCR signaling output on B-cell germinal center (GC) fate selection by constitutive, as well as inducible, monoallelic Syk kinase loss in the presence of a Zap-70 knock-in rescue allele. Cre-mediated Syk deletion in Syk(flox/Zap-70) B cells lowers pErk, but not pAkt-mediated signaling. Surprisingly, the use of a B-cell-specific constitutive mb1-cre deleter mouse model showed that a small cohort of peripheral Syk(flox/Zap-70);mb1-cre B cells efficiently circumvents deletion, which ultimately favors these Syk-sufficient cells to contribute to the GC reaction. Using a developmentally unbiased Syk(flox/Zap-70);mb1-creER(T2) approach in combination with an inducible tdRFP allele, we further demonstrate that this monoallelic deletion escape is not fully explained by leakiness of Cre expression, but is possibly the result of differential Syk locus accessibility in maturing B cells. Altogether, this underscores the importance of proper Syk kinase function not only during central and peripheral selection processes, but also during GC formation and maintenance.

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

    PubMed Central

    Park, Yoon; Jin, Hyung-seung; Lopez, Justine; Lee, Jeeho; Liao, Lujian; Elly, Chris; Liu, Yun-Cai

    2016-01-01

    SHARPIN forms a linear-ubiquitin-chain-assembly complex that promotes signaling via the transcription factor NF-κB. SHARPIN deficiency leads to progressive multi-organ inflammation and immune system malfunction, but how SHARPIN regulates T cell responses is unclear. Here we found that SHARPIN deficiency resulted in a substantial reduction in the number of and defective function of regulatory T cells (Treg cells). Transfer of SHARPIN-sufficient Treg cells into SHARPIN-deficient mice considerably alleviated their systemic inflammation. SHARPIN-deficient T cells displayed enhanced proximal signaling via the T cell antigen receptor (TCR) without an effect on the activation of NF-κB. SHARPIN conjugated with Lys63 (K63)-linked ubiquitin chains, which led to inhibition of the association of TCRζ with the signaling kinase Zap70; this affected the generation of Treg cells. Our study therefore identifies a role for SHARPIN in TCR signaling whereby it maintains immunological homeostasis and tolerance by regulating Treg cells. PMID:26829767

  18. Recognition of the Thomsen-Friedenreich Pancarcinoma Carbohydrate Antigen by a Lamprey Variable Lymphocyte Receptor*

    PubMed Central

    Luo, Ming; Velikovsky, C. Alejandro; Yang, Xinbo; Siddiqui, Maqbool A.; Hong, Xia; Barchi, Joseph J.; Gildersleeve, Jeffrey C.; Pancer, Zeev; Mariuzza, Roy A.

    2013-01-01

    Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs were recently shown to recognize glycans, such as the tumor-associated Thomsen-Friedenreich antigen (TFα; Galβ1–3GalNAcα), with a selectivity rivaling or exceeding that of lectins and antibodies. To understand the basis for TFα recognition by one such VLR (VLRB.aGPA.23), we measured thermodynamic parameters for the binding interaction and determined the structure of the VLRB.aGPA.23-TFα complex to 2.2 Å resolution. In the structure, four tryptophan residues form a tight hydrophobic cage encasing the TFα disaccharide that completely excludes buried water molecules. This cage together with hydrogen bonding of sugar hydroxyls to polar side chains explains the exquisite selectivity of VLRB.aGPA.23. The topology of the glycan-binding site of VLRB.aGPA.23 differs markedly from those of lectins or antibodies, which typically consist of long, convex grooves for accommodating the oligosaccharide. Instead, the TFα disaccharide is sandwiched between a variable loop and the concave surface of the VLR formed by the β-strands of the leucine-rich repeat modules. Longer oligosaccharides are predicted to extend perpendicularly across the β-strands, requiring them to bend to match the concavity of the VLR solenoid. PMID:23782692

  19. Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization.

    PubMed

    Beauregard, K E; Collier, R J; Swanson, J A

    2000-06-01

    Immunofluorescence and other methods have been used to probe the self-assembly and internalization of the binary toxin, anthrax lethal toxin (LeTx), in primary murine macrophages. Proteolytic activation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surface. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronase-resistant state, characteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation that PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nicked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowly from the cell surface. These studies show that proteolytic cleavage of PA, in addition to permitting oligomerization and LF binding, also promotes internalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this binary toxin that maximizes self-assembly.

  20. The receptor tyrosine kinase ROR1--an oncofetal antigen for targeted cancer therapy.

    PubMed

    Hojjat-Farsangi, Mohammad; Moshfegh, Ali; Daneshmanesh, Amir Hossein; Khan, Abdul Salam; Mikaelsson, Eva; Osterborg, Anders; Mellstedt, Håkan

    2014-12-01

    Targeted cancer therapies have emerged as new treatment options for various cancer types. Among targets, receptor tyrosine kinases (RTKs) are among the most promising. ROR1 is a transmembrane RTK of importance during the normal embryogenesis for the central nervous system, heart, lung and skeletal systems, but is not expressed in normal adult tissues. However, ROR1 is overexpressed in several human malignancies and may act as a survival factor for tumor cells. Its unique expression by malignant cells may provide a target for novel therapeutics including monoclonal antibodies (mAbs) and small molecule inhibitors of tyrosine kinases (TKI) for the treatment of cancer. Promising preclinical results have been reported in e.g. chronic lymphocytic leukemia, pancreatic carcinoma, lung and breast cancer. ROR1 might also be an interesting oncofetal antigen for active immunotherapy. In this review, we provide an overview of the ROR1 structure and functions in cancer and highlight emerging therapeutic options of interest for targeting ROR1 in tumor therapy.

  1. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation

    PubMed Central

    Au-Yeung, Byron B.; Zikherman, Julie; Mueller, James L.; Ashouri, Judith F.; Matloubian, Mehrdad; Cheng, Debra A.; Chen, Yiling; Shokat, Kevan M.; Weiss, Arthur

    2014-01-01

    T-cell antigen receptor (TCR) signaling is essential for activation, proliferation, and effector function of T cells. Modulation of both intensity and duration of TCR signaling can regulate these events. However, it remains unclear how individual T cells integrate such signals over time to make critical cell-fate decisions. We have previously developed an engineered mutant allele of the critical T-cell kinase zeta-chain-associated protein kinase 70 kDa (Zap70) that is catalytically inhibited by a small molecule inhibitor, thereby blocking TCR signaling specifically and efficiently. We have also characterized a fluorescent reporter Nur77–eGFP transgenic mouse line in which T cells up-regulate GFP uniquely in response to TCR stimulation. The combination of these technologies unmasked a sharp TCR signaling threshold for commitment to cell division both in vitro and in vivo. Further, we demonstrate that this threshold is independent of both the magnitude of the TCR stimulus and Interleukin 2. Similarly, we identify a temporal threshold of TCR signaling that is required for commitment to proliferation, after which T cells are able to proliferate in a Zap70 kinase-independent manner. Taken together, our studies reveal a sharp threshold for the magnitude and duration of TCR signaling required for commitment of T cells to proliferation. These results have important implications for understanding T-cell responses to infection and optimizing strategies for immunomodulatory drug delivery. PMID:25136127

  2. Review: Current clinical applications of chimeric antigen receptor (CAR) modified T cells.

    PubMed

    Geyer, Mark B; Brentjens, Renier J

    2016-11-01

    The past several years have been marked by extraordinary advances in clinical applications of immunotherapy. In particular, adoptive cellular therapy utilizing chimeric antigen receptor (CAR)-modified T cells targeted to CD19 has demonstrated substantial clinical efficacy in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) and durable clinical benefit in a smaller subset of patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or B-cell non-Hodgkin lymphoma (B-NHL). Early-phase clinical trials are currently assessing CAR T-cell safety and efficacy in additional malignancies. Here, we discuss clinical results from the largest series to date investigating CD19-targeted CAR T cells in B-ALL, CLL, and B-NHL, including discussion of differences in CAR T-cell design and production and treatment approach, as well as clinical efficacy, nature of severe cytokine release syndrome and neurologic toxicities, and CAR T-cell expansion and persistence. We additionally review the current and forthcoming use of CAR T cells in multiple myeloma and several solid tumors and highlight challenges and opportunities afforded by the current state of CAR T-cell therapies, including strategies to overcome inhibitory aspects of the tumor microenvironment and enhance antitumor efficacy.

  3. Dopamine receptor genes: new tools for molecular psychiatry.

    PubMed Central

    Niznik, H B; Van Tol, H H

    1992-01-01

    For over a decade it has been generally assumed that all the pharmacological and biochemical actions of dopamine within the central nervous system and periphery were mediated by two distinct dopamine receptors. These receptors, termed D1 and D2, were defined as those coupled to the stimulation or inhibition of adenylate cyclase, respectively, and by their selectivity and avidity for various drugs and compounds. The concept that two dopamine receptors were sufficient to account for all the effects mediated by dopamine was an oversimplification. Recent molecular biological studies have identified five distinct genes which encode at least eight functional dopamine receptors. The members of the expanded dopamine receptor family, however, can still be codifed by way of the original D1 and D2 receptor dichotomy. These include two genes encoding dopamine D1-like receptors (D1 [D1A]/D5 [D1B]) and three genes encoding D2-like receptors (D2/D3/D4). We review here our recent work on the cloning and characterization of some of the members of the dopamine receptor gene family (D1, D2, D4, D5), their relationship to neuropsychiatric disorders and their potential role in antipsychotic drug action. Images Fig. 1 PMID:1450188

  4. Induction of interferon-stimulated genes by Simian virus 40 T antigens

    SciTech Connect

    Rathi, Abhilasha V.; Cantalupo, Paul G.; Sarkar, Saumendra N.; Pipas, James M.

    2010-10-25

    Simian virus 40 (SV40) large T antigen (TAg) is a multifunctional oncoprotein essential for productive viral infection and for cellular transformation. We have used microarray analysis to examine the global changes in cellular gene expression induced by wild-type T antigen (TAg{sup wt}) and TAg-mutants in mouse embryo fibroblasts (MEFs). The expression profile of approximately 800 cellular genes was altered by TAg{sup wt} and a truncated TAg (TAg{sup N136}), including many genes that influence cell cycle, DNA-replication, transcription, chromatin structure and DNA repair. Unexpectedly, we found a significant number of immune response genes upregulated by TAg{sup wt} including many interferon-stimulated genes (ISGs) such as ISG56, OAS, Rsad2, Ifi27 and Mx1. Additionally, we also observed activation of STAT1 by TAg{sup wt}. Our genetic studies using several TAg-mutants reveal an unexplored function of TAg and indicate that the LXCXE motif and p53 binding are required for the upregulation of ISGs.

  5. Widespread balancing selection and pathogen-driven selection at blood group antigen genes

    PubMed Central

    Fumagalli, Matteo; Cagliani, Rachele; Pozzoli, Uberto; Riva, Stefania; Comi, Giacomo P.; Menozzi, Giorgia; Bresolin, Nereo; Sironi, Manuela

    2009-01-01

    Historically, allelic variations in blood group antigen (BGA) genes have been regarded as possible susceptibility factors for infectious diseases. Since host–pathogen interactions are major determinants in evolution, BGAs can be thought of as selection targets. In order to verify this hypothesis, we obtained an estimate of pathogen richness for geographic locations corresponding to 52 populations distributed worldwide; after correction for multiple tests and for variables different from selective forces, significant correlations with pathogen richness were obtained for multiple variants at 11 BGA loci out of 26. In line with this finding, we demonstrate that three BGA genes, namely CD55, CD151, and SLC14A1, have been subjected to balancing selection, a process, rare outside MHC genes, which maintains variability at a locus. Moreover, we identified a gene region immediately upstream the transcription start site of FUT2 which has undergone non-neutral evolution independently from the coding region. Finally, in the case of BSG, we describe the presence of a highly divergent haplotype clade and the possible reasons for its maintenance, including frequency-dependent balancing selection, are discussed. These data indicate that BGAs have been playing a central role in the host–pathogen arms race during human evolutionary history and no other gene category shows similar levels of widespread selection, with the only exception of loci involved in antigen recognition. PMID:18997004

  6. Widespread balancing selection and pathogen-driven selection at blood group antigen genes.

    PubMed

    Fumagalli, Matteo; Cagliani, Rachele; Pozzoli, Uberto; Riva, Stefania; Comi, Giacomo P; Menozzi, Giorgia; Bresolin, Nereo; Sironi, Manuela

    2009-02-01

    Historically, allelic variations in blood group antigen (BGA) genes have been regarded as possible susceptibility factors for infectious diseases. Since host-pathogen interactions are major determinants in evolution, BGAs can be thought of as selection targets. In order to verify this hypothesis, we obtained an estimate of pathogen richness for geographic locations corresponding to 52 populations distributed worldwide; after correction for multiple tests and for variables different from selective forces, significant correlations with pathogen richness were obtained for multiple variants at 11 BGA loci out of 26. In line with this finding, we demonstrate that three BGA genes, namely CD55, CD151, and SLC14A1, have been subjected to balancing selection, a process, rare outside MHC genes, which maintains variability at a locus. Moreover, we identified a gene region immediately upstream the transcription start site of FUT2 which has undergone non-neutral evolution independently from the coding region. Finally, in the case of BSG, we describe the presence of a highly divergent haplotype clade and the possible reasons for its maintenance, including frequency-dependent balancing selection, are discussed. These data indicate that BGAs have been playing a central role in the host-pathogen arms race during human evolutionary history and no other gene category shows similar levels of widespread selection, with the only exception of loci involved in antigen recognition.

  7. Antigen-affinity controls pre-germinal centser B cell selection by promoting Mcl-1 induction through BAFF receptor signaling

    PubMed Central

    Wensveen, Felix M.; Slinger, Erik; van Attekum, Martijn HA; Brink, Robert; Eldering, Eric

    2016-01-01

    Upon antigen encounter, the responsive B cell pool undergoes stringent selection which eliminates cells with low B cell receptor (BCR) affinity. Already before formation of the germinal center, activated B cells of low-affinity are negatively selected in a process that is molecularly not well understood. In this study, we investigated the mechanism behind pre-GC affinity-mediated B cell selection. We applied affinity mutants of HEL antigen and found that rapidly after activation B cells become highly dependent on the cytokine BAFF. Moreover, expression of BAFF receptor CD268 is regulated in a BCR-affinity dependent fashion. High affinity responses via BAFF correlated with PI3K activation, which controlled expression of the pro-survival protein Mcl-1, and thereby increased survival. In the presence of excess BAFF, or in absence of the Mcl-1 antagonist Noxa, more low-affinity B cells survived the first two days after antigen encounter. This resulted in increased numbers of antigen-specific B cells of low affinity upon immunization and reduced the overall affinity of cells that contributed to the germinal center reaction. Our findings elucidate a crucial molecular pathway of B cell selection in the earliest phases of activation by identifying a novel link between BCR affinity and BAFF-R signaling towards Mcl-1. PMID:27762293

  8. DC subset-specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo.

    PubMed

    Lehmann, Christian H K; Baranska, Anna; Heidkamp, Gordon F; Heger, Lukas; Neubert, Kirsten; Lühr, Jennifer J; Hoffmann, Alana; Reimer, Katharina C; Brückner, Christin; Beck, Simone; Seeling, Michaela; Kießling, Melissa; Soulat, Didier; Krug, Anne B; Ravetch, Jeffrey V; Leusen, Jeanette H W; Nimmerjahn, Falk; Dudziak, Diana

    2017-04-07

    Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4(+) and CD8(+) T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8(+) T cell responses was largely dependent on CD11c(+)CD8(+) DCs, whereas CD11c(+)CD8(-) DCs were critical for priming CD4(+) T cell responses.

  9. cap alpha. -chain locus of the T-cell antigen receptor is involved in the t(10; 14) chromosome translocation of T-cell acute lymphocytic leukemia

    SciTech Connect

    Kagan, J.; Finan, J.; Letofsky, J.; Besa, E.C.; Nowell, P.C.; Croce, C.M.

    1987-07-01

    Human leukemic T cells carrying a t(10;14)(q24;q11) chromosome translocation were fused with mouse leukemic T cells, and the hybrids were examined for genetic markers of human chromosomes 10 and 14. Hybrids containing the human 10q+ chromosome had the human genes for terminal deoxynucleotidyltransferase that has been mapped at 10q23-q25 and for C/sub ..cap alpha../ (the constant region of TCRA (the ..cap alpha..-chain locus of the T-cell antigen receptor gene)), but not for V/sub ..cap alpha../ (the variable region of TCRA). Hybrids containing the human 14q- chromosome retained the V/sub ..cap alpha../genes. Thus the 14q11 breakpoint in the t(10;14) chromosome translocation directly involves TCRA, splitting the locus in a region between the V/sub ..cap alpha../ and the C/sub ..cap alpha../ genes. These results suggest that the translocation of the C/sub ..cap alpha../ locus to a putative cellular protooncogene located proximal to the breakpoint at 10q24, for which the authors propose the name TCL3, results in its deregulation, leading to T-cell leukemia. Since hybrids with the 10q+ chromosome also retained the human terminal deoxynucleotidyltransferase gene, it is further concluded that the terminal deoxynucleotidyltransferase locus is proximal to the TCL3 gene, at band 10q23-q24.

  10. Genes encoding homologous antigens in taeniid cestode parasites: Implications for development of recombinant vaccines produced in Escherichia coli.

    PubMed

    Gauci, Charles; Lightowlers, Marshall W

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

  11. Adenovirus receptors and their implications in gene delivery

    PubMed Central

    Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

    2010-01-01

    Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

  12. Acute Myeloid Leukemia Targeting by Chimeric Antigen Receptor T Cells: Bridging the Gap from Preclinical Modeling to Human Studies.

    PubMed

    Rotiroti, Maria Caterina; Arcangeli, Silvia; Casucci, Monica; Perriello, Vincenzo; Bondanza, Attilio; Biondi, Andrea; Tettamanti, Sarah; Biagi, Ettore

    2017-03-01

    Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients, thus demanding advanced and personalized therapies. In this regard, different targeted immunotherapeutic approaches are available, ranging from naked monoclonal antibodies (mAb) to conjugated and multifunctional mAbs (i.e., BiTEs and DARTs). Recently, researchers have focused their attention on novel techniques of genetic manipulation specifically to redirect cytotoxic T cells endowed with chimeric antigen receptors (CARs) toward selected tumor associated antigens. So far, CAR T cells targeting the CD19 antigen expressed by B-cell origin hematological cancers have gained impressive clinical results, leading to the possibility of translating the CAR platform to treat other hematological malignancies such as AML. However, one of the main concerns in the field of AML CAR immunotherapy is the identification of an ideal target cell surface antigen, being highly expressed on tumor cells but minimally present on healthy tissues, together with the design of an anti-AML CAR appropriately balancing efficacy and safety profiles. The current review focuses mainly on AML target antigens and the related immunotherapeutic approaches developed so far, deeply dissecting methods of CAR T cell safety improvements, when designing novel CARs approaching human studies.

  13. Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

    SciTech Connect

    Xu, Rui; McBride, Ryan; Paulson, James C.; Basler, Christopher F.; Wilson, Ian A.

    2010-03-04

    The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.

  14. A gene cluster for the synthesis of serotype g-specific polysaccharide antigen in Aggregatibacter actinomycetemcomitans.

    PubMed

    Tsuzukibashi, Osamu; Saito, Masanori; Kobayashi, Taira; Umezawa, Koji; Nagahama, Fumio; Hiroi, Takachika; Hirasawa, Masatomo; Takada, Kazuko

    2014-04-01

    Aggregatibacter actinomycetemcomitans is an important pathogen related to aggressively progressive periodontal breakdown in adolescents and adults. The species can be divided into six serotypes (a-f) according to their surface carbohydrate antigens. Recently, a new serotype g of A. actinomycetemcomitans was proposed. The aim of the present study was to sequence the gene cluster associated with the biosynthesis of the serotype g-specific polysaccharide antigen and develop serotype-specific primers for PCR assay to identify serotype g strains of A. actinomycetemcomitans. The serotype-specific polysaccharide (SSPS) gene cluster of the NUM-Aa 4039 strain contained 21 genes in 21,842-bp nucleotides. The similarity of the SSPS gene cluster sequence was 96.7 % compared with that of the serotype e strain. Seventeen serotype g genes showed more than 90 % homology both in nucleotide and amino acids to the serotype e strain. Three additional genes with 1,579 bp in NUM-Aa 4039 were inserted into the corresponding ORF13 of the serotype e strain. The serotype g-specific primers were designed from the insertion region of NUM-Aa 4039. Serotypes of the a-f strains were not amplified by serotype-specific g primers; only NUM-Aa 4039 showed an amplicon band. The NUM-Aa 4039 strain was three genes in the SSPS gene cluster different from those of serotype e strain. The specific primers derived from these different regions are useful for identification and distribution of serotype g strain among A. actinomycetemcomitans from clinical samples.

  15. A Gene Encoding Antigenic Peptides of Human Squamous Cell Carcinoma Recognized by Cytotoxic T Lymphocytes

    PubMed Central

    Shichijo, Shigeki; Nakao, Masanobu; Imai, Yasuhisa; Takasu, Hideo; Kawamoto, Mayumi; Niiya, Fumihiko; Yang, Damu; Toh, Yuji; Yamana, Hideaki; Itoh, Kyogo

    1998-01-01

    Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601–restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients. PMID:9449708

  16. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens

    PubMed Central

    1992-01-01

    The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3- expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial

  17. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations

    PubMed Central

    Deniger, Drew C.; Yu, Jianqiang; Huls, M. Helen; Figliola, Matthew J.; Mi, Tiejuan; Maiti, Sourindra N.; Widhopf, George F.; Hurton, Lenka V.; Thokala, Radhika; Singh, Harjeet; Olivares, Simon; Champlin, Richard E.; Wierda, William G.; Kipps, Thomas J.; Cooper, Laurence J. N.

    2015-01-01

    T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire. PMID:26030772

  18. An altered repertoire of T cell receptor V gene expression by rheumatoid synovial fluid T lymphocytes.

    PubMed Central

    Lunardi, C; Marguerie, C; So, A K

    1992-01-01

    The pattern of T cell receptor V gene expression by lymphocytes from rheumatoid synovial fluid and paired peripheral blood samples was compared using a polymerase chain reaction (PCR)-based assay. Eight rheumatoid arthritis (RA) patients who had varying durations of disease (from 2 to 20 years) were studied. In all patients there was evidence of a different pattern of V gene expression between the two compartments. Significantly increased expression of at least one V alpha or V beta gene family by synovial fluid T cells was observed in all the patients studied. Three different V alpha (V alpha 10, 15 and 18) and three V beta (V beta 4, 5 and 13) families were commonly elevated. Sequencing of synovial V beta transcripts demonstrated that the basis of increased expression of selected V gene families in the synovial fluid was due to the presence of dominant clonotypes within those families, which constituted up to 53% of the sequences isolated from one particular synovial V gene family. There were considerable differences in the NDJ sequences found in synovial and peripheral blood T cell receptor (TCR) transcripts of the same V beta gene family. These data suggest that the TCR repertoire in the two compartments differs, and that antigen-driven expansion of particular synovial T cell populations is a component of rheumatoid synovitis, and is present in all stages of the disease. PMID:1458680

  19. Targeting Ewing sarcoma with activated and GD2-specific chimeric antigen receptor-engineered human NK cells induces upregulation of immune-inhibitory HLA-G.

    PubMed

    Kailayangiri, Sareetha; Altvater, Bianca; Spurny, Christian; Jamitzky, Silke; Schelhaas, Sonja; Jacobs, Andreas H; Wiek, Constanze; Roellecke, Katharina; Hanenberg, Helmut; Hartmann, Wolfgang; Wiendl, Heinz; Pankratz, Susann; Meltzer, Jutta; Farwick, Nicole; Greune, Lea; Fluegge, Maike; Rossig, Claudia

    2017-01-01

    Activated and in vitro expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their in vivo efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen GD2 in activated NK cells increased their responses to GD2+ allogeneic EwS cells in vitro and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of GD2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate GD2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, in vitro co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells in vitro. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

  20. Lenalidomide enhances antitumor functions of chimeric antigen receptor modified T cells.

    PubMed

    Otáhal, Pavel; Průková, Dana; Král, Vlastimil; Fabry, Milan; Vočková, Petra; Latečková, Lucie; Trněný, Marek; Klener, Pavel

    2016-04-01

    Tumor immunotherapy based on the use of chimeric antigen receptor modified T cells (CAR T cells) is a promising approach for the treatment of refractory hematological malignancies. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable.Lenalidomide (LEN) is an immunomodulatory drug currently approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma, while it is clinically tested in the therapy of diffuse large B-cell lymphoma of activated B cell immunophenotype. LEN was shown to increase antitumor immune responses at least partially by modulating the activity of E3 ubiquitin ligase Cereblon, which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors, which in turn results in changed expression of various receptors on the surface of tumor cells. In order to enhance the effectiveness of CAR-based immunotherapy, we assessed the anti-lymphoma efficacy of LEN in combination with CAR19 T cells or CAR20 T cells in vitro and in vivo using various murine models of aggressive B-cell non-Hodgkin lymphomas (B-NHL).Immunodeficient NSG mice were transplanted with various human B-NHL cells followed by treatment with CAR19 or CAR20 T cells with or without LEN. Next, CAR19 T cells were subjected to series of tests in vitro to evaluate their response and signaling capacity following recognition of B cell in the presence or absence of LEN.Our data shows that LEN significantly enhances antitumor functions of CAR19 and CAR20 T cells in vivo. Additionally, it enhances production of interferon gamma by CAR19 T cells and augments cell signaling via CAR19 protein in T cells in vitro. Our data further suggests that LEN works through direct effects on T cells but not on B-NHL cells. The biochemical events underlying this costimulatory effect of LEN are currently being investigated. In summary, our data supports the use of LEN for

  1. Lenalidomide enhances antitumor functions of chimeric antigen receptor modified T cells

    PubMed Central

    Otáhal, Pavel; Průková, Dana; Král, Vlastimil; Fabry, Milan; Vočková, Petra; Latečková, Lucie; Trněný, Marek; Klener, Pavel

    2016-01-01

    ABSTRACT Tumor immunotherapy based on the use of chimeric antigen receptor modified T cells (CAR T cells) is a promising approach for the treatment of refractory hematological malignancies. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable.Lenalidomide (LEN) is an immunomodulatory drug currently approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma, while it is clinically tested in the therapy of diffuse large B-cell lymphoma of activated B cell immunophenotype. LEN was shown to increase antitumor immune responses at least partially by modulating the activity of E3 ubiquitin ligase Cereblon, which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors, which in turn results in changed expression of various receptors on the surface of tumor cells. In order to enhance the effectiveness of CAR-based immunotherapy, we assessed the anti-lymphoma efficacy of LEN in combination with CAR19 T cells or CAR20 T cells in vitro and in vivo using various murine models of aggressive B-cell non-Hodgkin lymphomas (B-NHL).Immunodeficient NSG mice were transplanted with various human B-NHL cells followed by treatment with CAR19 or CAR20 T cells with or without LEN. Next, CAR19 T cells were subjected to series of tests in vitro to evaluate their response and signaling capacity following recognition of B cell in the presence or absence of LEN.Our data shows that LEN significantly enhances antitumor functions of CAR19 and CAR20 T cells in vivo. Additionally, it enhances production of interferon gamma by CAR19 T cells and augments cell signaling via CAR19 protein in T cells in vitro. Our data further suggests that LEN works through direct effects on T cells but not on B-NHL cells. The biochemical events underlying this costimulatory effect of LEN are currently being investigated. In summary, our data supports the use

  2. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

    PubMed Central

    Fraietta, Joseph A.; Beckwith, Kyle A.; Patel, Prachi R.; Ruella, Marco; Zheng, Zhaohui; Barrett, David M.; Lacey, Simon F.; Melenhorst, Jan Joseph; McGettigan, Shannon E.; Cook, Danielle R.; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B.; Cogdill, Alexandria P.; Gill, Saar; Porter, David L.; Woyach, Jennifer A.; Long, Meixiao; Johnson, Amy J.; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L.; June, Carl H.; Byrd, John C.

    2016-01-01

    Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. PMID:26813675

  3. Impact of estrogen receptor α gene and oxytocin receptor gene polymorphisms on female sexuality.

    PubMed

    Armeni, Anastasia K; Assimakopoulos, Konstantinos; Marioli, Dimitra; Koika, Vassiliki; Michaelidou, Euthychia; Mourtzi, Niki; Iconomou, Gregoris; Georgopoulos, Neoklis A

    2017-01-01

    Over the past decades, research attention has increasingly been paid to the neurobiological component of sexual behavior. The aim of the present study was to investigate the correlation of estrogen receptor α (ERA) gene polymorphism (rs2234693-PvuII) (T→C substitution) and oxytocin receptor gene polymorphism (rs53576) (G→A substitution) with sexuality parameters of young, healthy women. One hundred thirty-three Greek heterosexual women, students in higher education institutions, 20-25 years of age, sexually active, with normal menstrual cycles (28-35 days), were recruited in the study. Exclusion criteria were chronic and/or major psychiatric diseases, use of oral contraceptive pills (OCs), polycystic ovary syndrome (PCOS), thyroid diseases as well as drugs that are implicated in hypothalamus-pituitary-gonadal axis. T allele (wildtype) of rs2234693 (PvuII) polymorphism of ERA gene was correlated with increased levels of arousal and lubrication, whereas A allele (polymorphic) of rs53576 (OXTR) polymorphism was correlated with increased arousal levels. The simultaneous presence of both T allele of rs2234693 (PvuII) and A allele of rs53576 (OXTR) polymorphisms (T + A group) was correlated with increased arousal, orgasm levels as well as female sexual function index full score. To our knowledge, this is the first study to investigate the interaction between ERA and OXTR with regard to sexual function in women. Female sexuality is a complex behavioral trait that encompasses both biological and psychological components. It seems that variability in female sexual response stems from genetic variability that characterizes endocrine, neurotransmitter and central nervous system influences.

  4. Impact of estrogen receptor α gene and oxytocin receptor gene polymorphisms on female sexuality

    PubMed Central

    Armeni, Anastasia K; Assimakopoulos, Konstantinos; Marioli, Dimitra; Koika, Vassiliki; Michaelidou, Euthychia; Mourtzi, Niki; Iconomou, Gregoris

    2017-01-01

    Over the past decades, research attention has increasingly been paid to the neurobiological component of sexual behavior. The aim of the present study was to investigate the correlation of estrogen receptor α (ERA) gene polymorphism (rs2234693-PvuII) (T→C substitution) and oxytocin receptor gene polymorphism (rs53576) (G→A substitution) with sexuality parameters of young, healthy women. One hundred thirty-three Greek heterosexual women, students in higher education institutions, 20–25 years of age, sexually active, with normal menstrual cycles (28–35 days), were recruited in the study. Exclusion criteria were chronic and/or major psychiatric diseases, use of oral contraceptive pills (OCs), polycystic ovary syndrome (PCOS), thyroid diseases as well as drugs that are implicated in hypothalamus–pituitary–gonadal axis. T allele (wildtype) of rs2234693 (PvuII) polymorphism of ERA gene was correlated with increased levels of arousal and lubrication, whereas A allele (polymorphic) of rs53576 (OXTR) polymorphism was correlated with increased arousal levels. The simultaneous presence of both T allele of rs2234693 (PvuII) and A allele of rs53576 (OXTR) polymorphisms (T + A group) was correlated with increased arousal, orgasm levels as well as female sexual function index full score. To our knowledge, this is the first study to investigate the interaction between ERA and OXTR with regard to sexual function in women. Female sexuality is a complex behavioral trait that encompasses both biological and psychological components. It seems that variability in female sexual response stems from genetic variability that characterizes endocrine, neurotransmitter and central nervous system influences. PMID:28069897

  5. Human T-cell receptor v{beta} gene polymorphism and multiple sclerosis

    SciTech Connect

    Wei, S.; Charmley, P.; Birchfield, R.I.; Concannon, P.

    1995-04-01

    Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the {beta} chain of the T-cell receptor for antigen (RCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of {approximately}110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility. The TCRBV coding region-specific markers generated in these studies, as well as the approach of testing for associations with specific functionally relevant polymorphic sites within individual BV genes, should be useful in the evaluation of the many reported disease associations involving the human TCRB region. 22 refs., 1 fig., 3 tabs.

  6. Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent.

    PubMed

    Leskinen, Katarzyna; Varjosalo, Markku; Li, Zhilin; Li, Chun-Mei; Skurnik, Mikael

    2015-06-01

    The antiterminator RfaH is required for the expression of LPS, capsule, haemolysin, exotoxin, haemin uptake receptor and F pilus. As these structures are critical for bacterial virulence, loss of RfaH usually leads to attenuation. Here, we inactivated the rfaH gene of Yersinia enterocolitica O:3 to study its role in this enteropathogen. RNA sequencing of the WT and ΔrfaH strain transcriptomes revealed that RfaH acted as a highly specific regulator that enhanced the transcription of the operons involved in biosynthesis of LPS O-antigen and outer core (OC), but did not affect the expression of enterobacterial common antigen. Interestingly, the transcriptome of the ΔrfaH strain was very similar to that of an O-antigen-negative mutant. This indicated that some of the changes seen in the ΔrfaH strain, such as the genes involved in outer membrane homeostasis or in the stress-response-associated Cpx pathway, were actually due to indirect responses via the loss of O-antigen. The decreased amount of LPS on the ΔrfaH strain cell surface resulted in an attenuated stress response, and lower resistance to compounds such as SDS and polymyxin B. However, the ΔrfaH strain was significantly more resistant to complement-mediated killing by normal human serum. Taken together, our results revealed a novel role of RfaH acting as a highly specific regulator of O-antigen and OC of LPS in Y. enterocolitica O:3. It may be speculated that RfaH might have an in vivo role in controlling tissue-specific expression of bacterial surface oligo/polysaccharides.

  7. Estrogen increases renal oxytocin receptor gene expression.

    PubMed

    Ostrowski, N L; Young, W S; Lolait, S J

    1995-04-01

    Estrogens have been implicated in the sodium and fluid imbalances associated with the menstrual cycle and late pregnancy. An estrogen-dependent role for renal oxytocin receptors in fluid homeostasis is suggested by the present findings which demonstrate that estradiol benzoate treatment increases the expression of the oxytocin receptor messenger ribonucleic acid and 125I-OTA binding to oxytocin receptors in the renal cortex and medullary collecting ducts of ovariectomized female rats. Moreover, estradiol induced high levels of oxytocin receptor expression in outer stripe proximal tubules of ovariectomized female and adrenalectomized male rats. Proximal tubule induction was inhibited in a dose-dependent manner by the antiestrogen tamoxifen, but cortical expression of oxytocin receptors in macula densa cells was unaffected by tamoxifen. These data demonstrate cell-specific regulation of oxytocin receptor expression in macula densa and proximal tubule cells, and suggest a important role for these receptors in mediating estrogen-induced alterations in renal fluid dynamics by possibly affecting glomerular filtration and water and solute reabsorption during high estrogen states.

  8. Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum.

    PubMed

    Tonkin, Christopher J; Carret, Céline K; Duraisingh, Manoj T; Voss, Till S; Ralph, Stuart A; Hommel, Mirja; Duffy, Michael F; Silva, Liliana Mancio da; Scherf, Artur; Ivens, Alasdair; Speed, Terence P; Beeson, James G; Cowman, Alan F

    2009-04-14

    Cytoadherance of Plasmodium falciparum-infected erythrocytes in the brain, organs and peripheral microvasculature is linked to morbidity and mortality associated with severe malaria. Parasite-derived P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) molecules displayed on the erythrocyte surface are responsible for cytoadherance and undergo antigenic variation in the course of an infection. Antigenic variation of PfEMP1 is achieved by in situ switching and mutually exclusive transcription of the var gene family, a process that is controlled by epigenetic mechanisms. Here we report characterisation of the P. falciparum silent information regulator's A and B (PfSir2A and PfSir2B) and their involvement in mutual exclusion and silencing of the var gene repertoire. Analysis of P. falciparum parasites lacking either PfSir2A or PfSir2B shows that these NAD(+)-dependent histone deacetylases are required for silencing of different var gene subsets classified by their conserved promoter type. We also demonstrate that in the absence of either of these molecules mutually exclusive expression of var genes breaks down. We show that var gene silencing originates within the promoter and PfSir2 paralogues are involved in cis spreading of silenced chromatin into adjacent regions. Furthermore, parasites lacking PfSir2A but not PfSir2B have considerably longer telomeric repeats, demonstrating a role for this molecule in telomeric end protection. This work highlights the pivotal but distinct role for both PfSir2 paralogues in epigenetic silencing of P. falciparum virulence genes and the control of pathogenicity of malaria infection.

  9. Characteristics of the mouse genomic histamine H1 receptor gene

    SciTech Connect

    Inoue, Isao; Taniuchi, Ichiro; Kitamura, Daisuke

    1996-08-15

    We report here the molecular cloning of a mouse histamine H1 receptor gene. The protein deduced from the nucleotide sequence is composed of 488 amino acid residues with characteristic properties of GTP binding protein-coupled receptors. Our results suggest that the mouse histamine H1 receptor gene is a single locus, and no related sequences were detected. Interspecific backcross analysis indicated that the mouse histamine H1 receptor gene (Hrh1) is located in the central region of mouse Chromosome 6 linked to microphthalmia (Mitfmi), ras-related fibrosarcoma oncogene 1 (Raf1), and ret proto-oncogene (Ret) in a region of homology with human chromosome 3p. 12 refs., 3 figs.

  10. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    PubMed Central

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

  11. Molecular and biological interaction between major histocompatibility complex class I antigens and luteinizing hormone receptors or beta-adrenergic receptors triggers cellular response in mice.

    PubMed Central

    Solano, A R; Cremaschi, G; Sánchez, M L; Borda, E; Sterin-Borda, L; Podestá, E J

    1988-01-01

    Purified IgG from BALB/c mouse anti-C3H serum exerts positive inotropic and chronotropic effects in C3H mouse atria and induces testosterone synthesis in C3H mouse Leydig cells. The effect depends on IgG concentration and can be abolished by beta-adrenergic-receptor and luteinizing hormone-receptor antagonists. IgG interferes with the binding of dihydroalprenolol and luteinizing hormone. Monoclonal antibodies against major histocompatibility complex class I antigens were active on the Leydig cells of C3H and BALB/c mice. There was a parallelism between the effect of each individual monoclonal antibody with specificity for a particular haplotype and the response of the target cell from the strains carrying such haplotypes. These antibodies could precipitate the soluble luteinizing hormone-receptor complex. The results suggested that bound hormone triggers the association of major histocompatibility class I antigen with the receptor, thereby activating the respective target cells. PMID:2839829

  12. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection.

    PubMed

    Khakoo, Salim I; Thio, Chloe L; Martin, Maureen P; Brooks, Collin R; Gao, Xiaojiang; Astemborski, Jacquie; Cheng, Jie; Goedert, James J; Vlahov, David; Hilgartner, Margaret; Cox, Steven; Little, Ann-Margeret; Alexander, Graeme J; Cramp, Matthew E; O'Brien, Stephen J; Rosenberg, William M C; Thomas, David L; Carrington, Mary

    2004-08-06

    Natural killer (NK) cells provide a central defense against viral infection by using inhibitory and activation receptors for major histocompatibility complex class I molecules as a means of controlling their activity. We show that genes encoding the inhibitory NK cell receptor KIR2DL3 and its human leukocyte antigen C group 1 (HLA-C1) ligand directly influence resolution of hepatitis C virus (HCV) infection. This effect was observed in Caucasians and African Americans with expected low infectious doses of HCV but not in those with high-dose exposure, in whom the innate immune response is likely overwhelmed. The data strongly suggest that inhibitory NK cell interactions are important in determining antiviral immunity and that diminished inhibitory responses confer protection against HCV.

  13. Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis

    PubMed Central

    Jirholt, Pernilla; Turesson, Olof; Wing, Kajsa; Holmdahl, Rikard; Kihlberg, Jan; Stern, Anna; Mårtensson, Inga-Lill; Henningsson, Louise; Gustafsson, Kenth; Gjertsson, Inger

    2016-01-01

    Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases. PMID:27159398

  14. Cloning and characterization of the merozoite surface antigen 1 gene of Plasmodium berghei.

    PubMed

    Zhong, H; Fan, J Y; Yang, S; Davidson, E A

    1999-06-01

    Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome.

  15. Prolactin receptor and signal transduction to milk protein genes

    SciTech Connect

    Djiane, J.; Daniel, N.; Bignon, C.

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

  16. Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization.

    PubMed

    Porgador, A; Irvine, K R; Iwasaki, A; Barber, B H; Restifo, N P; Germain, R N

    1998-09-21

    Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50-100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000-30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.

  17. Targeting TARP, a novel breast and prostate tumor-associated antigen, with T cell receptor-like human recombinant antibodies.

    PubMed

    Epel, Malka; Carmi, Irit; Soueid-Baumgarten, Sharon; Oh, Sang Kon; Bera, Tapan; Pastan, Ira; Berzofsky, Jay; Reiter, Yoram

    2008-06-01

    MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptide-specific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRgamma alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes.

  18. T-cell receptor alpha chain plays a critical role in antigen-specific suppressor cell function.

    PubMed Central

    Kuchroo, V K; Byrne, M C; Atsumi, Y; Greenfield, E; Connolly, J B; Whitters, M J; O'Hara, R M; Collins, M; Dorf, M E

    1991-01-01

    Antigen-specific suppressor T-cell hybridomas release soluble suppressor factors (TsF) in the supernatant that modulate both in vivo delayed-type hypersensitivity and in vitro plaque-forming cell responses in an antigen-specific manner. To study the relationship between the T-cell receptor (TcR) and TsF, we developed a series of TcR alpha- or TcR beta- expression variants from suppressor T-cell hybridomas that expressed the CD3-TcR alpha/beta complex. We demonstrate that loss of TcR alpha but not TcR beta mRNA was accompanied by the concomitant loss of suppressor bioactivity. Homologous transfection of TcR alpha cDNA into a TcR alpha- beta+ clone reconstituted both CD3-TcR expression and suppressor function. Furthermore, suppressor activity from TcR beta- variants was specifically absorbed by antigen and anti-TcR alpha antibodies, but not by anti-CD3 or anti-TcR beta affinity columns. These data directly establish a role for the TcR alpha chain in suppressor T-cell function and suggest that the TcR alpha chain is part of the antigen-specific TsF molecule. Images PMID:1833764

  19. Polymorphism in the gene encoding the Pfs48/45 antigen of Plasmodium falciparum. XI. Asembo Bay Cohort Project.

    PubMed

    Escalante, Ananias A; Grebert, Heather M; Chaiyaroj, Sansanee C; Riggione, Flavia; Biswas, Sukla; Nahlen, Bernard L; Lal, Altaf A

    2002-01-01

    We have investigated the genetic diversity of the gene encoding the transmission-blocking vaccine antigen Pfs48/45 of Plasmodium falciparum parasites from western Kenya and compared it with parasite populations from Thailand, India, and Venezuela. We report 44 complete new sequences. Overall, the antigen is less polymorphic as compared with other pre-erythrocytic and blood stage antigens. Contrary to other P. falciparum antigens, the number of synonymous substitutions per synonymous site exceeds the number of non-synonymous substitutions per non-synonymous site. We have found that the Pfs48/45 gene of Kenyan parasites is more polymorphic than parasites from other geographic origins. Our analysis reveals that positive natural selection is involved in the maintenance of the observed polymorphism. No evidence of intragenic recombination was found. F(st) values reveal high levels of gene flow between India and Thailand, however, there are strong constraints in gene flow among Kenyan, Southeast Asian, and Venezuelan parasites. No alleles could be linked to a specific geographic region. The results of this study suggest that this gametocyte antigen, like other asexual blood stage antigens, is under selection pressure.

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

    PubMed

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

    2016-03-01

    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.

  1. 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

  2. Conserved structure of amphibian T-cell antigen receptor beta chain.

    PubMed Central

    Fellah, J S; Kerfourn, F; Guillet, F; Charlemagne, J

    1993-01-01

    All jawed vertebrates possess well-differentiated thymuses and elicit T-cell-like cell-mediated responses; however, no surface T-cell receptor (TCR) molecules or TCR genes have been identified in ectothermic vertebrate species. Here we describe cDNA clones from an amphibian species, Ambystoma mexicanum (the Mexican axolotl), that have sequences highly homologous to the avian and mammalian TCR beta chains. The cloned amphibian beta chain variable region (V beta) shares most of the structural characteristics with the more evolved vertebrate V beta and presents approximately 56% amino acid identities with the murine V beta 14 and human V beta 18 families. The two different cloned axolotl beta chain joining regions (J beta) were found to have conserved all the invariant mammalian J beta residues, and in addition, the presence of a conserved glycine at the V beta-J beta junction suggests the existence of diversity elements. The extracellular domains of the two axolotl beta chain constant region isotypes C beta 1 and C beta 2 show an impressively high degree of identity, thus suggesting that a very efficient mechanism of gene correction has been in operation to preserve this structure at least from the early tetrapod evolution. The transmembrane axolotl C beta domains have been less well conserved when compared to the mammalian C beta but they do maintain the lysine residue that is thought to be involved in the charged interaction between the TCR alpha beta heterodimer and the CD3 complex. Images Fig. 1 PMID:8341702

  3. Consistent quantitative gene product expression: #2. Antigen intensities on bone marrow cells are invariant between individuals

    PubMed Central

    Voigt, Andrew P.; Eidenschink Brodersen, Lisa; Fritschle, Wayne; Menssen, Andrew J.; Meshinchi, Soheil; Wells, Denise A.

    2016-01-01

    Abstract Five reference populations in bone marrow specimens were identified by flow cytometry using specific combinations of reagents in order define the variation of gene product expression intensities both within and between individuals. Mature lymphocytes, uncommitted progenitor cells, promyelocytes, mature monocytes and mature neutrophils can be reproducibly identified as distinct clusters of events in heterogeneous, maturing bone marrow specimens. Support Vector Machines were used to identify the reference populations in order to reduce subjective bias in manually defining boundaries of these populations since they were not discretely separated from the remainder of the cells. Reference populations were identified in 50 randomly selected bone marrow aspirates obtained over a period spanning 3 years and 6 months from pediatric patients following chemotherapy for acute myeloid leukemia (AML). The quantitative expression of gene products (cell surface antigens) and light scattering characteristics on these stressed specimens were demonstrated to be tightly regulated both within individuals and between individuals. Within an individual most gene products (CD45, CD34, CD14, CD16, CD64, CD33) demonstrated limited variability with a standard deviation of <0.20 log units while CD13 and CD36 exhibited broader variation >0.25 log units. Surprisingly, with the exception of CD33, the variation of the mean intensities of each antigen between individuals was even less than the variation within an individual. These data confirm that the amounts of gene products expressed on normal developing cells are highly regulated but differ in intensities between different lineages and during the maturational pathway of those lineages. The amounts of gene products expressed at specific stages of development of each lineage are a biologic constant with minimal variation within or between individuals. © 2016 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of

  4. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio )

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  5. T Cell Receptor-Independent Basal Signaling via Erk and Abl Kinases Suppresses RAG Gene Expression

    PubMed Central

    Roose, Jeroen P; Diehn, Maximilian; Tomlinson, Michael G; Lin, Joseph; Alizadeh, Ash A; Botstein, David; Brown, Patrick O

    2003-01-01

    Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation. PMID:14624253

  6. Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

    NASA Technical Reports Server (NTRS)

    Chen, J.; Fabry, B.; Schiffrin, E. L.; Wang, N.; Ingber, D. E. (Principal Investigator)

    2001-01-01

    A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

  7. The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens.

    PubMed

    Lindsten, T; Yaffe, L J; Thompson, C B; Guelde, G; Berning, A; Scher, I; Kenny, J J

    1985-05-01

    Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.

  8. Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses.

    PubMed

    Wang, Xiaoquan; Ilyushina, Natalia A; Lugovtsev, Vladimir Y; Bovin, Nicolai V; Couzens, Laura K; Gao, Jin; Donnelly, Raymond P; Eichelberger, Maryna C; Wan, Hongquan

    2017-01-15

    Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the United States in recent years, originated from human seasonal H3N2 viruses that were introduced into North American swine in the mid-1990s, but they are antigenically distinct from both the ancestral and current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 156, 158, 189, and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype to that of BJ/92. Mutations at residues 156, 157, 158, 189, and 193 of BJ/92 HA to amino acids present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/10. The HA amino acid substitutions responsible for switching the antigenic phenotype also impacted HA binding to sialyl receptors that are usually present in the human respiratory tract. Our study demonstrates that antigenic site B residues play a critical role in determining both the unique antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate future surveillance and risk assessment of novel influenza viruses.

  9. Structure of the human histamine H1 receptor gene.

    PubMed Central

    De Backer, M D; Loonen, I; Verhasselt, P; Neefs, J M; Luyten, W H

    1998-01-01

    Histamine H1 receptor expression has been reported to change in disorders such as allergic rhinitis, autoimmune myocarditis, rheumatoid arthritis and atherosclerosis. Here we report the isolation and characterization of genomic clones containing the 5' flanking (regulatory) region of the human histamine H1 receptor gene. An intron of approx. 5.8 kb was identified in the 5' untranslated region, which suggests that an entire subfamily of G-protein-coupled receptors may contain an intron immediately upstream of the start codon. The transcription initiation site was mapped by 5' rapid amplification of cDNA ends to a region 6.2 kb upstream of the start codon. Immediately upstream of the transcription start site a fragment of 1.85 kb was identified that showed promoter activity when placed upstream of a luciferase reporter gene and transiently transfected into cells expressing the histamine H1 receptor. The promoter sequence shares a number of characteristics with the promoter sequences of other G-protein-coupled receptor encoding genes, including binding sites for several transcription factors, and the absence of TATA and CAAT sequences at the appropriate locations. The promoter sequence described here differs from that reported previously [Fukui, Fujimoto, Mizuguchi, Sakamoto, Horio, Takai, Yamada and Ito (1994) Biochem. Biophys. Res. Commun. 201, 894-901] because the reported genomic clone was chimaeric. Furthermore our study provides evidence that the 3' untranslated region of the H1 receptor mRNA is much longer than previously accepted. Together, these findings provide a complete view of the structure of the human histamine H1 receptor gene. Both the coding region of the H1 receptor gene and its promoter region were independently mapped to chromosome 3p25. PMID:9794809

  10. Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation

    PubMed Central

    Kouzel, Nadzeya; Oldewurtel, Enno R.

    2015-01-01

    ABSTRACT Extracellular DNA is an important structural component of many bacterial biofilms. It is unknown, however, to which extent external DNA is used to transfer genes by means of transformation. Here, we quantified the acquisition of multidrug resistance and visualized its spread under selective and nonselective conditions in biofilms formed by Neisseria gonorrhoeae. The density and architecture of the biofilms were controlled by microstructuring the substratum for bacterial adhesion. Horizontal transfer of antibiotic resistance genes between cocultured strains, each carrying a single resistance, occurred efficiently in early biofilms. The efficiency of gene transfer was higher in early biofilms than between planktonic cells. It was strongly reduced after 24 h and independent of biofilm density. Pilin antigenic variation caused a high fraction of nonpiliated bacteria but was not responsible for the reduced gene transfer at later stages. When selective pressure was applied to dense biofilms using antibiotics at their MIC, the double-resistant bacteria did not show a significant growth advantage. In loosely connected biofilms, the spreading of double-resistant clones was prominent. We conclude that multidrug resistance readily develops in early gonococcal biofilms through horizontal gene transfer. However, selection and spreading of the multiresistant clones are heavily suppressed in dense biofilms. IMPORTANCE Biofilms are considered ideal reaction chambers for horizontal gene transfer and development of multidrug resistances. The rate at which genes are exchanged within biofilms is unknown. Here, we quantified the acquisition of double-drug resistance by gene transfer between gonococci with single resistances. At early biofilm stages, the transfer efficiency was higher than for planktonic cells but then decreased with biofilm age. The surface topography affected the architecture of the biofilm. While the efficiency of gene transfer was independent of the

  11. Engineering NK Cells Modified With an EGFRvIII-specific Chimeric Antigen Receptor to Overexpress CXCR4 Improves Immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma.

    PubMed

    Müller, Nadja; Michen, Susanne; Tietze, Stefanie; Töpfer, Katrin; Schulte, Alexander; Lamszus, Katrin; Schmitz, Marc; Schackert, Gabriele; Pastan, Ira; Temme, Achim

    2015-06-01

    Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell-resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII glioblastoma cells in vitro and to established subcutaneous U87-MG tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor-engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.

  12. A Survey of ABO, Rhesus (D) Antigen and Haemoglobin Genes Variants in Oyo State, Nigeria.

    PubMed

    Omotosho, Ishiaq

    2015-12-20

    A survey of ABO and Rhesus (Rh D) antigens and variants of haemoglobin genes (HbGen) in Oyo state was carried out. This longitudinal study involved the determination of ABO and Rh(D) antigens in 3241 and HbGen in 2622 male and female adults (aged 26-65years) respectively using standard methods. 94.5% of the subjects were Rh(D) positive while 5.5% were Rh(D) negative respectively based on the detection (Positive) or absence (Negative) of Rh(D) antigen. 22.8% of the subjects had ABO blood group A, 26.4% were group B, 4.1% were group AB while 46.7% were group O. Further analysis revealed that 695 (21.4%) of the group A were Apositive while 44 (1.4%) were Anegative. 800 of these subjects (24.7%) were Bpositive while 56 (1.7%) were group Bnegative. 133 (4.1%) showed group AB out of which 125 (3.8%) were ABpositive and 8 (0.3%) were ABnegative. 1513 (46.7%) were group O out of which 1444 (44.6%) were Opositive while 69 (2.1%) were Onegative. HbGen determination showed that 1933 of the subjects (73.7%) had HbGen AA; 553 (21.1%) were AS; 119 (4.5%) were AC; 11 (0.4%) were SC while 3 subjects representing 0.1% and 0.2% each had HbGen SS and CC respectively. Although the results were similar to earlier ones; however, the need for sustained counselling towards eradication of SS genes and increased research towards identifying artificial blood substitutes was highlighted in this work. The increasing need for blood transfusion especially with the increase in various politically/communally motivated emergency situations underscores this fact.

  13. Structure and gene cluster of the O-antigen of Enterobacter cloacae G3421.

    PubMed

    Perepelov, Andrei V; Filatov, Andrei V; Wang, Min; Shashkov, Alexander S; Wang, Lei; Knirel, Yuriy A

    2016-06-02

    The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G3421 and studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. In addition, partial solvolysis with anhydrous trifluoroacetic acid was applied, which cleaved selectively the α-l-rhamnopyranosidic linkages. The following structure of the branched hexasaccharide repeating unit was established. The O-polysaccharide studied shares the β-l-Rhap-(1→4)-α-l-Rhap-(1→2)-α-l-Rhap trisaccharide fragment with the O-polysaccharide of Shigella boydii type 18. The O-antigen gene cluster of E. cloacae G3421 was sequenced. Functions of genes in the cluster, including those for glycosyltransferases, were tentatively assigned by a comparison with sequences in the available databases and found to be consistent with the O-polysaccharide structure.

  14. Structure elucidation and gene cluster characterization of the O-antigen of Escherichia coli O80.

    PubMed

    Senchenkova, Sof'ya N; Guo, Xi; Filatov, Andrei V; Perepelov, Andrei V; Liu, Bin; Shashkov, Alexander S; Knirel, Yuriy A

    2016-09-02

    Mild alkaline degradation of the lipopolysaccharide of Escherichia coli O80 afforded a polysaccharide, which was studied by sugar analysis, selective cleavage of glycosidic linkages, and (1)H and (13)C NMR spectroscopy. Solvolysis of the polysaccharide with CF3CO2H cleaved the linkages of α-Fuc and β-linked GlcNAc and GalNAc residues to give two disaccharides. The following structure of the hexasaccharide repeating unit of the O-polysaccharide was established: The polysaccharide repeat also contains a minor O-acetyl group but its position was not determined. The O-antigen gene cluster of E. coli O80 between the conserved galF and gnd genes was analyzed and found to be consistent with the O-polysaccharide structure established.

  15. Cloning, sequencing, and expression of the gene coding for an antigenic 120-kilodalton protein of Rickettsia conorii.

    PubMed Central

    Schuenke, K W; Walker, D H

    1994-01-01

    Several high-molecular-mass (above 100 kDa) antigens are recognized by sera from humans infected with spotted fever group rickettsiae and may be important stimulators of the host immune response. Molecular cloning techniques were used to make genomic Rickettsia conorii (Malish 7 strain) libraries in expression vector lambda gt11. The 120-kDa R. conorii antigen was identified by monospecific antibodies to the recombinant protein expressed on construct lambda 4-7. The entire gene DNA sequence was obtained by using this construct and two other overlapping constructs. An open reading frame of 3,068 bp with a calculated molecular mass of approximately 112 kDa was identified. Promoters and a ribosome-binding site were identified on the basis of their DNA sequence homology to other rickettsial genes and their relative positions in the sequence. The DNA coding region shares no significant homology with other spotted fever group rickettsial antigen genes (i.e., the R. rickettsii 190-, 135-, and 17-kDa antigen-encoding genes). The PCR technique was used to amplify the gene from eight species of spotted fever group rickettsiae. A 75-kDa portion of the 120-kDa antigen was overexpressed in and purified from Escherichia coli. This polypeptide was recognized by antirickettsial antibodies and may be a useful diagnostic reagent for spotted fever group rickettsioses. Images PMID:8112862

  16. The inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides-mathematical modeling.

    PubMed

    Cheng, B; Fournier, R L; Relue, P A

    2000-11-20

    Gene transcription is regulated by transcription factors that can bind to specific regions on DNA. Antigene oligonucleotides (oligos) can bind to specific regions on DNA and form a triplex with the double-stranded DNA. The triplex can competitively inhibit the binding of transcription factors and, as a result, transcription can be inhibited. A genetically structured model has been developed to quantitatively describe the inhibition of the Escherichia coli lac operon gene expression by triplex-forming oligos. The model predicts that the effect of triplex-forming oligos on the lac operon gene expression depends on their target sites. Oligonucleotides targeted to the operator are much more effective than those targeted to other regulatory sites on the lac operon. In some cases, the effect of oligo binding is similar to that of a mutation in the lac operon. The model provides insight as to the specific binding site to be targeted to achieve the most effective inhibition of gene expression. The model is also capable of predicting the oligo concentration needed to inhibit gene expression, which is in general agreement with results reported by other investigators.

  17. T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen

    PubMed Central

    1995-01-01

    Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self- reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area

  18. Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma.

    PubMed

    Chen, Cheng; Li, Kesang; Jiang, Hua; Song, Fei; Gao, Huiping; Pan, Xiaorong; Shi, Bizhi; Bi, Yanyu; Wang, Huamao; Wang, Hongyang; Li, Zonghai

    2017-04-01

    Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1(+)GPC3(-) tumor cells, but they exhibited a similar cytotoxicity against GPC3(+)ASGR1(-) and GPC3(+)ASGR1(+) HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3(+)ASGR1(+) HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3(+)ASGR1(+) HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3(+)ASGR1(+) HCC.

  19. Functional responses of human neonatal B lymphocytes to antigen receptor cross-linking and CpG DNA

    PubMed Central

    TASKER, L; MARSHALL-CLARKE, S

    2003-01-01

    Human neonates are immunologically immature and consequently are highly susceptible to infection. The cellular basis for the dysfunctional immune responses of neonates is not clear, but is likely to reflect the immaturity of both B and T cell populations. Here we have examined the ability of human cord blood B cells to respond to antigen receptor cross-linking and also to CpG containing oligodeoxynucleotides (ODN), and compared their responses with those of adult peripheral blood B cells. Antigen receptor cross-linking with soluble F(ab′)2 anti-IgM antibodies, induced HLA-DR and CD86 up-regulation and proliferation to a similar extent in adult and cord blood B cells. Both interleukin (IL)-2 and IL-4 co-stimulated anti-IgM-induced proliferation, but cord blood B cells were less sensitive than adult B cells to the co-stimulatory effects of IL-2. Antigen receptor cross-linking induced secretion of the chemokines macrophage inflammatory protein-1α (MIP-1α) and MIP-1β in adult and cord blood B cells, and secretion was enhanced by IL-2 or IL-4. CpG-ODN induced up-regulation of HLA-DR and CD86 expression and proliferation of adult and cord blood B cells, and anti-IgM and CPG-ODN synergized in the induction of proliferation. CpG-ODN also induced MIP-1α and MIP-1α secretion in adult and cord blood B cells. In addition to functional studies we examined the expression of CD62L (l-selectin), CCR7 and CXCR5. Our data show that surface expression of CD62L and CCR7 is lower on cord blood B cells than on adult B cells, suggesting that human cord blood B cells may exhibit homing defects. PMID:14632745

  20. Analysis of T cell receptor repertoire of muscle-infiltrating T lymphocytes in polymyositis. Restricted V alpha/beta rearrangements may indicate antigen-driven selection.

    PubMed Central

    Mantegazza, R; Andreetta, F; Bernasconi, P; Baggi, F; Oksenberg, J R; Simoncini, O; Mora, M; Cornelio, F; Steinman, L

    1993-01-01

    Polymyositis is an inflammatory myopathy characterized by mononuclear cell infiltration of muscle tissue. Myocytotoxic T lymphocytes have been recognized in the infiltrates, but the muscle antigen, target of the immune attack, has not been identified. Molecular characterization of the variable regions of T cell receptors (TCRs) on the infiltrating lymphocytes can be expected to provide insights into the pathogenic process. The V alpha/beta TCR repertoire was investigated by RNA-PCR in muscle biopsies from 15 polymyositis patients and 16 controls (6 Duchenne muscular dystrophy and 10 with no inflammatory or dystrophic myopathy). A variety of rearranged variable TCR genes was found in polymyositis, V alpha 1, V alpha 5, V beta 1, and V beta 15 being the most common (present in 60-100% of patients). In Duchenne muscular dystrophy patients TCR V alpha or beta rearrangements were found although no restriction was observed; no rearrangements were found in muscles from the other controls. Sequence analysis revealed the presence of the J beta 2.1 region in 90% of the V beta 15 clones studied, no random N additions in the diversity region, and a common motif within the CDR3 region. These results suggest that selection of muscle-infiltrating T lymphocytes is antigen driven in polymyositis. Images PMID:8514895

  1. The T Cell Response to the Contact Sensitizer Paraphenylenediamine Is Characterized by a Polyclonal Diverse Repertoire of Antigen-Specific Receptors

    PubMed Central

    Oakes, Theres; Popple, Amy Lee; Williams, Jason; Best, Katharine; Heather, James M.; Ismail, Mazlina; Maxwell, Gavin; Gellatly, Nichola; Dearman, Rebecca J.; Kimber, Ian; Chain, Benny

    2017-01-01

    Paraphenylenediamine (PPD) is a common component of hair dyes and black henna tattoos and can cause skin sensitization and allergic contact dermatitis (ACD). The cutaneous inflammatory reaction associated with ACD is driven by both CD4+ and CD8+ T cells. However, the characteristics of such responses with respect to clonal breadth and magnitude are poorly defined. In this study, we have characterized the in vitro recall response of peripheral blood T cells prepared from PPD-allergic individuals to a PPD–human serum albumin (HSA) conjugate (PPD–HSA). Quantitative high throughput sequencing was used to characterize the changes in the repertoire of T cell receptor (TCR) α and β genes after exposure to antigen in vitro. The PPD conjugate induced expansion of T cells carrying selected TCRs, with around 800 sequences (around 1%) being 8 or more times as abundant after culture than before. The expanded sequences showed strong skewing of V and J usage, consistent with an antigen-driven clonal expansion. The complementarity-determining region 3 sequences of the expanded TCRs could be grouped into several families of related amino acid sequence, but the overall diversity of the expanded sample was not much less than that of a random sample of the same size. The results suggest a model in which PPD–HSA conjugate stimulates a broad diversity of TCRs, with a wide range of stimulation strengths, which manifest as different degrees of in vitro expansion. PMID:28261218

  2. The T Cell Response to the Contact Sensitizer Paraphenylenediamine Is Characterized by a Polyclonal Diverse Repertoire of Antigen-Specific Receptors.

    PubMed

    Oakes, Theres; Popple, Amy Lee; Williams, Jason; Best, Katharine; Heather, James M; Ismail, Mazlina; Maxwell, Gavin; Gellatly, Nichola; Dearman, Rebecca J; Kimber, Ian; Chain, Benny

    2017-01-01

    Paraphenylenediamine (PPD) is a common component of hair dyes and black henna tattoos and can cause skin sensitization and allergic contact dermatitis (ACD). The cutaneous inflammatory reaction associated with ACD is driven by both CD4+ and CD8+ T cells. However, the characteristics of such responses with respect to clonal breadth and magnitude are poorly defined. In this study, we have characterized the in vitro recall response of peripheral blood T cells prepared from PPD-allergic individuals to a PPD-human serum albumin (HSA) conjugate (PPD-HSA). Quantitative high throughput sequencing was used to characterize the changes in the repertoire of T cell receptor (TCR) α and β genes after exposure to antigen in vitro. The PPD conjugate induced expansion of T cells carrying selected TCRs, with around 800 sequences (around 1%) being 8 or more times as abundant after culture than before. The expanded sequences showed strong skewing of V and J usage, consistent with an antigen-driven clonal expansion. The complementarity-determining region 3 sequences of the expanded TCRs could be grouped into several families of related amino acid sequence, but the overall diversity of the expanded sample was not much less than that of a random sample of the same size. The results suggest a model in which PPD-HSA conjugate stimulates a broad diversity of TCRs, with a wide range of stimulation strengths, which manifest as different degrees of in vitro expansion.

  3. pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc-receptor.

    PubMed

    Zav'yalov, V P; Abramov, V M; Cherepanov, P G; Spirina, G V; Chernovskaya, T V; Vasiliev, A M; Zav'yalova, G A

    1996-05-01

    It was found that recombinant pH6 antigen (rPsaA protein) forming virulence-associated fimbriae on the surface of Yersinia pestis at pH 6.7 in host macrophage phagolysosomes or extracellularly in abscesses such as buboes, is a novel bacterial Fc-receptor. rPsaA protein displays reactivity with human IgG1, IgG2 and IgG3 subclasses but does not react with rabbit, mouse and sheep IgG.

  4. Characterization of the Aspergillus nidulans aspnd1 gene demonstrates that the ASPND1 antigen, which it encodes, and several Aspergillus fumigatus immunodominant antigens belong to the same family.

    PubMed Central

    Calera, J A; Ovejero, M C; López-Medrano, R; Segurado, M; Puente, P; Leal, F

    1997-01-01

    For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive with serum samples from aspergilloma patients has been purified and characterized, and its coding gene (aspnd1) has been cloned and sequenced. ASPND1 is a glycoprotein with four N-glycosidically-bound sugar chains (around 2.1 kDa each) which are not necessary for reactivity with immune human sera. The polypeptide part is synthesized as a 277-amino-acid precursor of 30.6 kDa that after cleavage of a putative signal peptide of 16 amino acids, affords a mature protein of 261 amino acids with a molecular mass of 29 kDa and a pI of 4.24 (as deduced from the sequence). The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen. All of the cysteine residues and most of the glycosylation sites are perfectly conserved in the three proteins, suggesting a similar but yet unknown function. Analysis of the primary structure of the ASPND1 coding gene (aspnd1) has allowed the establishment of a clear relationship between several previously reported A. fumigatus and A. nidulans immunodominant antigens. PMID:9119471

  5. Androgen receptor gene mutation, rearrangement, polymorphism

    PubMed Central

    Eisermann, Kurtis; Wang, Dan; Jing, Yifeng; Pascal, Laura E.

    2013-01-01

    Genetic aberrations of the androgen receptor (AR) caused by mutations, rearrangements, and polymorphisms result in a mutant receptor that has varied functions compared to wild type AR. To date, over 1,000 mutations have been reported in the AR with most of these being associated with androgen insensitivity syndrome (AIS). While mutations of AR associated with prostate cancer occur less often in early stage localized disease, mutations in castration-resistant prostate cancer (CRPC) patients treated with anti-androgens occur more frequently with 10-30% of these patients having some form of mutation in the AR. Resistance to anti-androgen therapy usually results from gain-of-function mutations in the LBD such as is seen with bicalutamide and more recently with enzalutamide (MDV3100). Thus, it is crucial to investigate these new AR mutations arising from drug resistance to anti-androgens and other small molecule pharmacological agents. PMID:25045626

  6. BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems.

    PubMed

    Patnaik, Santosh Kumar; Helmberg, Wolfgang; Blumenfeld, Olga O

    2012-01-01

    Analogous to human leukocyte antigens, blood group antigens are surface markers on the erythrocyte cell membrane whose structures differ among individuals and which can be serologically identified. The Blood Group Antigen Gene Mutation Database (BGMUT) is an online repository of allelic variations in genes that determine the antigens of various human blood group systems. The database is manually curated with allelic information collated from scientific literature and from direct submissions from research laboratories. Currently, the database documents sequence variations of a total of 1251 alleles of all 40 gene loci that together are known to affect antigens of 30 human blood group systems. When available, information on the geographic or ethnic prevalence of an allele is also provided. The BGMUT website also has general information on the human blood group systems and the genes responsible for them. BGMUT is a part of the dbRBC resource of the National Center for Biotechnology Information, USA, and is available online at http://www.ncbi.nlm.nih.gov/projects/gv/rbc/xslcgi.fcgi?cmd=bgmut. The database should be of use to members of the transfusion medicine community, those interested in studies of genetic variation and related topics such as human migrations, and students as well as members of the general public.

  7. Mouse T-cell receptor variable gene segment families

    SciTech Connect

    Arden, B.; Kabelitz, D.; Clark, S.P.; Mak, T.W.

    1995-10-01

    All mouse T-cell receptor {alpha}/{delta}, {beta}, and {gamma} variable (Tcra/d-, b-, and g-V) gene segments were aligned to compare the sequences with one another, to group them into subfamilies, and to derive a name which complies with the standard nomenclature. it was necessary to change the names of some V gene segments because they conflicted with those of other segments. The traditional classification into subfamilies was re-evaluated using a much larger pool of sequences. In the mouse, most V gene segments can be grouped into subfamilies of closely related genes with significantly less similarity between different subfamilies. 118 refs., 11 figs., 4 tabs.

  8. Isolation and characterization of a human sperm antigen gene h-Sp-1.

    PubMed

    Kanazawa, Ri-Ichiro; Komori, Shinji; Sakata, Kazuko; Tanaka, Hiroyuki; Sawai, Hideaki; Tsuji, Yoshiyuki; Koyama, Koji

    2003-08-01

    We isolated and characterized a human sperm antigen gene (h-Sp-1) from human testis complementary DNA using antiserum against the human sperm membrane. Northern blot analysis detected two transcripts (2.3 and 1.1 kb) of the h-Sp-1 gene. The 2.3-kb transcript is ubiquitous, whereas the 1.1-kb transcript is specific to the human testis with a high level of expression. Determination of the base sequence of h-Sp-1 showed a size of 2170 bp and 43.4% homology with human synaptophysin. The base sequence indicates a molecule consisting of 259 amino acids, with four hydrophilic and four hydrophobic regions. In order to further characterize the h-Sp-1 molecule, we synthesized the probable region of amino acids with high antigenicity based on the amino acid sequence (amino acid nos. 174-198) and immunized rabbits to prepare an antiserum. In our experimental model of fertilization between human sperm and zona pellucida-free hamster ova, partial inhibition of fertilization was observed. We were able to synthesize a large quantity of recombinant protein by inserting the h-Sp-1 gene into a baculovirus vector and infecting spodoptera frugiperda culture cells (sf9 insect cells). The synthesized protein had a molecular weight of 30 kDa. We then immunized Balb/c mice with this protein to prepare a monoclonal antibody (G3G9), which was used to localize the h-Sp-1 molecule in sperm and tissues (e.g. testis). The h-Sp-1 molecule was present in the cell membrane from the head to tail of human sperm. Staining of the testis and epididymis also showed h-Sp-1 to be present in spermatogonia, spermatocyte, sperm and epididymal duct epithelium. These findings suggest that the h-Sp-1 molecule is expressed in sperm and testes and plays a role in fertilization.

  9. Identification of a Receptor-Binding Region within Domain 4 of the Protective Antigen Component of Anthrax Toxin

    PubMed Central

    Varughese, Mini; Teixeira, Avelino V.; Liu, Shihui; Leppla, Stephen H.

    1999-01-01

    Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF are the catalytic components of the toxin, whereas PA is the receptor-binding component. To identify residues of PA that are involved in interaction with the cellular receptor, two solvent-exposed loops of domain 4 of PA (amino acids [aa] 679 to 693 and 704 to 723) were mutagenized, and the altered proteins purified and tested for toxicity in the presence of LF. In addition to the intended substitutions, novel mutations were introduced by errors that occurred during PCR. Substitutions within the large loop (aa 704 to 723) had no effect on PA activity. A mutated protein, LST-35, with three substitutions in the small loop (aa 679 to 693), bound weakly to the receptor and was nontoxic. A mutated protein, LST-8, with changes in three separate regions did not bind to receptor and was nontoxic. Toxicity was greatly decreased by truncation of the C-terminal 3 to 5 aa, but not by their substitution with nonnative residues or the extension of the terminus with nonnative sequences. Comparison of the 28 mutant proteins described here showed that the large loop (aa 704 to 722) is not involved in receptor binding, whereas residues in and near the small loop (aa 679 to 693) play an important role in receptor interaction. Other regions of domain 4, in particular residues at the extreme C terminus, appear to play a role in stabilizing a conformation needed for receptor-binding activity. PMID:10085028

  10. Effect of Genetic Diversity in Swine Leukocyte Antigen-DRA Gene on Piglet Diarrhea

    PubMed Central

    Huang, Xiaoyu; Yang, Qiaoli; Yuan, Junhu; Liu, Lixia; Sun, Wenyang; Jiang, Yingdi; Zhao, Shengguo; Zhang, Shengwei; Huang, Wangzhou; Gun, Shuangbao

    2016-01-01

    The swine leukocyte antigens (SLAs) are the multigene families related to immune responses. Little is known about the effect of the DRA gene on diarrheal disease. This study reported the genetic diversity of the DRA gene in exons 1, 3 and 4 in 290 Chinese Yantai black pigs. No variation was identified in exon 3. In exon 1, three genotypes and two alleles were identified, generated by two single nucleotide polymorphisms (SNPs). In exon 4, there were eight genotypes and five alleles containing seven SNPs were detected with four SNPs being novel SNPs. The low polymorphism found in swine DRA is consistent with the concept that the DRA gene is highly conserved among all mammalian species. Statistical analyses indicated that the genotypes of exon 1 were not significantly associated with piglet diarrhea (p > 0.05); however, genotypes C4C4 (1.80 ± 0.33) and A4E4 (1.66 ± 0.25) of exon 4 were significantly susceptible to diarrhea (p < 0.01). These indicate that the particular genotypes of the DRA gene are susceptible to diarrheal disease, which provides valuable information for disease-resistance breeding in swine. PMID:27429004

  11. Suppression of colitis by adoptive transfer of helminth antigen-treated dendritic cells requires interleukin-4 receptor-α signaling

    PubMed Central

    Matisz, C. E.; Faz-López, B.; Thomson, E.; Al Rajabi, A.; Lopes, F.; Terrazas, L. I.; Wang, A.; Sharkey, K. A.; McKay, D. M.

    2017-01-01

    Infection with helminth parasites has been explored as a treatment for autoimmune and inflammatory diseases. As helminth antigens have potent immunomodulation properties capable of inducing regulatory programs in a variety of cell types, transferring cells treated with helminth antigens represents a novel extension to helminth therapy. Previous work determined that transfer of bone marrow-derived dendritic cells (DC) pulsed with a crude extract of the tapeworm Hymenolepis diminuta (HD) can suppress colitis in recipient mice. The present study explored the mechanism of disease suppression and the importance of interleukin (IL)-4 signaling. Transfer of HD-DCs suppressed dinitrobenzene sulfonic acid (DNBS)-induced colitis through activation of recipient IL-4 receptor-α. The transferred HD-DCs required IL-4Rα and the capacity to secrete IL-10 to drive IL-4 and IL-10 production and to suppress colitis in recipient mice. Treatment of DCs with IL-4 evokes an alternatively activated phenotype, but adoptive transfer of these cells did not affect the outcome of colitis. Collectively, these studies demonstrate the complexity between IL-4 and IL-10 in donor cells and recipient, and the requirement for parasite- and host-derived factors in this novel form of cell therapy. Thus IL-4Rα signaling is revealed as a pathway that could be exploited for helminth antigen cell-based therapy. PMID:28094779

  12. The Novel Toll-Like Receptor 2 Agonist SUP3 Enhances Antigen Presentation and T Cell Activation by Dendritic Cells

    PubMed Central

    Guo, Xueheng; Wu, Ning; Shang, Yingli; Liu, Xin; Wu, Tao; Zhou, Yifan; Liu, Xin; Huang, Jiaoyan; Liao, Xuebin; Wu, Li

    2017-01-01

    Dendritic cells (DCs) are highly specialized antigen-presenting cells that play crucial roles in innate and adaptive immunity. Previous studies suggested that Toll-like receptor (TLR) agonists could be used as potential adjuvants, as activation of TLRs can boost DC-induced immune responses. TLR2 agonists have been shown to enhance DC-mediated immune responses. However, classical TLR2 agonists such as Pam3CSK4 are not stable enough in vivo, which limits their clinical applications. In this study, a novel structurally stable TLR2 agonist named SUP3 was designed. Functional analysis showed that SUP3 induced much stronger antitumor response than Pam3CSK4 by promoting cytotoxic T lymphocytes activation in vivo. This effect was achieved through the following mechanisms: SUP3 strongly enhanced the ability of antigen cross-presentation by DCs and subsequent T cell activation. SUP3 upregulated the expression of costimulatory molecules on DCs and increased antigen deposition in draining lymph nodes. More interestingly, SUP3 induced less amount of pro-inflammatory cytokine production in vivo compared to other TLR agonists such as lipopolysaccharide. Taken together, SUP3 could serve as a novel promising immune adjuvant in vaccine development and immune modulations. PMID:28270814

  13. At the Bench: Chimeric antigen receptor (CAR) T cell therapy for the treatment of B cell malignancies.

    PubMed

    Daniyan, Anthony F O; Brentjens, Renier J

    2016-12-01

    The chimeric antigen receptor (CAR) represents the epitome of cellular engineering and is one of the best examples of rational biologic design of a synthetic molecule. The CAR is a single polypeptide with modular domains, consisting of an antibody-derived targeting moiety, fused in line with T cell-derived signaling domains, allowing for T cell activation upon ligand binding. T cells expressing a CAR are able to eradicate selectively antigen-expressing tumor cells in a MHC-independent fashion. CD19, a tumor-associated antigen (TAA) present on normal B cells, as well as most B cell-derived malignancies, was an early target of this technology. Through years of experimental refinement and preclinical optimization, autologously derived CD19-targeting CAR T cells have been successfully, clinically deployed, resulting in dramatic and durable antitumor responses but not without therapy-associated toxicity. As CD19-targeted CAR T cells continue to show clinical success, work at the bench continues to be undertaken to increase further the efficacy of this therapy, while simultaneously minimizing the risk for treatment-related morbidities. In this review, we cover the history and evolution of CAR technology and its adaptation to targeting CD19. Furthermore, we discuss the future of CAR T cell therapy and the need to ask, as well as answer, critical questions as this treatment modality is being translated to the clinic.

  14. 4-1BB Costimulation Ameliorates T Cell Exhaustion Induced by Tonic Signaling of Chimeric Antigen Receptors

    PubMed Central

    Long, Adrienne H.; Haso, Waleed M.; Shern, Jack F.; Wanhainen, Kelsey M.; Murgai, Meera; Ingaramo, Maria; Smith, Jillian P.; Walker, Alec J.; Kohler, M. Eric; Venkateshwara, Vikas R.; Kaplan, Rosandra N.; Patterson, George H.; Fry, Terry J.; Orentas, Rimas J.; Mackall, Crystal L.

    2015-01-01

    Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic anti-tumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We discovered that tonic CAR CD3ζ phosphorylation, triggered by antigen-independent clustering of CAR scFvs, can induce early exhaustion of CAR T cells that limits anti-tumor efficacy. Such activation is present to varying degrees in all CARs studied, with the exception of the highly effective CD19 CAR. We further identify that CD28 costimulation augments, while 4-1BB costimulation ameliorates, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the dramatic anti-tumor effects of CD19 CARs and for the observations that CD19.BBz CAR T cells are more persistent than CD19.28z CAR T cells in clinical trials. PMID:25939063

  15. Peroxisome proliferator-activated receptor alpha target genes.

    PubMed

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  16. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    PubMed Central

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well. PMID:20936127

  17. In vivo gene transfer targeting in pancreatic adenocarcinoma with cell surface antigens

    PubMed Central

    2012-01-01

    Background Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector. Results Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down. Conclusion This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors

  18. Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: A potential therapeutic treatment

    SciTech Connect

    Taylor, R.P.; Sutherland, W.M.; Reist, C.J.; Webb, D.J.; Wright, E.L.; Labuguen, R.H. )

    1991-04-15

    The authors prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine {gamma} globulin) to human RBCs under conditions that either allow or preclude complement activation. Radioimmuno-assay analyses of this binding agree well with the number of complement receptors per RBC. In vitro whole-blood model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37C. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  19. Use of Heteropolymeric Monoclonal Antibodies to Attach Antigens to the C3b Receptor of Human Erythrocytes: A Potential Therapeutic Treatment

    NASA Astrophysics Data System (ADS)

    Taylor, Ronald P.; Sutherland, William M.; Reist, Craig J.; Webb, Donna J.; Wright, Eleanor L.; Labuguen, Ronald H.

    1991-04-01

    We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine γ globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37^circC. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  20. SH3P7 is a cytoskeleton adapter protein and is coupled to signal transduction from lymphocyte antigen receptors.

    PubMed

    Larbolette, O; Wollscheid, B; Schweikert, J; Nielsen, P J; Wienands, J

    1999-02-01

    Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

  1. Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

    PubMed

    Johnson, Laura A; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R; McGettigan, Shannon E; Nace, Arben K; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C; Cogdill, Alexandria P; Chen, Taylor; Fraietta, Joseph A; Kloss, Christopher C; Posey, Avery D; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

    2015-02-18

    Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

  2. Allelic association of the D2 dopamine receptor gene with receptor-binding characteristics in alcoholism

    SciTech Connect

    Noble, E.P.; Blum, K.; Ritchie, T.; Montgomery, A.; Sheridan, P.J. )

    1991-07-01

    The allelic association of the human D2 dopamine receptor gene with the binding characteristics of the D2 dopamine receptor was determined in 66 brains of alcoholic and non-alcoholic subjects. In a blinded experiment, DNA from the cerebral cortex was treated with the restriction endonuclease Taql and probed with a 1.5-kilobase (kb) digest of a clone (lambda hD2G1) of the human D2 dopamine receptor gene. The binding characteristics (Kd (binding affinity) and Bmax (number of binding sites)) of the D2 dopamine receptor were determined in the caudate nuclei of these brains using tritiated spiperone as the ligand. The adjusted Kd was significantly lower in alcoholic than in nonalcoholic subjects. In subjects with the A1 allele, in whom a high association with alcoholism was found, the Bmax was significantly reduced compared with the Bmax of subjects with the A2 allele. Moreover, a progressively reduced Bmax was found in subjects with A2/A2, A1/A2, and A1/A1 alleles, with subjects with A2/A2 having the highest mean values, and subjects with A1/A1, the lowest. The polymorphic pattern of the D2 dopamine receptor gene and its differential expression of receptors suggests the involvement of the dopaminergic system in conferring susceptibility to at least one subtype of severe alcoholism.

  3. Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.

    PubMed Central

    Evans, C M; Fryer, A D; Jacoby, D B; Gleich, G J; Costello, R W

    1997-01-01

    In antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs. PMID:9410903

  4. Altered transcription of genes coding for class I histocompatibility antigens in murine tumor cells

    PubMed Central

    1983-01-01

    Three murine tumors induced by Moloney murine leukemia virus (M-MLV) which exhibited loss of some or all H-2 class I antigens at the cell surface were analyzed at the DNA and RNA level with molecular probes specific of H-2 heavy chains and beta 2-microglobulin sequences. No observable difference could be detected at the DNA level between the tumors and the parent animals. However, a decrease in H-2 mRNA was observed, especially in phenotypically H-2 negative tumor, BM5R, where H-2 transcripts were at least 30-fold less abundant. These results show that an H-2-negative character may result from a general alteration in the transcription of H-2 genes, which could reflect some kind of regulatory process. PMID:6311935

  5. Crosstalk between ABO and Forssman (FORS) blood group systems: FORS1 antigen synthesis by ABO gene-encoded glycosyltransferases

    PubMed Central

    Yamamoto, Miyako; Cid, Emili; Yamamoto, Fumiichiro

    2017-01-01

    A and B alleles at the ABO genetic locus specify A and B glycosyltransferases that catalyze the biosynthesis of A and B oligosaccharide antigens, respectively, of blood group ABO system which is important in transfusion and transplantation medicine. GBGT1 gene encodes Forssman glycolipid synthase (FS), another glycosyltransferase that produces Forssman antigen (FORS1). Humans are considered to be Forssman antigen-negative species without functional FS. However, rare individuals exhibiting Apae phenotype carry a dominant active GBGT1 gene and express Forssman antigen on RBCs. Accordingly, FORS system was recognized as the 31st blood group system. Mouse ABO gene encodes a cis-AB transferase capable of producing both A and B antigens. This murine enzyme contains the same GlyGlyAla tripeptide sequence as FSs at the position important for the determination of sugar specificity. We, therefore, transfected the expression construct into appropriate recipient cells and examined whether mouse cis-AB transferase may also exhibit FS activity. The result was positive, confirming the crosstalk between the ABO and FORS systems. Further experiments have revealed that the introduction of this tripeptide sequence to human A transferase conferred some, although weak, FS activity, suggesting that it is also involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugars. PMID:28134301

  6. Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen

    PubMed Central

    Real, Catherine Isabell; Lu, Mengji; Liu, Jia; Huang, Xuan; Trippler, Martin; Hossbach, Markus; Deckert, Jochen; Jahn-Hofmann, Kerstin; Ickenstein, Ludger Markus; John, Matthias Johannes; Gibbert, Kathrin; Dittmer, Ulf; Vornlocher, Hans-Peter; Schirmbeck, Reinhold; Gerken, Guido; Schlaak, Joerg Friedrich; Broering, Ruth

    2016-01-01

    The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV. PMID:27121087

  7. Antibodies to the GABAB receptor in limbic encephalitis with seizures: case series and characterisation of the antigen

    PubMed Central

    Lancaster, Eric; Lai, Meizan; Peng, Xiaoyu; Hughes, Ethan; Constantinescu, Radu; Raizer, Jeffrey; Friedman, Daniel; Skeen, Mark B; Grisold, Wolfgang; Kimura, Akio; Ohta, Kouichi; Iizuka, Takahiro; Guzman, Miguel; Graus, Francesc; Moss, Stephen J; Balice-Gordon, Rita; Dalmau, Josep

    2010-01-01

    Summary Background Some encephalitides or seizure disorders once thought idiopathic now seem to be immune mediated. We aimed to describe the clinical features of one such disorder and to identify the autoantigen involved. Methods 15 patients who were suspected to have paraneoplastic or immune-mediated limbic encephalitis were clinically assessed. Confocal microscopy, immunoprecipitation, and mass spectrometry were used to characterise the autoantigen. An assay of HEK293 cells transfected with rodent GABAB1 or GABAB2 receptor subunits was used as a serological test. 91 patients with encephalitis suspected to be paraneoplastic or immune mediated and 13 individuals with syndromes associated with antibodies to glutamic acid decarboxylase 65 were used as controls. Findings All patients presented with early or prominent seizures; other symptoms, MRI, and electroencephalography findings were consistent with predominant limbic dysfunction. All patients had antibodies (mainly IgG1) against a neuronal cell-surface antigen; in three patients antibodies were detected only in CSF. Immunoprecipitation and mass spectrometry showed that the antibodies recognise the B1 subunit of the GABAB receptor, an inhibitory receptor that has been associated with seizures and memory dysfunction when disrupted. Confocal microscopy showed colocalisation of the antibody with GABAB receptors. Seven of 15 patients had tumours, five of which were small-cell lung cancer, and seven patients had non-neuronal autoantibodies. Although nine of ten patients who received immunotherapy and cancer treatment (when a tumour was found) showed neurological improvement, none of the four patients who were not similarly treated improved (p=0.005). Low levels of GABAB1 receptor antibodies were identified in two of 104 controls (p<0.0001). Interpretation GABAB receptor autoimmune encephalitis is a potentially treatable disorder characterised by seizures and, in some patients, associated with small-cell lung cancer and

  8. Translational control of beta2-adrenergic receptor mRNA by T-cell-restricted intracellular antigen-related protein.

    PubMed

    Kandasamy, Karthikeyan; Joseph, Kusumam; Subramaniam, Kothandharaman; Raymond, John R; Tholanikunnel, Baby G

    2005-01-21

    Cellular expression of the beta(2)-adrenergic receptor (beta(2)-AR) is suppressed at the translational level by 3'-untranslated region (UTR) sequences. To test the possible role of 3'-UTR-binding proteins in translational suppression of beta(2)-AR mRNA, we expressed the full-length 3'-UTR or the adenylate/uridylate-rich (A+U-rich element (ARE)) RNA from the 3'-UTR sequences of beta(2)-AR in cell lines that endogenously express this receptor. Reversal of beta(2)-adrenergic receptor translational repression by retroviral expression of 3'-UTR sequences suggested that ARE RNA-binding proteins are involved in translational suppression of beta(2)-adrenergic receptor expression. Using a 20-nucleotide ARE RNA from the receptor 3'-UTR as an affinity ligand, we purified the proteins that bind to these sequences. T-cell-restricted intracellular antigen-related protein (TIAR) was one of the strongly bound proteins identified by this method. UV-catalyzed cross-linking experiments using in vitro transcribed 3'-UTR RNA and glutathione S-transferase-TIAR demonstrated multiple binding sites for this protein on beta(2)-AR 3'-UTR sequences. The distal 340-nucleotide region of the 3'-UTR was identified as a target RNA motif for TIAR binding by both RNA gel shift analysis and immunoprecipitation experiments. Overexpression of TIAR resulted in suppression of receptor protein synthesis and a significant shift in endogenously expressed beta(2)-AR mRNA toward low molecular weight fractions in sucrose gradient polysome fractionation. Taken together, our results provide the first evidence for translational control of beta(2)-AR mRNA by TIAR.

  9. Improving antigenic peptide vaccines for cancer immunotherapy using a dominant tumor-specific T cell receptor.

    PubMed

    Buhrman, Jonathan D; Jordan, Kimberly R; Munson, Daniel J; Moore, Brandon L; Kappler, John W; Slansky, Jill E

    2013-11-15

    Vaccines that incorporate peptide mimics of tumor antigens, or mimotope vaccines, are commonly used in cancer immunotherapy and function by eliciting increased numbers of T cells that cross-react with the native tumor antigen. Unfortunately, they often elicit T cells that do not cross-react with or that have low affinity for the tumor antigen. Using a high affinity tumor-specific T cell clone, we identified a panel of mimotope vaccines for the dominant peptide antigen from a mouse colon tumor that elicits a range of tumor protection following vaccination. The TCR from this high affinity T cell clone was rarely identified in ex vivo evaluation of tumor-specific T cells elicited by mimotope vaccination. Conversely, a low affinity clone found in the tumor and following immunization was frequently identified. Using peptide libraries, we determined if this frequently identified TCR improved the discovery of efficacious mimotopes. We demonstrated that the representative TCR identified more protective mimotopes than the high affinity TCR. These results suggest that targeting a dominant fraction of tumor-specific T cells generates potent immunity and that consideration of the available T cell repertoire is necessary for targeted T cell therapy. These results have important implications when optimizing mimotope vaccines for cancer immunotherapy.

  10. Chimeric Antigen Receptors to CD276 for Treating Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    This licensing opportunity from the National Cancer Institute concerns the development of CARs comprising an antigen-binding fragment derived from the MGA271 antibody. The resulting CARs can be used in adoptive cell therapy treatment for neuroblastoma and other tumors that express CD276.

  11. Selection for Genes Encoding Secreted Proteins and Receptors

    NASA Astrophysics Data System (ADS)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  12. Characterization of the "CCR5" Chemokine Receptor Gene

    ERIC Educational Resources Information Center

    Thomas, John C.

    2004-01-01

    The life cycle of retroviruses is an essential topic of modern cell biology instruction. Furthermore, the process of HIV viral entry into the cell is a question of great interest in basic and clinical biology. This paper describes how students can easily recover their own DNA, amplify a portion of the "CCR5" chemokine receptor gene, characterize…

  13. Gene for the major antigenic structural protein (p100) of human herpesvirus 6.

    PubMed Central

    Neipel, F; Ellinger, K; Fleckenstein, B

    1992-01-01

    A human herpesvirus 6 (HHV-6) structural protein of 100 kDa (p100) is the polypeptide most frequently and intensively reactive in immunoblotting analyses with human sera on HHV-6-infected cells or partially purified virions. The gene for p100 was identified by screening a bacteriophage lambda library with monospecific rabbit antisera. The gene codes for a polypeptide of 870 amino acids with a calculated molecular size of 97 kDa. Its amino-terminal third is weakly homologous to the immunogenic basic matrix phosphoprotein pp150 of human cytomegalovirus. Five fragments representing more than 93% of HHV-6 p100 were prokaryotically expressed. The antigenic epitopes of p100 were preliminary mapped by immunoblotting with human sera. They are located within the carboxy-terminal part which is neither homologous nor cross-reactive to pp150 of human cytomegalovirus. Availability of the gene for the immunodominant structural protein should provide tools for studies of pathogenesis by HHV-6. Images PMID:1374813

  14. Pasteurella haemolytica serotype 2 contains the gene for a noncapsular serotype 1-specific antigen.

    PubMed Central

    Gonzalez, C T; Maheswaran, S K; Murtaugh, M P

    1995-01-01

    An ssa1-homologous genomic fragment cloned from Pasteurella haemolytica serotype 2 (ST2) enabled transformation of Escherichia coli DH5 alpha to a serotype 1 (ST1) phenotype through expression of the ST1-specific antigen (Ssa1). The Ssa1 protein expressed by ssa1-transformed E. coli was susceptible to heat and protease treatment and was distinct from P. haemolytica ST1-specific capsular polysaccharide. Electrophoretic analysis of in vitro-translated proteins, as well as the predicted amino acid sequence, demonstrated that Ssa1 proteins encoded from either ST1- or ST2-derived ssa1 genes were essentially identical. A comparison of the nucleotide sequences of ssa1 genes derived from P. haemolytica ST1 and ST2 revealed greater than 99% homology. Amino acid sequence homology of the predicted products of ST1- and ST2-derived ssa1 genes was greater than 98%. Northern (RNA) blot studies revealed that the presence of an increased level of ssa1 transcript in P. haemolytica ST1 grown as surface-adherent cultures on solid medium was correlated with a serologically detectable Ssa1 protein. Expression of the ssa1 transcript in ST1 was similarly upregulated by a high iron concentration in the growth medium. PMID:7890392

  15. T-cell receptor analysis in Omenn's syndrome: evidence for defects in gene rearrangement and assembly.

    PubMed

    Brooks, E G; Filipovich, A H; Padgett, J W; Mamlock, R; Goldblum, R M

    1999-01-01

    Patients with Omenn's syndrome have a form of severe immune deficiency that is associated with pathological features of graft-versus-host disease, except for the lack of foreign engraftment. It has been hypothesized that the disease's unique clinical features are mediated by an expanded population of autologous self-reactive T cells of limited clonality. In the current study, an investigation of the T-cell receptor (TCR) repertoire was undertaken to identify defects in T-cell rearrangement and development. The TCR repertoire in this group of patients was exquisitely restricted in the number of different TCR clonotypes, and some of these clonotypes seemed to have similar recognition motifs in the antigen-binding region, indicating antigen-driven proliferation of T lymphocytes. The TCRs from some patients lacked N- or P-nucleotide insertions and used proximal variable and joining gene segments, suggesting abnormal intrathymic T-cell development. Finally, abnormal assembly of gene segments and truncated rearrangements within nonproductive alleles suggested abnormalities in TCR rearrangement mechanisms. Overall, the findings suggest that inefficient and/or abnormal generation of TCRs may be a consistent feature of this disease.

  16. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases

    PubMed Central

    Shiu, Shin-Han; Bleecker, Anthony B.

    2001-01-01

    Plant receptor-like kinases (RLKs) are proteins with a predicted signal sequence, single transmembrane region, and cytoplasmic kinase domain. Receptor-like kinases belong to a large gene family with at least 610 members that represent nearly 2.5% of Arabidopsis protein coding genes. We have categorized members of this family into subfamilies based on both the identity of the extracellular domains and the phylogenetic relationships between the kinase domains of subfamily members. Surprisingly, this structurally defined group of genes is monophyletic with respect to kinase domains when compared with the other eukaryotic kinase families. In an extended analysis, animal receptor kinases, Raf kinases, plant RLKs, and animal receptor tyrosine kinases form a well supported group sharing a common origin within the superfamily of serine/threonine/tyrosine kinases. Among animal kinase sequences, Drosophila Pelle and related cytoplasmic kinases fall within the plant RLK clade, which we now define as the RLK/Pelle family. A survey of expressed sequence tag records for land plants reveals that mosses, ferns, conifers, and flowering plants have similar percentages of expressed sequence tags representing RLK/Pelle homologs, suggesting that the size of this gene family may have been close to the present-day level before the diversification of land plant lineages. The distribution pattern of four RLK subfamilies on Arabidopsis chromosomes indicates that the expansion of this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. PMID:11526204

  17. Adhesion of immunomagnetic particles targeted to antigens and cytokine receptors on tumor cells determined by magnetophoresis

    NASA Astrophysics Data System (ADS)

    Tchikov, V.; Winoto-Morbach, S.; Kabelitz, D.; Krönke, M.; Schütze, S.

    2001-01-01

    The adhesion of immunomagnetic particles to surfaces of target cells based on ligand-receptor interactions was evaluated by counting the number of magnetically labeled cells using magnetophoresis and analyzed by the Langmuir adsorption theory. As few as 200 tumor necrosis factor (TNF) receptor molecules per cell could be detected with streptavidin-coated magnetic particles coupled to biotinylated TNF as ligand.

  18. Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities

    PubMed Central

    Van Caeneghem, Yasmine; De Munter, Stijn; Tieppo, Paola; Goetgeluk, Glenn; Weening, Karin; Verstichel, Greet; Bonte, Sarah; Taghon, Tom; Leclercq, Georges; Kerre, Tessa; Debets, Reno; Vermijlen, David; Abken, Hinrich; Vandekerckhove, Bart

    2017-01-01

    ABSTRACT Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

  19. Interspecies variations in Bordetella catecholamine receptor gene regulation and function.

    PubMed

    Brickman, Timothy J; Suhadolc, Ryan J; Armstrong, Sandra K

    2015-12-01

    Bordetella bronchiseptica can use catecholamines to obtain iron from transferrin and lactoferrin via uptake pathways involving the BfrA, BfrD, and BfrE outer membrane receptor proteins, and although Bordetella pertussis has the bfrD and bfrE genes, the role of these genes in iron uptake has not been demonstrated. In this study, the bfrD and bfrE genes of B. pertussis were shown to be functional in B. bronchiseptica, but neither B. bronchiseptica bfrD nor bfrE imparted catecholamine utilization to B. pertussis. Gene fusion analyses found that expression of B. bronchiseptica bfrA was increased during iron starvation, as is common for iron receptor genes, but that expression of the bfrD and bfrE genes of both species was decreased during iron limitation. As shown previously for B. pertussis, bfrD expression in B. bronchiseptica was also dependent on the BvgAS virulence regulatory system; however, in contrast to the case in B. pertussis, the known modulators nicotinic acid and sulfate, which silence Bvg-activated genes, did not silence expression of bfrD in B. bronchiseptica. Further studies using a B. bronchiseptica bvgAS mutant expressing the B. pertussis bvgAS genes revealed that the interspecies differences in bfrD modulation are partly due to BvgAS differences. Mouse respiratory infection experiments determined that catecholamine utilization contributes to the in vivo fitness of B. bronchiseptica and B. pertussis. Additional evidence of the in vivo importance of the B. pertussis receptors was obtained from serologic studies demonstrating pertussis patient serum reactivity with the B. pertussis BfrD and BfrE proteins.

  20. Interspecies Variations in Bordetella Catecholamine Receptor Gene Regulation and Function

    PubMed Central

    Brickman, Timothy J.; Suhadolc, Ryan J.

    2015-01-01

    Bordetella bronchiseptica can use catecholamines to obtain iron from transferrin and lactoferrin via uptake pathways involving the BfrA, BfrD, and BfrE outer membrane receptor proteins, and although Bordetella pertussis has the bfrD and bfrE genes, the role of these genes in iron uptake has not been demonstrated. In this study, the bfrD and bfrE genes of B. pertussis were shown to be functional in B. bronchiseptica, but neither B. bronchiseptica bfrD nor bfrE imparted catecholamine utilization to B. pertussis. Gene fusion analyses found that expression of B. bronchiseptica bfrA was increased during iron starvation, as is common for iron receptor genes, but that expression of the bfrD and bfrE genes of both species was decreased during iron limitation. As shown previously for B. pertussis, bfrD expression in B. bronchiseptica was also dependent on the BvgAS virulence regulatory system; however, in contrast to the case in B. pertussis, the known modulators nicotinic acid and sulfate, which silence Bvg-activated genes, did not silence expression of bfrD in B. bronchiseptica. Further studies using a B. bronchiseptica bvgAS mutant expressing the B. pertussis bvgAS genes revealed that the interspecies differences in bfrD modulation are partly due to BvgAS differences. Mouse respiratory infection experiments determined that catecholamine utilization contributes to the in vivo fitness of B. bronchiseptica and B. pertussis. Additional evidence of the in vivo importance of the B. pertussis receptors was obtained from serologic studies demonstrating pertussis patient serum reactivity with the B. pertussis BfrD and BfrE proteins. PMID:26371128

  1. Immunoglobulin class switching appears to be regulated by B cell antigen receptor-specific T cell action

    PubMed Central

    Lange, Hans; Hecht, Oliver; Zemlin, Michael; Trad, Ahmad; Tanasa, Radu I.; Schroeder, Harry W.; Lemke, Hilmar

    2013-01-01

    Summary Antigen affinity is commonly viewed as the driving force behind the selection for dominant clonotypes that can occur during the T cell-dependent processes of class switch recombination (CSR) and immune maturation. To test this view, we analyzed the variable gene repertoires of natural monoclonal antibodies to the hapten 2-phenyloxazolone (phOx) as well as those generated after phOx protein carrier-induced thymus-dependent or Ficoll-induced thymus independent antigen stimulation. In contrast to expectations, the extent of IgM heterogeneity proved similar and many IgM from these three populations exhibited similar or even greater affinities than the classic Ox1 clonotype that dominates only after CSR among primary and memory IgG. The population of clones that were selected during CSR exhibited a reduced VH/VL repertoire that was enriched for variable domains with shorter and more uniform CDR-H3 lengths and almost completely stripped of variable domains encoded by the large VH1 family. Thus, contrary to the current paradigm, T-cell dependent clonal selection during CSR appeared to select for VH family and CDR-H3 loop content even when the affinity provided by alternative clones exhibited similar to increased affinity for antigen. PMID:22531925

  2. The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

    PubMed

    Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

    2015-02-01

    Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

  3. Proliferating cell nuclear antigen is a novel inhibitory ligand for the natural cytotoxicity receptor NKp44.

    PubMed

    Rosental, Benyamin; Brusilovsky, Michael; Hadad, Uzi; Oz, Dafna; Appel, Michael Y; Afergan, Fabian; Yossef, Rami; Rosenberg, Lior Ann; Aharoni, Amir; Cerwenka, Adelheid; Campbell, Kerry S; Braiman, Alex; Porgador, Angel

    2011-12-01

    NK cells play an important role in the early immune response to cancer. The NKp44 activating receptor is the only natural cytotoxicity receptor that is expressed exclusively by primate NK cells, yet its cellular ligands remain largely unknown. Proliferating cell nuclear Ag (PCNA) is overexpressed in cancer cells. In this study, we show that the NKp44 receptor recognizes PCNA. Their interaction inhibits NK cell function through NKp44/ITIM. The physical interaction of NKp44 and PCNA is enabled by recruitment of target cell PCNA to the NK immunological synapse. We demonstrate that PCNA promotes cancer survival by immune evasion through inhibition of NKp44-mediated NK cell attack.

  4. A protective antigen mutation increases the pH threshold of anthrax toxin receptor 2-mediated pore formation.

    PubMed

    Dennis, Melissa K; Mogridge, Jeremy

    2014-04-08

    Anthrax toxin protective antigen (PA) binds cellular receptors and self-assembles into oligomeric prepores. A prepore converts to a protein translocating pore after it has been transported to an endosome where the low pH triggers formation of a membrane-spanning β-barrel channel. Formation of this channel occurs after some PA-receptor contacts are broken to allow pore formation, while others are retained to preserve receptor association. The interaction between PA and anthrax toxin receptor 1 (ANTXR1) is weaker than its interaction with ANTXR2 such that the pH threshold of ANTXR1-mediated pore formation is higher by 1 pH unit. Here we examine receptor-specific differences in toxin binding and pore formation by mutating PA residue G342 that selectively abuts ANTXR2. Mutation of G342 to valine, leucine, isoleucine, or tryptophan increased the amount of PA bound to ANTXR1-expressing cells and decreased the amount of PA bound to ANTXR2-expressing cells. The more conservative G342A mutation did not affect the level of binding to ANTXR2, but ANTXR2-bound PA-G342A prepores exhibited a pH threshold higher than that of wild-type prepores. Mixtures of wild-type PA and PA-G342A were functional in toxicity assays, and the pH threshold of ANTXR2-mediated pore formation was dictated by the relative amounts of the two proteins in the hetero-oligomers. These results suggest that PA subunits within an oligomer do not have to be triggered simultaneously for a productive membrane insertion event to occur.

  5. Anti-colorectal cancer effect of interleukin-2 and interferon-β fusion gene driven by carcinoembryonic antigen promoter

    PubMed Central

    Wang, Yan; Wang, Mengchun; Li, Yan

    2016-01-01

    This study was designed to investigate the antitumor effects of combined interleukin-2/interferon-β-based gene therapy in colorectal cancer. Transfection of the fusion gene expression plasmid induced significant apoptosis of Lovo cells. Additionally, the fusion gene exhibited strong inhibitory activity against tumor growth and apoptosis when being injected into the nude mice implanted with human colon cancer cells. Furthermore, the tail-vein injection showed a more notable effect than direct injection into tumor. These results suggest that the combined interleukin-2/interferon-β-based gene therapy with the carcinoembryonic antigen promoter might be an effective antitumor strategy. PMID:27313471

  6. Folate receptor gene variants and neural tube defect occurrence

    SciTech Connect

    Finnell, R.; Greer, K.; Lammer, E.

    1994-09-01

    Recent epidemiological evidence shows that periconceptional use of folic acid supplements may prevent 40-50% of neural tube defects (NTDs). The FDA has subsequently recommended folic acid supplementation of all women of childbearing potential, even though the mechanism by which folic acid prevents NTDs is unknown. We investigated genetic variation of a candidate gene, the 5-methyltetrahydrofolate (5-MeTHF) receptor, that may mediate this preventive effect. The receptor concentrates folate within cells and we have localized its mRNA to neuroepithelial cells during neurulation. Our hypothesis is that dysfunctional 5-MeTHF receptors inadequately concentrate folate intracellularly, predisposing infants to NTDs. We have completed SSCP analysis on 3 of the 4 coding exons of the 5-MeTHF receptor gene of 474 infants participating in a large population-based epidemiological case-control study of NTDs in California; genotyping of another 500 infants is ongoing. Genomic DNA was extracted from residual blood spots from newborn screening samples of cases and controls. Genotyping was done blinded to case status. Polymorphisms have been detected for exons 4 and 5; fourteen percent of the infants have exon 5 polymorphisms. Data will be presented on the prevalence of 5-MeTHF receptor polymorphisms among cases and controls. Relationships among the polymorphisms and NTD occurrence may shed light on how folic acid supplementation prevents NTDs.

  7. Functions of Cell Surface-Anchored Antigen I/II Family and Hsa Polypeptides in Interactions of Streptococcus gordonii with Host Receptors

    PubMed Central

    Jakubovics, Nicholas S.; Kerrigan, Steven W.; Nobbs, Angela H.; Strömberg, Nicklas; van Dolleweerd, Craig J.; Cox, Dermot M.; Kelly, Charles G.; Jenkinson, Howard F.

    2005-01-01

    Streptococcus gordonii colonizes multiple sites within the human oral cavity. This colonization depends upon the initial interactions of streptococcal adhesins with host receptors. The adhesins that bind salivary agglutinin glycoprotein (gp340) and human cell surface receptors include the antigen I/II (AgI/II) family polypeptides SspA and SspB and a sialic acid-binding surface protein designated Hsa or GspB. In this study we determined the relative functions of the AgI/II polypeptides and Hsa in interactions of S. gordonii DL1 (Challis) with host receptors. For an isogenic mutant with the sspA and sspB genes deleted the levels of adhesion to surface-immobilized gp340 were reduced 40%, while deletion of the hsa gene alone resulted in >80% inhibition of bacterial cell adhesion to gp340. Adhesion of S. gordonii DL1 cells to gp340 was sialidase sensitive, verifying that Hsa has a major role in mediating sialic acid-specific adhesion to gp340. Conversely, aggregation of S. gordonii cells by fluid-phase gp340 was not affected by deletion of hsa but was eliminated by deletion of the sspA and sspB genes. Deletion of the AgI/II polypeptide genes had no measurable effect on hsa mRNA levels or Hsa surface protein expression, and deletion of hsa did not affect AgI/II polypeptide expression. Further analysis of mutant phenotypes showed that the Hsa and AgI/II proteins mediated adhesion of S. gordonii DL1 to human HEp-2 epithelial cells. Hsa was also a principal streptococcal cell surface component promoting adhesion of human platelets to immobilized streptococci, but Hsa and AgI/II polypeptides acted in concert in mediating streptococcal cell-platelet aggregation. The results suggest that Hsa directs primary adhesion events for S. gordonii DL1 (Challis) with immobilized gp340, epithelial cells, and platelets. AgI/II polypeptides direct gp340-mediated aggregation, facilitate multimodal interactions necessary for platelet aggregation, and modulate S. gordonii-host engagements

  8. CRDB: Database of Chemosensory Receptor Gene Families in Vertebrate

    PubMed Central

    Wu, Xiaoli; Zhong, Yang

    2012-01-01

    Chemosensory receptors (CR) are crucial for animals to sense the environmental changes and survive on earth. The emergence of whole-genome sequences provides us an opportunity to identify the entire CR gene repertoires. To completely gain more insight into the evolution of CR genes in vertebrates, we identified the nearly all CR genes in 25 vertebrates using homology-based approaches. Among these CR gene repertoires, nearly half of them were identified for the first time in those previously uncharacterized species, such as the guinea pig, giant panda and elephant, etc. Consistent with previous findings, we found that the numbers of CR genes vary extensively among different species, suggesting an extreme form of ‘birth-and-death’ evolution. For the purpose of facilitating CR gene analysis, we constructed a database with the goals to provide a resource for CR genes annotation and a web tool for exploring their evolutionary patterns. Besides a search engine for the gene extraction from a specific chromosome region, an easy-to-use phylogenetic analysis tool was also provided to facilitate online phylogeny study of CR genes. Our work can provide a rigorous platform for further study on the evolution of CR genes in vertebrates. PMID:22393364

  9. Immunoglobulin gene repertoire in ocular adnexal lymphomas: hints on the nature of the antigenic stimulation.

    PubMed

    Dagklis, A; Ponzoni, M; Govi, S; Cangi, M G; Pasini, E; Charlotte, F; Vino, A; Doglioni, C; Davì, F; Lossos, I S; Ntountas, I; Papadaki, T; Dolcetti, R; Ferreri, A J M; Stamatopoulos, K; Ghia, P

    2012-04-01

    Evidence from certain geographical areas links lymphomas of the ocular adnexa marginal zone B-cell lymphomas (OAMZL) with Chlamydophila psittaci (Cp) infection, suggesting that lymphoma development is dependent upon chronic stimulation by persistent infections. Notwithstanding that, the actual immunopathogenetical mechanisms have not yet been elucidated. As in other B-cell lymphomas, insight into this issue, especially with regard to potential selecting ligands, could be provided by analysis of the immunoglobulin (IG) receptors of the malignant clones. To this end, we studied the molecular features of IGs in 44 patients with OAMZL (40% Cp-positive), identifying features suggestive of a pathogenic mechanism of autoreactivity. Herein, we show that lymphoma cells express a distinctive IG repertoire, with electropositive antigen (Ag)-binding sites, reminiscent of autoantibodies (auto-Abs) recognizing DNA. Additionally, five (11%) cases of OAMZL expressed IGs homologous with autoreactive Abs or IGs of patients with chronic lymphocytic leukemia, a disease known for the expression of autoreactive IGs by neoplastic cells. In contrast, no similarity with known anti-Chlamydophila Abs was found. Taken together, these results strongly indicate that OAMZL may originate from B cells selected for their capability to bind Ags and, in particular, auto-Ags. In OAMZL associated with Cp infection, the pathogen likely acts indirectly on the malignant B cells, promoting the development of an inflammatory milieu, where auto-Ags could be exposed and presented, driving proliferation and expansion of self-reactive B cells.

  10. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    SciTech Connect

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo; Kim, Myoung Hee

    2010-02-19

    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  11. A common gene pool for the Neisseria FetA antigen.

    PubMed

    Bennett, Julia S; Thompson, Emily A L; Kriz, Paula; Jolley, Keith A; Maiden, Martin C J

    2009-02-01

    Meningococcal FetA is an iron-regulated, immunogenic outer membrane protein and vaccine component. The most diverse region of this protein is a previously defined variable region (VR) that has been shown to be immunodominant. In this analysis, a total of 275 Neisseria lactamica isolates, collected during studies of nasopharyngeal bacterial carriage in infants, were examined for the presence of a fetA gene. The fetA VR nucleotide sequence was determined for 217 of these isolates, with fetA apparently absent from 58 isolates, the majority of which belonged to the ST-624 clonal complex. The VR in N. lactamica was compared to the same region in N. meningitidis, N. gonorrhoeae, and a number of other commensal Neisseria. Identical fetA variable region sequences were identified among commensal and pathogenic Neisseria, suggesting a common gene pool, differing from other antigens in this respect. Carriage of commensal Neisseria species, such as N. lactamica, that express FetA may be involved in the development of natural immunity to meningococcal disease.

  12. A common gene pool for the Neisseria FetA antigen

    PubMed Central

    Bennett, Julia S.; Thompson, Emily A. L.; Kriz, Paula; Jolley, Keith A.; Maiden, Martin C. J.

    2014-01-01

    Meningococcal FetA is an iron-regulated, immunogenic outer membrane protein and vaccine component. The most diverse region of this protein is a previously defined variable region (VR) that has been shown to be immunodominant. In this analysis, a total of 275 Neisseria lactamica isolates, collected during studies of nasopharyngeal bacterial carriage in infants were examined for the presence of a fetA gene. The fetA VR nucleotide sequence was determined for 217 of these isolates, with fetA apparently absent from 58 isolates, the majority of which belonged to the ST-624 clonal complex. The VR in N. lactamica was compared to the same region in Neisseria meningitidis, Neisseria gonorrhoeae and a number of other commensal Neisseria. Identical fetA variable region sequences were identified among commensal and pathogenic Neisseria, suggesting a common gene pool, differing from other antigens in this respect. Carriage of commensal Neisseria species, such as N. lactamica, that express FetA may be involved in the development of natural immunity to meningococcal disease. PMID:18718812

  13. An experimental and theoretical study of the inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides.

    PubMed

    Cheng, B; Fournier, R L; Relue, P A; Schisler, J

    2001-08-05

    Previously, we have developed a genetically structured mathematical model to describe the inhibition of Escherichia coli lac operon gene expression by antigene oligos. Our model predicted that antigene oligos targeted to the operator region of the lac operon would have a significant inhibitory effect on beta-galactosidase production. In this investigation, the E. coli lac operon gene expression in the presence of antigene oligos was studied experimentally. A 21-mer oligo, which was designed to form a triplex with the operator, was found to be able to specifically inhibit beta-galactosidase production in a dose-dependent manner. In contrast to the 21-mer triplex-forming oligonucleotide (TFO), several control oligos showed no inhibitory effect. The ineffectiveness of the various control oligos, along with the fact that the 21-mer oligo has no homology sequence with lacZYA, and no mRNA is transcribed from the operator, suggests that the 21-mer oligo inhibits target gene expression by an antigene mechanism. To simulate the kinetics of lac operon gene expression in the presence of antigene oligos, a genetically structured kinetic model, which includes transport of oligo into the cell, growth of bacteria cells, and lac operon gene expression, was developed. Predictions of the kinetic model fit the experimental data quite well after adjustment of the value of the oligonucleotide transport rate constant (9.0 x 10(-)(3) min(-)(1)) and oligo binding affinity constant (1.05 x 10(6) M(-)(1)). Our values for these two adjusted parameters are in the range of reported literature values.

  14. Olfactory receptor gene expression in tiger salamander olfactory epithelium.

    PubMed

    Marchand, James E; Yang, Xinhai; Chikaraishi, Dona; Krieger, Jurgen; Breer, Heinz; Kauer, John S

    2004-06-28

    Physiological studies of odor-elicited responses from the olfactory epithelium and bulb in the tiger salamander, Ambystoma tigrinum, have elucidated a number of features of olfactory coding that appear to be conserved across several vertebrate species. This animal model has provided an accessible in vivo system for observing individual and ensemble olfactory responses to odorant stimulation using biochemical, neurophysiological, and behavioral assays. In this paper we have complemented these studies by characterizing 35 candidate odorant receptor genes. These receptor sequences are similar to those of the large families of olfactory receptors found in mammals and fish. In situ hybridization, using RNA probes to 20 of these sequences, demonstrates differential distributions of labeled cells across the extent and within the depth of the olfactory epithelium. The distributions of cells labeled with probes to different receptors show spatially restricted patterns that are generally localized to different degrees in medial-lateral and anterior-posterior directions. The patterns of receptor expression in the ventral olfactory epithelium (OE) are mirrored in the dorsal OE. We present a hypothesis as to how the sensory neuron populations expressing different receptor types responding to a particular odorant may relate to the distribution patterns of epithelial and bulbar responses previously characterized using single-unit and voltage-sensitive dye recording methods.

  15. Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes.

    PubMed

    McGrath, Patrick T; Xu, Yifan; Ailion, Michael; Garrison, Jennifer L; Butcher, Rebecca A; Bargmann, Cornelia I

    2011-08-17

    Evolution can follow predictable genetic trajectories, indicating that discrete environmental shifts can select for reproducible genetic changes. Conspecific individuals are an important feature of an animal's environment, and a potential source of selective pressures. Here we show that adaptation of two Caenorhabditis species to growth at high density, a feature common to domestic environments, occurs by reproducible genetic changes to pheromone receptor genes. Chemical communication through pheromones that accumulate during high-density growth causes young nematode larvae to enter the long-lived but non-reproductive dauer stage. Two strains of Caenorhabditis elegans grown at high density have independently acquired multigenic resistance to pheromone-induced dauer formation. In each strain, resistance to the pheromone ascaroside C3 results from a deletion that disrupts the adjacent chemoreceptor genes serpentine receptor class g (srg)-36 and -37. Through misexpression experiments, we show that these genes encode redundant G-protein-coupled receptors for ascaroside C3. Multigenic resistance to dauer formation has also arisen in high-density cultures of a different nematode species, Caenorhabditis briggsae, resulting in part from deletion of an srg gene paralogous to srg-36 and srg-37. These results demonstrate rapid remodelling of the chemoreceptor repertoire as an adaptation to specific environments, and indicate that parallel changes to a common genetic substrate can affect life-history traits across species.

  16. The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

    PubMed Central

    Alarcón, B; Ley, S C; Sánchez-Madrid, F; Blumberg, R S; Ju, S T; Fresno, M; Terhorst, C

    1991-01-01

    The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development. Images PMID:1826255

  17. Plasmodium simium, a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines

    PubMed Central

    Camargos Costa, Daniela; Pereira de Assis, Gabriela Maíra; de Souza Silva, Flávia Alessandra; Araújo, Flávia Carolina; de Souza Junior, Júlio César; Braga Hirano, Zelinda Maria; Satiko Kano, Flora; Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Ferreira Alves de Brito, Cristiana

    2015-01-01

    Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells. PMID:26107662

  18. Plasmodium simium, a Plasmodium vivax-related malaria parasite: genetic variability of Duffy binding protein II and the Duffy antigen/receptor for chemokines.

    PubMed

    Camargos Costa, Daniela; Pereira de Assis, Gabriela Maíra; de Souza Silva, Flávia Alessandra; Araújo, Flávia Carolina; de Souza Junior, Júlio César; Braga Hirano, Zelinda Maria; Satiko Kano, Flora; Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Ferreira Alves de Brito, Cristiana

    2015-01-01

    Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells.

  19. Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen.

    PubMed Central

    Klena, J D; Ashford, R S; Schnaitman, C A

    1992-01-01

    The rfp gene of Shigella dysenteriae 1 and the rfa genes of Escherichia coli K-12 and Salmonella typhimurium LT2 have been studied to determine their relationship to lipopolysaccharide (LPS) core heterogeneity and their role in the attachment of O antigen to LPS. It has been inferred from the nucleotide sequence that the rfp gene encodes a protein of 41,864 Da which has a structure similar to that of RfaG protein. Expression of this gene in E. coli K-12 results in the loss of one of the three bands seen in gel analysis of the LPS and in the appearance of a new, more slowly migrating band. This is consistent with the hypothesis that Rfp is a sugar transferase which modifies a subset of core molecules so that they become substrates for attachment of S. dysenteriae O antigen. A shift in gel migration of the bands carrying S. dysenteriae O antigen and disappearance of the Rfp-modified band in strains producing O antigen suggest that the core may be trimmed or modified further before attachment of O antigen. Mutation of rfaL results in a loss of the rough LPS band which appears to be modified by Rfp and prevents the appearance of the Rfp-modified band. Thus, RfaL protein is involved in core modification and is more than just a component of the O-antigen ligase. The products of rfaK and rfaQ also appear to be involved in modification of the core prior to attachment of O antigen, and the sites of rfaK modification are different in E. coli K-12 and S. typhimurium. In contrast, mutations in rfaS and rfaZ result in changes in the LPS core but do not affect the attachment of O antigen. We propose that these genes are involved in an alternative pathway for the synthesis of rough LPS species which are similar to lipooligosaccharides of other species and which are not substrates for O-antigen attachment. All of these studies indicate that the apparent heterogeneity of E. coli K-12 LPS observed on gels is not an artifact but instead a reflection of functional differences among

  20. Extensive Variation in the O-Antigen Gene Cluster within One Salmonella enterica Serogroup Reveals an Unexpected Complex History

    PubMed Central

    Wang, Lei; Andrianopoulos, Kanella; Liu, Dan; Popoff, Michel Y.; Reeves, Peter R.

    2002-01-01

    The 46 serogroups of Salmonella enterica have different O-antigens, and each is thought to have a specific form of the O-antigen cluster. Comparison of the 145 serovars of serogroup B revealed much more intraserogroup genetic diversity than expected. The O27 factor, due to an α 1-6 linkage between O units in place of the more common α 1-2 linkage and previously thought to be due to a converting bacteriophage, is now shown to be due to a wzyα(1-6) gene located within the major gene cluster. Surprisingly a remnant of this gene in all O27− serovars shows that the ancestor was O27+. There are six distinct gene cluster forms, five apparently derived by a series of deletions and one by an insertion from an ancestral O27+ form present in 57 serovars. The history of the gene cluster and movement between subspecies I and II can be traced. Two of the derivative forms still have a functional wzyα(1-6) gene, while in three it has been inactivated by deletion or insertion. Two of the forms lacking a functional wzyα(1-6) gene have the wzyα(1-2) gene first described for strain LT2 as rfc, whereas for the third the wzy gene has not been located. PMID:11872718

  1. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    PubMed

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges.

  2. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor

    PubMed Central

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A.; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges. PMID:27124285

  3. Cloning and Expression of Genes for Dengue Virus Type-2 Encoded Antigens for Rapid Diagnosis and Vaccine Development

    DTIC Science & Technology

    1986-11-26

    SIE cop AD nCloning and Expression of Genes for Dengue Virus ,4. CJ Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 0ANNUAL...Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 12. PERSONAL AUTHOR(S) Radha K. Padmanabhan, Ph.D. 13a. TYPE OF REPORT 13b. TIME...pVVI and pVVI7 cDNA clones, synthetic peptides homologous to NS5 and NSI regions were synthesized. These peptides are being used at Walter Reed Army

  4. Membrane immunoglobulin expressed by retroviral vector gene transfer mimics partial function of the B-cell receptor in vivo.

    PubMed

    Lu, Jing; Chen, Feng; Xu, Zhen; Zhang, Lingling; Xu, Peng; Liu, Depei; Liang, Chihchuan

    2016-01-01

    Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell receptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, resulting in the phosphorylation of Syk and Erk1/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.

  5. Chimeric Antigen Receptor T Cells and Hematopoietic Cell Transplantation: How Not to Put the CART Before the Horse.

    PubMed

    Kenderian, Saad S; Porter, David L; Gill, Saar

    2017-02-01

    Hematopoietic cell transplantation (HCT) remains an important and potentially curative option for most hematologic malignancies. As a form of immunotherapy, allogeneic HCT (allo-HCT) offers the potential for durable remissions but is limited by transplantation- related morbidity and mortality owing to organ toxicity, infection, and graft-versus-host disease. The recent positive outcomes of chimeric antigen receptor T (CART) cell therapy in B cell malignancies may herald a paradigm shift in the management of these disorders and perhaps other hematologic malignancies as well. Clinical trials are now needed to address the relative roles of CART cells and HCT in the context of transplantation-eligible patients. In this review, we summarize the state of the art of the development of CART cell therapy for leukemia, lymphoma, and myeloma and discuss our perspective of how CART cell therapy can be applied in the context of HCT.

  6. Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma.

    PubMed

    Chmielewski, Markus; Hombach, Andreas A; Abken, Hinrich

    2014-01-01

    Adoptive T-cell therapy recently achieved impressive efficacy in early phase trials, in particular in hematologic malignancies, strongly supporting the notion that the immune system can control cancer. A current strategy of favor is based on ex vivo-engineered patient T cells, which are redirected by a chimeric antigen receptor (CAR) and recognize a predefined target by an antibody-derived binding domain. Such CAR T cells can substantially reduce the tumor burden as long as the targeted antigen is present on the cancer cells. However, given the tremendous phenotypic diversity in solid tumor lesions, a reasonable number of cancer cells are not recognized by a given CAR, considerably reducing the therapeutic success. This article reviews a recently described strategy for overcoming this shortcoming of the CAR T-cell therapy by modulating the tumor stroma by a CAR T-cell-secreted transgenic cytokine like interleukin-12 (IL-12). The basic process is that CAR T cells, when activated by their CAR, deposit IL-12 in the targeted tumor lesion, which in turn attracts an innate immune cell response toward those cancer cells that are invisible to CAR T cells. Such TRUCKs, T cells redirected for universal cytokine-mediated killing, exhibited remarkable efficacy against solid tumors with diverse cancer cell phenotypes, suggesting their evaluation in clinical trials.

  7. Mesothelin-specific Chimeric Antigen Receptor mRNA-Engineered T cells Induce Anti-Tumor Activity in Solid Malignancies

    PubMed Central

    Beatty, Gregory L.; Haas, Andrew R.; Maus, Marcela V.; Torigian, Drew A.; Soulen, Michael C.; Plesa, Gabriela; Chew, Anne; Zhao, Yangbing; Levine, Bruce L.; Albelda, Steven M.; Kalos, Michael; June, Carl H.

    2014-01-01

    Off-target toxicity due to the expression of target antigens in normal tissue represents a major obstacle to the use of chimeric antigen receptor (CAR)-engineered T cells for treatment of solid malignancies. To circumvent this issue, we established a clinical platform for engineering T cells with transient CAR expression by using in vitro transcribed mRNA encoding a CAR that includes both the CD3-ζ and 4-1BB co-stimulatory domains. We present two case reports from ongoing trials indicating that adoptive transfer of mRNA CAR T cells that target mesothelin (CARTmeso cells) is feasible and safe without overt evidence of off-tumor on-target toxicity against normal tissues. CARTmeso cells persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites. Clinical and laboratory evidence of antitumor activity was demonstrated in both patients and the CARTmeso cells elicited an antitumor immune response revealed by the development of novel anti-self antibodies. These data demonstrate the potential of utilizing mRNA engineered T cells to evaluate, in a controlled manner, potential off-tumor on-target toxicities and show that short-lived CAR T cells can induce epitope-spreading and mediate antitumor activity in patients with advanced cancer. Thus, these findings support the development of mRNA CAR-based strategies for carcinoma and other solid tumors. PMID:24579088

  8. Realism and pragmatism in developing an effective chimeric antigen receptor T-cell product for solid cancers.

    PubMed

    Gad, Ahmed Z; El-Naggar, Shahenda; Ahmed, Nabil

    2016-11-01

    Over the last two decades, harnessing the power of the immune system has shown substantial promise. Specifically, the successes that chimeric antigen receptor (CAR) T cells achieved in the treatment of hematologic malignancies provided a concrete platform for further development in solid tumors. Considering that the latter contribute more than three quarters of cancer-related deaths in humans makes it clear that solid tumors represent the larger medical challenge, but also the larger developmental promise in the market. In solid tumors though, the more is achieved the more challenges are unveiled. The mere fact that engineered T cells are personalized therapies rather than a mass product has been a main constraint for clinical outspread. Further, the complexity of the hostile solid tumor microenvironment, antigenic diversity and dynamicity and the presence of a tenacious stem cell population rendered the effective development to the clinic questionable. In this article we shed light on the importance of a realistic understanding of challenges faced in solid tumors and some very innovative efforts to overcome these challenges in a manner that paves a pragmatic yet realistic road toward effective development at the discovery level and beyond.

  9. A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies.

    PubMed

    Mamonkin, Maksim; Rouce, Rayne H; Tashiro, Haruko; Brenner, Malcolm K

    2015-08-20

    Options for targeted therapy of T-cell malignancies remain scarce. Recent clinical trials demonstrated that chimeric antigen receptors (CARs) can effectively redirect T lymphocytes to eradicate lymphoid malignancies of B-cell origin. However, T-lineage neoplasms remain a more challenging task for CAR T cells due to shared expression of most targetable surface antigens between normal and malignant T cells, potentially leading to fratricide of CAR T cells or profound immunodeficiency. Here, we report that T cells transduced with a CAR targeting CD5, a common surface marker of normal and neoplastic T cells, undergo only limited fratricide and can be expanded long-term ex vivo. These CD5 CAR T cells effectively eliminate malignant T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma lines in vitro and significantly inhibit disease progression in xenograft mouse models of T-ALL. These data support the therapeutic potential of CD5 CAR in patients with T-cell neoplasms.

  10. Fluorine substitutions in an antigenic peptide selectively modulate T-cell receptor binding in a minimally perturbing manner

    SciTech Connect

    Piepenbrink, Kurt H.; Borbulevych, Oleg Y.; Sommese, Ruth F.; Clemens, John; Armstrong, Kathryn M.; Desmond, Clare; Do, Priscilla; Baker, Brian M.

    2010-08-17

    TCR (T-cell receptor) recognition of antigenic peptides bound and presented by MHC (major histocompatibility complex) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide - MHC complexes weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Furthermore, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. In the present study, we examined how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the Tax peptide [HTLV (human T-cell lymphotropic virus)-1 Tax] enhanced binding by the Tax-specific TCR A6, yet weakened binding by the Tax-specific TCR B7. The changes in affinity were consistent with crystallographic structures and fluorine chemistry, and with the A6 TCR independent of other substitutions in the interface. Peptide fluorination thus provides a means to selectively modulate TCR binding affinity without significantly perturbing peptide composition or structure. Lastly, we probed the mechanism of fluorine's effect on TCR binding and we conclude that our results were most consistent with a 'polar hydrophobicity' mechanism, rather than a purely hydrophobic- or electrostatic-based mechanism. This finding should have an impact on other attempts to alter molecular recognition with fluorine.

  11. Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen

    PubMed Central

    Sprott, G. Dennis; Sad, Subash; Fleming, L. Perry; DiCaire, Chantal J.; Patel, Girishchandra B.; Krishnan, Lakshmi

    2003-01-01

    Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis ofarchaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation. PMID:15803661

  12. Cytotoxic T Lymphocyte Antigen-4 Gene Variants in Type 2 Diabetic Patients with or without Neuropathy.

    PubMed

    Kiani, Javad; Khadempar, Saedeh; Hajilooi, Mehrdad; Rezaei, Hamzeh; Keshavarzi, Fatemeh; Solgi, Ghasem

    2016-06-01

    Many studies have shown that cytotoxic T lymphocyte antigen-4 (CTLA-4) gene variants are associated with several autoimmune diseases particularly type 1 diabetes. Due to the lack of consistent data for this association with type 2 diabetes (T2D), this study explored the possible influence of CTLA-4 gene polymorphisms at -1722 (T/C), -318 (C/T), and +49 (G/A) positions for susceptibility to T2D in relation with neuropathy. One hundred and eleven unrelated patients with T2D [49 patients with diabetic peripheral neuropathy (DPN) and 62 patients without PDN] and 100 healthy ethnic- and gender-matched controls were included in this study. The dimorphisms at -1722 (C/T), -318 (C/T) and +49 (A/G) for CTLA-4 gene were determined using ARMS-PCR. The CTLA-4 (+49 G/G) and (+49 A/A) genotypes were found to be positively and negatively associated with T2D, respectively (p=0.03). The -318 C/T and T/T genotypes were more frequent in patients than controls and -318 C/C genotype was shown to be protective for T2D (p=0.003). ACT and GTT Haplotypes were less and more frequent in controls and patients, respectively (p=3.86×10-7 and p=2.29×10-5). Genotypes distribution among T2D patients with and without DPN compared to healthy controls showed significantly lower frequencies for -318 C/C and +49 A/A genotypes and significantly higher frequencies for -318 C/T and T/T genotypes as well. Our findings indicate that CTLA-4 (+49 A/G) and (-318 C/T) genotypes could be considered as genetic risk factors associated with susceptibility or protection for T2D.

  13. Structure and antigenicity analysis of the IgG gene for Nyctereutes procyonoides

    PubMed Central

    Zhao, Cui; Guo, Shuyuan; Pang, Xiaoru; Song, Daozhen; Fu, Shijun

    2015-01-01

    Objective Nyctereutes procyonoides immunoglobulin G (IgG) gene is partially cloned. Material and methods In order to obtain a certain length (966bp) of Nyctereutes procyonoides immunoglobulin G (IgG), two pairs of primers are designed according to the conserved nucleotide sequence of canine (GenBank:AF354265, AF354265, AF354266, AF354267) and mink (GenBank: L07789). Using Bioinformatics technology and Western-blot to analyze antigenicity of Nyctereutes procyonoides IgG-B gene. Results The homology for nucleotide sequence of IgG between Nyctereutes procyonoides and canine (IgG A, IgG B, IgG C, IgG D), mink, Homo sapiens, Oryctolagus cuniculus, Mus musculus, Anas platyrhynchos and gallus were respectively (88.1%, 93.6%, 85.4%, 87.2%), 83.7%, 74.8%, 71.8%, 69.2%, 51.6%, 48.4%. It can be seen that there was high homology of aminoacid sequence between IgG of Nyctereutes procyonoides and IgG (A, B, C, D) of canine. And the serum antibody of Nyctereutes procyonoides had obviously cross-reaction with HRP conjugated rabbit anti-dog IgG, compared with those of canine, oryctolagus cuniculus, mus musculus, mink, gallus. Conclusions We successfully got Nyctereutes procyonoides immuneglobulin G (IgG) gene (Gen- Bank: KM010191). There is the closest ties of consanguinity of IgG exist between Nyctereutes procyonoides and canine among the mammal through the genetic evolution. The detection and treament of canine distemper can be used on Nyctereutes procyonoides. PMID:26648768

  14. Association of Polymorphisms in HLA Antigen Presentation-Related Genes with the Outcomes of HCV Infection

    PubMed Central

    Lu, Xiaomei; Xu, Yin; Wang, Jie; Zhang, Yun; Yu, Rongbin; Su, Jing

    2015-01-01

    Antigen-presentation genes play a vital role in the pathogenesis of HCV infection. However, the relationship of variants of these genes with spontaneous outcomes of HCV infection has not been fully investigated. To explore novel loci in the Chinese population, 34 tagging-SNPs in 9 candidate genes were genotyped for their associations with the outcomes of HCV infection. The distributions of different genotypes and haplotypes were compared among 773 HCV-negative controls, 246 subjects with HCV natural clearance, and 218 HCV persistent carriers recruited from hemodialysis patients and intravenous drug users. Our study implicated that TAP2, HLA-DOA, HLA-DOB, and tapasin loci were novel candidate regions for susceptibility to HCV infection and viral clearance in the Chinese population. Logistic regression analyses showed that TAP2 rs1800454 A (OR = 1.48, P = 0.002) and HLA-DOB rs2071469 G (OR = 1.23, P = 0.048) were significantly associated with increased susceptibility to establishment of HCV infection. However, high-risk behavior exposure and age were stronger predictors of HCV infection. Mutation of tapasin rs9277972 T (OR = 1.57, P =0.043) increased the risk of HCV chronicity, and HLA-DOA rs3128935 C (OR = 0.62, P = 0.019) increased the chance of viral resolution. With regards to the effect of rs3128925, interactions were found with high-risk behavior (P = 0.013) and age (P = 0.035). The risk effect of rs3128925 T for persistent HCV infection was higher in injecting drug users (vs. dialysis patients) and in subjects ≥ 40 years old (vs. < 40 years old). PMID:25874709

  15. Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

    PubMed Central

    Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Bruno, Silvia; Ghiotto, Fabio; Tenca, Claudya; Stamatopoulos, Kostas; Hadzidimitriou, Anastasia; Ceccarelli, Jenny; Salvi, Sandra; Boccardo, Simona; Calevo, Maria Grazia; De Santanna, Amleto; Truini, Mauro; Fais, Franco; Ferrarini, Manlio

    2013-01-01

    Marginal zone (MZ) B cells, identified as surface (s)IgMhighsIgDlowCD23low/−CD21+CD38− B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27+ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli. PMID:23877718

  16. Identification of novel androgen receptor target genes in prostate cancer

    PubMed Central

    Jariwala, Unnati; Prescott, Jennifer; Jia, Li; Barski, Artem; Pregizer, Steve; Cogan, Jon P; Arasheben, Armin; Tilley, Wayne D; Scher, Howard I; Gerald, William L; Buchanan, Grant; Coetzee, Gerhard A; Frenkel, Baruch

    2007-01-01

    Background The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression. Results Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells – D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1) – most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. Conclusion AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general

  17. The Role of Metabotropic Glutamate Receptor Genes in Schizophrenia

    PubMed Central

    Maj, Carlo; Minelli, Alessandra; Giacopuzzi, Edoardo; Sacchetti, Emilio; Gennarelli, Massimo

    2016-01-01

    Genomic studies revealed two main components in the genetic architecture of schizophrenia, one constituted by common variants determining a distributed polygenic effect and one represented by a large number of heterogeneous rare and highly disruptive mutations. These gene modifications often affect neural transmission and different studies proved an involvement of metabotropic glutamate receptors in schizophrenia phenotype. Through the combination of literature information with genomic data from public repositories, we analyzed the current knowledge on the involvement of genetic variations of the human metabotropic glutamate receptors in schizophrenia and related endophenotypes. Despite the analysis did not reveal a definitive connection, different suggestive associations have been identified and in particular a relevant role has emerged for GRM3 in affecting specific schizophrenia endophenotypes. This supports the hypothesis that these receptors are directly involved in schizophrenia disorder. PMID:27296644

  18. The Role of Metabotropic Glutamate Receptor Genes in Schizophrenia.

    PubMed

    Maj, Carlo; Minelli, Alessandra; Giacopuzzi, Edoardo; Sacchetti, Emilio; Gennarelli, Massimo

    2016-01-01

    Genomic studies revealed two main components in the genetic architecture of schizophrenia, one constituted by common variants determining a distributed polygenic effect and one represented by a large number of heterogeneous rare and highly disruptive mutations. These gene modifications often affect neural transmission and different studies proved an involvement of metabotropic glutamate receptors in schizophrenia phenotype. Through the combination of literature information with genomic data from public repositories, we analyzed the current knowledge on the involvement of genetic variations of the human metabotropic glutamate receptors in schizophrenia and related endophenotypes. Despite the analysis did not reveal a definitive connection, different suggestive associations have been identified and in particular a relevant role has emerged for GRM3 in affecting specific schizophrenia endophenotypes. This supports the hypothesis that these receptors are directly involved in schizophrenia disorder.

  19. Genomic analysis of isolates from the United Kingdom 2012 pertussis outbreak reveals that vaccine antigen genes are unusually fast evolving.

    PubMed

    Sealey, Katie L; Harris, Simon R; Fry, Norman K; Hurst, Laurence D; Gorringe, Andrew R; Parkhill, Julian; Preston, Andrew

    2015-07-15

    A major outbreak of whooping cough, or pertussis, occurred in 2012 in the United Kingdom (UK), with nearly 10 000 laboratory-confirmed cases and 14 infant deaths attributed to pertussis. A worldwide resurgence of pertussis has been linked to switch to the use of acellular pertussis vaccines and the evolution of Bordetella pertussis away from vaccine-mediated immunity. We have conducted genomic analyses of multiple strains from the UK outbreak. We show that the UK outbreak was polyclonal in nature, caused by multiple distinct but closely related strains. Importantly, we demonstrate that acellular vaccine antigen-encoding genes are evolving at higher rates than other surface protein-encoding genes. This was true even prior to the introduction of pertussis vaccines but has become more pronounced since the introduction of the current acellular vaccines. The fast evolution of vaccine antigen-encoding genes has serious consequences for the ability of current vaccines to continue to control pertussis.

  20. Asymmetrical phosphorylation and function of immunoreceptor tyrosine-based activation motif tyrosines in B cell antigen receptor signal transduction.

    PubMed

    Pao, L I; Famiglietti, S J; Cambier, J C

    1998-04-01

    CD79a and CD79b function as transducers of B cell antigen receptor signals via a cytoplasmic sequence, termed the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs contain two conserved tyrosines that may become phosphorylated upon receptor aggregation and bind distinct effectors by virtue of the distinct preference of phosphotyrosyl-containing sequences for SH2 domains. To explore the function of CD79a and CD79b ITAM tyrosines, we created membrane molecules composed of MHC class II I-Ak extracellular and transmembrane domains, and CD79a or CD79b cytoplasmic domains in which one or both of the ITAM tyrosines were mutated to phenylalanine. Functional analysis revealed that both ITAM tyrosines are required for ligand-induced Syk phosphorylation. However CD79a-ITAM and CD79b-ITAM tyrosine phosphorylations were asymmetrical, with >80% of phosphorylation occurring on the N-terminal tyrosine (Y-E-G-L). Thus, these findings suggest that following receptor ligation, only a minor proportion of phosphorylated ITAMs are doubly phosphorylated and thus can engage Syk. Only the N-terminal ITAM tyrosine of CD79a was required for ligand-mediated phosphorylation of the receptor and a subset of downstream substrates, including p62, p110, and Shc, and for Ca2+ mobilization. However, responses mediated through CD79b exhibited a greater dependence on the presence of both tyrosines. Neither tyrosine in CD79a or CD79b appeared absolutely essential for Src family kinase phosphorylation. These results indicate that phosphorylations of the tyrosines in CD79a and CD79b occur with very different stoichiometry, and the respective tyrosyl residues have distinct functions.

  1. Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

    PubMed Central

    Deniger, Drew C; Switzer, Kirsten; Mi, Tiejuan; Maiti, Sourindra; Hurton, Lenka; Singh, Harjeet; Huls, Helen; Olivares, Simon; Lee, Dean A; Champlin, Richard E; Cooper, Laurence JN

    2013-01-01

    Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR+γδ T cells were expanded on CD19+ artificial antigen-presenting cells (aAPC), which resulted in >109 CAR+γδ T cells from <106 total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR+γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19+ tumor cell lines compared with CARnegγδ T cells. CD19+ leukemia xenografts in mice were reduced with CAR+γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells. PMID:23295945

  2. Patterns of human genetic variation inferred from comparative analysis of allelic mutations in blood group antigen genes.

    PubMed

    Patnaik, Santosh Kumar; Blumenfeld, Olga O

    2011-03-01

    Comparative analysis of allelic variation of a gene sheds light on the pattern and process of its diversification at the population level. Gene families for which a large number of allelic forms have been verified by sequencing provide a useful resource for such studies. In this regard, human blood group-encoding genes are unique in that differences of cell surface traits among individuals and populations can be readily detected by serological screening, and correlation between the variant cell surface phenotype and the genotype is, in most cases, unequivocal. Here, we perform a comprehensive analysis of allelic forms, compiled in the Blood Group Antigen Gene Mutation database, of ABO, RHD/CE, GYPA/B/E and FUT1/2 gene families that encode the ABO, RH, MNS, and H/h blood group system antigens, respectively. These genes are excellent illustrative examples showing distinct mutational patterns among the alleles, and leading to speculation on how their origin may have been driven by recurrent but different molecular mechanisms. We illustrate how alignment of alleles of a gene may provide an additional insight into the DNA variation process and its pathways, and how this approach may serve to catalog alleles of a gene, simplifying the task and content of mutation databases.

  3. Replicated Risk Nicotinic Cholinergic Receptor Genes for Nicotine Dependence

    PubMed Central

    Zuo, Lingjun; Garcia-Milian, Rolando; Guo, Xiaoyun; Zhong, Chunlong; Tan, Yunlong; Wang, Zhiren; Wang, Jijun; Wang, Xiaoping; Kang, Longli; Lu, Lu; Chen, Xiangning; Li, Chiang-Shan R.; Luo, Xingguang

    2016-01-01

    It has been hypothesized that the nicotinic acetylcholine receptors (nAChRs) play important roles in nicotine dependence (ND) and influence the number of cigarettes smoked per day (CPD) in smokers. We compiled the associations between nicotinic cholinergic receptor genes (CHRNs) and ND/CPD that were replicated across different studies, reviewed the expression of these risk genes in human/mouse brains, and verified their expression using independent samples of both human and mouse brains. The potential functions of the replicated risk variants were examined using cis-eQTL analysis or predicted using a series of bioinformatics analyses. We found replicated and significant associations for ND/CPD at 19 SNPs in six genes in three genomic regions (CHRNB3-A6, CHRNA5-A3-B4 and CHRNA4). These six risk genes are expressed in at least 18 distinct areas of the human/mouse brain, with verification in our independent human and mouse brain samples. The risk variants might influence the transcription, expression and splicing of the risk genes, alter RNA secondary or protein structure. We conclude that the replicated associations between CHRNB3-A6, CHRNA5-A3-B4, CHRNA4 and ND/CPD are very robust. More research is needed to examine how these genetic variants contribute to the risk for ND/CPD. PMID:27827986

  4. Replicated Risk Nicotinic Cholinergic Receptor Genes for Nicotine Dependence.

    PubMed

    Zuo, Lingjun; Garcia-Milian, Rolando; Guo, Xiaoyun; Zhong, Chunlong; Tan, Yunlong; Wang, Zhiren; Wang, Jijun; Wang, Xiaoping; Kang, Longli; Lu, Lu; Chen, Xiangning; Li, Chiang-Shan R; Luo, Xingguang

    2016-11-07

    It has been hypothesized that the nicotinic acetylcholine receptors (nAChRs) play important roles in nicotine dependence (ND) and influence the number of cigarettes smoked per day (CPD) in smokers. We compiled the associations between nicotinic cholinergic receptor genes (CHRNs) and ND/CPD that were replicated across different studies, reviewed the expression of these risk genes in human/mouse brains, and verified their expression using independent samples of both human and mouse brains. The potential functions of the replicated risk variants were examined using cis-eQTL analysis or predicted using a series of bioinformatics analyses. We found replicated and significant associations for ND/CPD at 19 SNPs in six genes in three genomic regions (CHRNB3-A6, CHRNA5-A3-B4 and CHRNA4). These six risk genes are expressed in at least 18 distinct areas of the human/mouse brain, with verification in our independent human and mouse brain samples. The risk variants might influence the transcription, expression and splicing of the risk genes, alter RNA secondary or protein structure. We conclude that the replicated associations between CHRNB3-A6, CHRNA5-A3-B4,CHRNA4 and ND/CPD are very robust. More research is needed to examine how these genetic variants contribute to the risk for ND/CPD.

  5. Mechanisms of oestrogen receptor (ER) gene regulation in breast cancer

    PubMed Central

    2016-01-01

    Most breast cancers are driven by a transcription factor called oestrogen receptor (ER). Understanding the mechanisms of ER activity in breast cancer has been a major research interest and recent genomic advances have revealed extraordinary insights into how ER mediates gene transcription and what occurs during endocrine resistance. This review discusses our current understanding on ER activity, with an emphasis on several evolving, but important areas of ER biology. PMID:26884552

  6. Complete structural characterisation of the human aryl hydrocarbon receptor gene

    PubMed Central

    Bennett, P; Ramsden, D B; Williams, A C

    1996-01-01

    Aims—To clone and characterise the complete structural gene for the human aryl hydrocarbon receptor (AhR). This gene, located on chromosome 7, encodes a cytosolic receptor protein which, upon activation by various xenobiotic ligands, translocates to the nucleus, where it acts as a specific transcription factor. Methods—Primers, based on the AhR cDNA sequence, were used in conjunction with recently developed long range PCR techniques to amplify contiguous sections of the cognate gene. The amplicons produced were then cloned and characterised. A cDNA probe was also used to screen a human P1 library. Results—Using the cDNA primers, DNA fragments which mapped the entire coding region of the gene were amplified and cloned. All but one of these fragments were amplified directly from human genomic DNA. The remaining fragment was amplified using DNA prepared from a P1 clone as the PCR template. This P1 clone, obtained by screening a human P1 library, also contained the entire Ah locus. Characterisation of amplified and cloned DNA fragments provided sufficient information for the construction of a complete structural map of the gene. This also included 150 base pairs of nucleotide sequence data at all intronic termini. Conclusions—These data indicate that the human AhR gene is about 50 kilobases long and contains 11 exons. The overall intron/exon structure of the human gene is homologous to that of the previously characterised mouse gene; however, it is probably some 20 kilobases larger. These results demonstrate the need for further characterisation and provide the data to facilitate this. Images PMID:16696038

  7. Construction of a multicolor GeneScan analytical system to detect clonal rearrangements of immunoglobulin and T cell receptor genes in canine lymphoid tumors.

    PubMed

    Goto-Koshino, Yuko; Mochizuki, Hiroyuki; Sato, Masahiko; Nakashima, Ko; Hiyoshi, Saaya; Fujiwara-Igarashi, Aki; Maeda, Shingo; Nakamura, Kenji; Uchida, Kazuyuki; Fujino, Yasuhito; Ohno, Koichi; Tsujimoto, Hajime

    2015-05-15

    Polymerase chain reaction (PCR) amplification to detect immunoglobulin heavy chain (IgH) and T cell receptor γ-chain (TCRγ) gene rearrangements has recently become widely used as part of the diagnostic strategy for lymphoid tumors in dogs. In this study, we constructed a multicolor GeneScan analytical system to improve the sensitivity and resolution of the clonality analysis of antigen receptor gene rearrangements in dogs. We used 7 reactions per sample, with 2 PCR conditions, to amplify IgH/TCRγ and control genes. By using multicolor-labeled primers, these 7 PCR products could be combined into 3 tubes before capillary electrophoresis. Clonal rearrangement of the IgH/TCRγ genes was detected in 93.3% of dogs with multicentric lymphoma and 84.6% of dogs with gastrointestinal lymphoma. Detection sensitivity of the clonally expanded cells in the background of normal peripheral blood mononuclear cells was 1-10%. The multicolor GeneScan analytical system developed here may prove to be helpful for the diagnosis of lymphoid tumors in dogs.

  8. Allergic rhinitis and genetic components: focus on Toll-like receptors (TLRs) gene polymorphism

    PubMed Central

    Gao, Zhiwei; Rennie, Donna C; Senthilselvan, Ambikaipakan

    2010-01-01

    Allergic rhinitis represents a global health issue affecting 10% to 25% of the population worldwide. Over the years, studies have found that allergic diseases, including allergic rhinitis, are associated with immunological responses to antigens driven by a Th2-mediated immune response. Because Toll-like receptors (TLRs) are involved in both innate and adaptive immune responses to a broad variety of antigens, the association between polymorphisms of TLRs and allergic diseases has been the focus in many animal and human studies. Although the etiology of allergic rhinitis is still unknown, extensive research over the years has confirmed that the underlying causes of allergic diseases are due to many genetic and environmental factors, along with the interactions among them, which include gene–environment, gene–gene, and environment–environment interactions. Currently, there is great inconsistency among studies mainly due to differences in genetic background and unique gene–environment interactions. This paper reviews studies focusing on the association between TLR polymorphisms and allergic diseases, including allergic rhinitis, which would help researchers better understand the role of TLR polymorphisms in the development of allergic rhinitis, and ultimately lead to more efficient therapeutic interventions being developed. PMID:23776356

  9. Analysis of the role of human leukocyte antigen class-I genes to understand the etiopathology of schizophrenia

    PubMed Central

    Singh, Bisu; Banerjee, Sikta; Bera, Nirmal K.; Nayak, Chitta R.; Chaudhuri, Tapas K.

    2008-01-01

    Background: Schizophrenia is the paradigmatic illness of psychiatry. The involvement of immunological and immunopathological mechanisms in the etiopathogenesis of schizophrenia has been a matter of research, with recently increasing effort. Aims: In this study, we investigated the incidence of human leukocyte antigen (HLA) Class I antigens to understand the role of HLA genes in schizophrenia. Materials and Methods: India born schizophrenic patients in and around Siliguri who attended outpatient department (OPD) of Department of Psychiatry, North Bengal Medical College and Hospital were considered for the present study. After the longitudinal follow up, 50 patients were enrolled for the study. The same number of age, sex and ethnically matched healthy subjects were considered as control. Low resolution polymerase chain reaction-sequence specific primer method was applied for typing the HLA antigens. Statistics: The phenotype frequencies were calculated by direct count. χ2 test was done to compare the frequency of each antigen among the patients and control group and it was followed by Fisher's exact test. Relative risk was estimated by using Haldane's method. Results: The result showed that some of the HLA antigens are associated with the schizophrenia and significant increase were observed for HLA A*03 antigen along with the significant decrease for HLA A*25, A*31 and HLA B*51. Conclusions: The study provides the evidence for the possible existence of susceptibility locus for schizophrenia within the HLA region. This preliminary observation may help to understand the etiological basis of this disorder and the study may further strengthen the HLA antigens as the marker for schizophrenia. PMID:19742184

  10. Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

    PubMed Central

    Biedroń, Rafał; Konopiński, Maciej K.; Marcinkiewicz, Janusz; Józefowski, Szczepan

    2015-01-01

    The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect

  11. Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

    SciTech Connect

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng . E-mail: Dongfeng_pan@yahoo.com

    2005-09-09

    PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10{sup 9} PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.

  12. Expressed var gene repertoire and variant surface antigen diversity in a shrinking Plasmodium falciparum population.

    PubMed

    Carlos, Bianca C; Fotoran, Wesley L; Menezes, Maria J; Cabral, Fernanda J; Bastos, Marcele F; Costa, Fabio T M; Sousa-Neto, Jayme A; Ribolla, Paulo E M; Wunderlich, Gerhard; Ferreira, Marcelo U

    2016-11-01

    The var gene-encoded erythrocyte membrane protein-1 of Plasmodium falciparum (PfEMP-1) is the main variant surface antigen (VSA) expressed on infected erythrocytes. The rate at which antibody responses to VSA expressed by circulating parasites are acquired depends on the size of the local VSA repertoire and the frequency of exposure to new VSA. Because parasites from areas with declining malaria endemicity, such as the Amazon, typically express a restricted PfEMP-1 repertoire, we hypothesized that Amazonians would rapidly acquire antibodies to most locally circulating VSA. Consistent with our expectations, the analysis of 5878 sequence tags expressed by 10 local P. falciparum samples revealed little PfEMP-1 DBL1α domain diversity. Among the most commonly expressed DBL1α types, 45% were shared by two or more independent parasite lines. Nevertheless, Amazonians displayed major gaps in their repertoire of anti-VSA antibodies, although the breadth of anti-VSA antibody responses correlated positively with their cumulative exposure to malaria. We found little antibody cross-reactivity even when testing VSA from related parasites expressing the same dominant DBL1α types. We conclude that variant-specific immunity to P. falciparum VSAs develops slowly despite the relatively restricted PfEMP-1 repertoire found in low-endemicity settings.

  13. The enterobacterial common antigen-like gene cluster of Haemophilus ducreyi contributes to virulence in humans.

    PubMed

    Banks, Keith E; Fortney, Kate R; Baker, Beth; Billings, Steven D; Katz, Barry P; Munson, Robert S; Spinola, Stanley M

    2008-06-01

    Haemophilus ducreyi 35000HP contains a cluster of homologues of genes required for the synthesis of enterobacterial common antigen (ECA), suggesting that H. ducreyi may express a putative ECA-like glycoconjugate. WecA initiates the synthesis of ECA by transferring N-acetylglucosamine to undecaprenyl-P, to form lipid I. A wecA mutant (35000HPwecA) was constructed, and 5 volunteers were inoculated at 3 sites with fixed doses of 35000HP on one arm and at 3 sites with varying doses of 35000HPwecA on the other arm. 35000HPwecA caused pustules to form at 3 sites inoculated with a dose 2.5-fold higher than that of 35000HP. However, at sites inoculated with similar doses of 35000HP and 35000HPwecA, pustules developed at 46.7% (95% confidence interval [CI], 23.3%-70.0%) of 15 parent-strain sites and at 8.3% (95% CI, 0.01%-23.6%) of 12 mutant-strain sites (P = .013). Thus, the expression of wecA contributes to the ability of H. ducreyi to cause pustules in humans.

  14. Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

    PubMed

    Ong, E K; Knox, R B; Singh, M B

    1995-03-01

    The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

  15. Role of CD4 molecule in the induction of interleukin 2 and interleukin 2 receptor in class II major histocompatibility complex-restricted antigen-specific T helper clones. T cell receptor/CD3 complex transmits CD4-dependent and CD4-independent signals.

    PubMed Central

    Oyaizu, N; Chirmule, N; Pahwa, S

    1992-01-01

    The CD4 molecule plays an essential role in antigen-induced activation of T helper (Th) cells, but its contribution to signal transduction events resulting in physiologic T cell function is ill defined. By utilizing anti-CD4 monoclonal antibodies (MAbs) that recognize distinct epitopes of CD4, we have investigated the role of CD4 molecule in antigen-induced interleukin 2 (IL-2) and IL-2 receptor (IL-2R) alpha chain expression in class II major histocompatibility complex-restricted antigen-specific human Th clones. Pretreatment of the Th clones with Leu3a resulted in a dose-dependent suppression of antigen-induced proliferative responses, inositol phosphate accumulation, increase in free cytoplasmic calcium ions ([Ca2+]i), IL-2 mRNA accumulation, IL-2 secretion, and membrane IL-2R expression. IL-2R mRNA accumulation, however, was unaffected even at highest Leu3a concentrations. Leu3a treatment did not affect bypass activation of T cells with PMA plus ionomycin or activation via CD2 molecule. The MAb OKT4, which binds another domain of CD4, was not inhibitory. These results suggest that after T cell antigen receptor-CD3 activation, IL-2 gene induction, IL-2 secretion, and membrane IL-2R expression are absolutely dependent upon participation of CD4 molecules, phosphatidylinositol (PI) hydrolysis, and increase in [Ca2+]i. The requirement for IL-2R gene induction, however, occurs independently of CD4 molecule participation and PI hydrolysis. Images PMID:1534818

  16. Epidermal growth factor receptor protects proliferating cell nuclear antigen from cullin 4A protein-mediated proteolysis.

    PubMed

    Lo, Yuan-Hung; Ho, Po-Chun; Wang, Shao-Chun

    2012-08-03

    Proliferating cell nuclear antigen (PCNA) is an essential component for DNA synthesis upon growth stimulation. It has been shown that phosphorylation of PCNA at Tyr-211 by the EGF receptor (EGFR) protects PCNA from polyubiquitylation and degradation, whereas blocking phosphorylation induces ubiquitylation-mediated degradation of the chromatin-bound, but not the -unbound, PCNA, and suppresses cell proliferation. However, the ubiquitin E3 ligase linking growth signaling to the proteolysis of PCNA and the underlying regulatory mechanism remain to be identified. Here we show that, in the absence of Tyr-211 phosphorylation, PCNA is subject to polyubiquitylation at Lys-164 by the CUL4A E3 ligase, resulting in the degradation of PCNA. Mutation of Lys-164 to arginine prevents PCNA ubiquitylation and rescues the degradation of the K164R/Y211F PCNA double mutant. Activation of EGFR inhibits the interaction of PCNA with CUL4A, whereas inhibition of EGFR leads to increased CUL4A-PCNA interaction and CUL4A-dependent ubiquitin-mediated degradation of PCNA. Substitution of endogenous PCNA with the Y211F mutant PCNA conveys enhanced sensitization to EGFR inhibition. Our findings identify CUL4A as the ubiquitin ligase linking the down-regulation of cell surface receptor tyrosine kinase to the nuclear DNA replication machinery in cancer cells.

  17. A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing.

    PubMed

    Prapa, Malvina; Caldrer, Sara; Spano, Carlotta; Bestagno, Marco; Golinelli, Giulia; Grisendi, Giulia; Petrachi, Tiziana; Conte, Pierfranco; Horwitz, Edwin M; Campana, Dario; Paolucci, Paolo; Dominici, Massimo

    2015-09-22

    Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.

  18. Proliferation of thymic stem cells with and without receptors for interleukin 2. Implications for intrathymic antigen recognition

    PubMed Central

    1985-01-01

    We have tested the dividing cells in the mouse thymus for expression of interleukin 2 (IL-2) receptors (IL-2-R) using the rat monoclonal antibody 7D4. A discrete subpopulation of the lymphoblasts clearly expressed IL-2-R at levels comparable to those on mitogen-activated peripheral T cells. This subpopulation, however, represented a small minority of the proliferating cells. IL-2-R-bearing cells were depleted from the PNA+ (peanut agglutinin) lymphoblast population, which contains the direct precursors of most of the cells in the thymus. The majority of receptor-bearing cells were found in the PNA- lymphoblast population, where they constituted only approximately 12% of the cells. Thus, virtually all the PNA+ and most of the PNA- blast cells were in cycle without detectable IL-2-R expression. This indicates that they were not dividing in response to IL-2, and implies that they were not dividing in response to antigen, but rather to novel thymus-specific mitogenic stimuli. On the other hand, the proliferating cells that do express IL-2-R were enriched 4-5-fold in the rapidly growing neonatal thymus, suggesting that they may also play a key role in T cell development. PMID:3921650

  19. The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host–parasite interaction

    PubMed Central

    Jackson, Andrew P.; Otto, Thomas D.; Darby, Alistair; Ramaprasad, Abhinay; Xia, Dong; Echaide, Ignacio Eduardo; Farber, Marisa; Gahlot, Sunayna; Gamble, John; Gupta, Dinesh; Gupta, Yask; Jackson, Louise; Malandrin, Laurence; Malas, Tareq B.; Moussa, Ehab; Nair, Mridul; Reid, Adam J.; Sanders, Mandy; Sharma, Jyotsna; Tracey, Alan; Quail, Mike A.; Weir, William; Wastling, Jonathan M.; Hall, Neil; Willadsen, Peter; Lingelbach, Klaus; Shiels, Brian; Tait, Andy; Berriman, Matt; Allred, David R.; Pain, Arnab

    2014-01-01

    Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct. PMID:24799432

  20. Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12.

    PubMed Central

    Rick, P D; Hubbard, G L; Barr, K

    1994-01-01

    The Escherichia coli O8 antigen is a mannan composed of the trisaccharide repeat unit -->3)-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1--> (K. Reske and K. Jann, Eur. J. Biochem. 67:53-56, 1972), and synthesis of the O8 antigen is rfe dependent (G. Schmidt, H. Mayer, and P. H. Mäkelä, J. Bacteriol. 127:755-762, 1976). The rfe gene has recently been identified as encoding a tunicamycin-sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase (U. Meier-Dieter, K. Barr, R. Starman, L. Hatch, and P. D. Rick, J. Biol. Chem. 267:746-753, 1992). However, the role of rfe in O8 side chain synthesis is not understood. Thus, the role of the rfe gene in the synthesis of the O8 antigen was investigated in an rfbO8+ (rfb genes encoding O8 antigen) derivative of E. coli K-12 mutant possessing a defective phosphoglucose isomerase (pgi). The in vivo synthesis of O8 side chains was inhibited by the antibiotic tunicamycin. In addition, putative lipid carrier-linked O8 side chains accumulated in vivo when lipopolysaccharide outer core synthesis was precluded by growing cells in the absence of exogenously supplied glucose. The lipid carrier-linked O8 antigen was extracted from cells and treated with mild acid in order to release free O8 side chains. The water-soluble O8 side chains were then purified by affinity chromatography using Sepharose-bound concanavalin A. Characterization of the affinity-purified O8 side chains revealed the occurrence of glucosamine in the reducing terminal position of the polysaccharide chains. The data presented suggest that GlcNAc-pyrophosphorylundecaprenol functions as the acceptor of mannose residues for the in vivo synthesis of O8 side chains in E. coli K-12. Images PMID:7514591

  1. Constitutive androstane receptor activation evokes the expression of glycolytic genes.

    PubMed

    Yarushkin, Andrei A; Kazantseva, Yuliya A; Prokopyeva, Elena A; Markova, Diana N; Pustylnyak, Yuliya A; Pustylnyak, Vladimir O

    2016-09-23

    It is well-known that constitutive androstane receptor (CAR) activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases the liver-to-body weight ratio. CAR-mediated liver growth is correlated with increased expression of the pleiotropic transcription factor cMyc, which stimulates cell cycle regulatory genes and drives proliferating cells into S phase. Because glycolysis supports cell proliferation and cMyc is essential for the activation of glycolytic genes, we hypothesized that CAR-mediated up-regulation of cMyc in mouse livers might play a role in inducing the expression of glycolytic genes. The aim of the present study was to examine the effect of long-term CAR activation on glycolytic genes in a mouse model not subjected to metabolic stress. We demonstrated that long-term CAR activation by TCPOBOP increases expression of cMyc, which was correlated with reduced expression of gluconeogenic genes and up-regulation of glucose transporter, glycolytic and mitochondrial pyruvate metabolising genes. These changes in gene expression after TCPOBOP treatment were strongly correlated with changes in levels of glycolytic intermediates in mouse livers. Moreover, we demonstrated a significant positive regulatory effect of TCPOBOP-activated CAR on both mRNA and protein levels of Pkm2, a master regulator of glucose metabolism and cell proliferation. Thus, our findings provide evidence to support the conclusion that CAR activation initiates a transcriptional program that facilitates the coordinated metabolic activities required for cell proliferation.

  2. PD-1– and CTLA-4–Based Inhibitory Chimeric Antigen Receptors (iCARs) Divert Off-Target Immunotherapy Responses

    PubMed Central

    Fedorov, Victor D.; Themeli, Maria; Sadelain, Michel

    2014-01-01

    T cell therapies have demonstrated long-term efficacy and curative potential for the treatment of some cancers. However, their use is limited by damage to bystander tissues, as seen in graft-versus-host disease after donor lymphocyte infusion, or “on-target, off-tumor” toxicities incurred in some engineered T cell therapies. Non-specific immunosuppression and irreversible T cell elimination are currently the only means to control such deleterious responses, but at the cost of abrogating therapeutic benefits or causing secondary complications. On the basis of the physiological paradigm of immune inhibitory receptors, we designed antigen-specific inhibitory chimeric antigen receptors (iCARs) to preemptively constrain T cell responses. We demonstrate that CTLA-4– or PD-1–based iCARs can selectively limit cytokine secretion, cytotoxicity, and proliferation induced through the endogenous T cell receptor or an activating chimeric receptor. The initial effect of the iCAR is temporary, thus enabling T cells to function upon a subsequent encounter with the antigen recognized by their activating receptor. iCARs thus provide a dynamic, self-regulating safety switch to prevent, rather than treat, the consequences of inadequate T cell specificity. PMID:24337479

  3. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease.

    PubMed

    Ellebrecht, Christoph T; Bhoj, Vijay G; Nace, Arben; Choi, Eun Jung; Mao, Xuming; Cho, Michael Jeffrey; Di Zenzo, Giovanni; Lanzavecchia, Antonio; Seykora, John T; Cotsarelis, George; Milone, Michael C; Payne, Aimee S

    2016-07-08

    Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease.

  4. Chimeric Antigen Receptor- and TCR-Modified T Cells Enter Main Street and Wall Street.

    PubMed

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

    2015-08-01

    The field of adoptive cell transfer (ACT) is currently comprised of chimeric Ag receptor (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 it 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. In this overview, we discuss some of the challenges and opportunities that face the field of ACT.

  5. Toll-like receptor 3 gene polymorphisms in South African Blacks with type 1 diabetes.

    PubMed

    Pirie, F J; Pegoraro, R; Motala, A A; Rauff, S; Rom, L; Govender, T; Esterhuizen, T M

    2005-08-01

    Type 1 diabetes is the consequence of exposure of genetically susceptible individuals to specific environmental precipitants. The innate immune system provides the initial response to exogenous antigen and links with the adaptive immune system. The aim of this study was to assess the role of polymorphisms occurring in the cytoplasmic region of toll-like receptor (TLR) 3 gene and immediate 5' sequence, in subjects of Zulu descent with type 1 diabetes in KwaZulu-Natal, South Africa. Seventy-nine subjects with type 1 diabetes and 74 healthy normal glucose tolerant gender-matched control subjects were studied. Parts of exon 4 and exon 3/intron 3 of the TLR3 gene were studied by polymerase chain reaction, direct sequencing and restriction enzyme digestion with Bts 1. Of the nine polymorphisms studied, a significant association with type 1 diabetes was found for the major allele in the 2593 C/T polymorphism and for the minor alleles in the 2642 C/A and 2690 A/G polymorphisms, which were found to be in complete linkage disequilibrium. Correction of the P-values for the number of alleles studied, however, rendered the results no longer significant. These results suggest that polymorphisms in the TLR3 gene, which is part of the innate immune system, may be associated with type 1 diabetes in this population.

  6. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor.

    PubMed

    Foucault, Isabelle; Le Bras, Séverine; Charvet, Céline; Moon, Chéol; Altman, Amnon; Deckert, Marcel

    2005-02-01

    Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function. The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells. However, its involvement in BCR signaling is completely unknown. Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk. To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins. The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms. 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma). Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs). Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs. Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins.

  7. CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy.

    PubMed

    Bridgeman, J S; Ladell, K; Sheard, V E; Miners, K; Hawkins, R E; Price, D A; Gilham, D E

    2014-02-01

    Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions.

  8. Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer.

    PubMed

    Feng, Kaichao; Guo, Yelei; Dai, Hanren; Wang, Yao; Li, Xiang; Jia, Hejin; Han, Weidong

    2016-05-01

    The successes achieved by chimeric antigen receptor-modified T (CAR-T) cells in hematological malignancies raised the possibility of their use in non-small lung cancer (NSCLC). In this phase I clinical study (NCT01869166), patients with epidermal growth factor receptor (EGFR)-positive (>50% expression), relapsed/refractory NSCLC received escalating doses of EGFR-targeted CAR-T cell infusions. The EGFR-targeted CAR-T cells were generated from peripheral blood after a 10 to 13-day in vitro expansion. Serum cytokines in peripheral blood and copy numbers of CAR-EGFR transgene in peripheral blood and in tissue biopsy were monitored periodically. Clinical responses were evaluated with RECIST1.1 and immune- related response criteria, and adverse events were graded with CTCAE 4.0. The EGFR-targeted CAR-T cell infusions were well-tolerated without severe toxicity. Of 11 evaluable patients, two patients obtained partial response and five had stable disease for two to eight months. The median dose of transfused CAR(+) T cells was 0.97×10(7) cells kg(-1) (interquartile range (IQR), 0.45 to 1.09×10(7) cells kg(-1)). Pathological eradication of EGFR positive tumor cells after EGFR-targeted CAR-T cell treatment can be observed in tumor biopsies, along with the CAR-EGFR gene detected in tumor-infiltrating T cells in all four biopsied patients. The EGFR-targeted CAR-T cell therapy is safe and feasible for EGFR-positive advanced relapsed/refractory NSCLC.

  9. Dopamine Receptor Genes Modulate Associative Memory in Old Age.

    PubMed

    Papenberg, Goran; Becker, Nina; Ferencz, Beata; Naveh-Benjamin, Moshe; Laukka, Erika J; Bäckman, Lars; Brehmer, Yvonne

    2017-02-01

    Previous research shows that associative memory declines more than item memory in aging. Although the underlying mechanisms of this selective impairment remain poorly understood, animal and human data suggest that dopaminergic modulation may be particularly relevant for associative binding. We investigated the influence of dopamine (DA) receptor genes on item and associative memory in a population-based sample of older adults (n = 525, aged 60 years), assessed with a face-scene item associative memory task. The effects of single-nucleotide polymorphisms of DA D1 (DRD1; rs4532), D2 (DRD2/ANKK1/Taq1A; rs1800497), and D3 (DRD3/Ser9Gly; rs6280) receptor genes were examined and combined into a single genetic score. Individuals carrying more beneficial alleles, presumably associated with higher DA receptor efficacy (DRD1 C allele; DRD2 A2 allele; DRD3 T allele), performed better on associative memory than persons with less beneficial genotypes. There were no effects of these genes on item memory or other cognitive measures, such as working memory, executive functioning, fluency, and perceptual speed, indicating a selective association between DA genes and associative memory. By contrast, genetic risk for Alzheimer disease (AD) was associated with worse item and associative memory, indicating adverse effects of APOE ε4 and a genetic risk score for AD (PICALM, BIN1, CLU) on episodic memory in general. Taken together, our results suggest that DA may be particularly important for associative memory, whereas AD-related genetic variations may influence overall episodic memory in older adults without dementia.

  10. Carbon dioxide receptor genes in cotton bollworm Helicoverpa armigera

    NASA Astrophysics Data System (ADS)

    Xu, Wei; Anderson, Alisha

    2015-04-01

    Carbon dioxide (CO2) is important in insect ecology, eliciting a range of behaviours across different species. Interestingly, the numbers of CO2 gustatory receptors (GRs) vary among insect species. In the model organism Drosophila melanogaster, two GRs (DmelGR21a and DmelGR63a) have been shown to detect CO2. In the butterfly, moth, beetle and mosquito species studied so far, three CO2 GR genes have been identified, while in tsetse flies, four CO2 GR genes have been identified. In other species including honeybees, pea aphids, ants, locusts and wasps, no CO2 GR genes have been identified from the genome. These genomic differences may suggest different mechanisms for CO2 detection exist in different insects but, with the exception of Drosophila and mosquitoes, limited attention has been paid to the CO2 GRs in insects. Here, we cloned three putative CO2 GR genes from the cotton bollworm Helicoverpa armigera and performed phylogenetic and expression analysis. All three H. armigera CO2 GRs (HarmGR1, HarmGR2 and HarmGR3) are specifically expressed in labial palps, the CO2-sensing tissue of this moth. HarmGR3 is significantly activated by NaHCO3 when expressed in insect Sf9 cells but HarmGR1 and HarmGR2 are not. This is the first report characterizing the function of lepidopteran CO2 receptors, which contributes to our general understanding of the molecular mechanisms of insect CO2 gustatory receptors.

  11. Carbon dioxide receptor genes in cotton bollworm Helicoverpa armigera.

    PubMed

    Xu, Wei; Anderson, Alisha

    2015-04-01

    Carbon dioxide (CO2) is important in insect ecology, eliciting a range of behaviours across different species. Interestingly, the numbers of CO2 gustatory receptors (GRs) vary among insect species. In the model organism Drosophila melanogaster, two GRs (DmelGR21a and DmelGR63a) have been shown to detect CO2. In the butterfly, moth, beetle and mosquito species studied so far, three CO2 GR genes have been identified, while in tsetse flies, four CO2 GR genes have been identified. In other species including honeybees, pea aphids, ants, locusts and wasps, no CO2 GR genes have been identified from the genome. These genomic differences may suggest different mechanisms for CO2 detection exist in different insects but, with the exception of Drosophila and mosquitoes, limited attention has been paid to the CO2 GRs in insects. Here, we cloned three putative CO2 GR genes from the cotton bollworm Helicoverpa armigera and performed phylogenetic and expression analysis. All three H. armigera CO2 GRs (HarmGR1, HarmGR2 and HarmGR3) are specifically expressed in labial palps, the CO2-sensing tissue of this moth. HarmGR3 is significantly activated by NaHCO3 when expressed in insect Sf9 cells but HarmGR1 and HarmGR2 are not. This is the first report characterizing the function of lepidopteran CO2 receptors, which contributes to our general understanding of the molecular mechanisms of insect CO2 gustatory receptors.

  12. Engagement of the B cell receptor for antigen differentially affects B cell responses to Toll-like receptor-7 agonists and antagonists in BXSB mice

    PubMed Central

    Layer, T; Steele, A; Goeken, J A; Fleenor, S; Lenert, P

    2011-01-01

    Nucleic acid sensors of the Toll-like receptor (TLR) family play a well-established role in the pathogenesis of lupus. This is particularly true for a single-stranded RNA-sensing TLR-7 receptor, as lupus mice lacking TLR-7 show ameliorated disease. Cytosine–guanosine dinucleotide (CpG)-DNA-sensing TLR-9, conversely, has a complex regulatory role in systemic lupus erythematosus (SLE). Much less is known about whether signals through the B cell receptor for antigen (BCR) may affect the ability of B cells to respond to suboptimal TLR-7 agonists and antagonists. We studied this question in prediseased BXSB male and female B cells. We found that male B cells responded more vigorously to numerous TLR-7 ligands and this responsiveness was enhanced further upon co-engagement of the BCR. This synergy was seen primarily with the interleukin (IL)-6 secretion. A number of 32-mer inhibitory oligonucleotides (INH-ODNs) with a nuclease-resistant phosphorothioate backbone were capable of blocking TLR-7, but not BCR-induced B cell activation, with an inhibitory concentration (IC)50 of approximately 100 nm. Surprisingly, while the presence of a single TGC motif at the 5′ end of an ODN did not increase its inhibitory capacity, INH-ODNs containing multiple TGC motifs had greater inhibitory potency. When BCR and TLR-7 were co-engaged, INH-ODNs showed a differential effect on B cell activation. Whereas apoptosis protection and G1-M entry completely escaped suppression, IL-6 secretion remained sensitive to inhibition, although with a 10-fold lower potency. Our results suggest that while TLR-7 antagonists may be considered as lupus therapeutics, simultaneous co-engagement of the TLR-7 and BCR might favour autoreactive B cell survival. This hypothesis needs further experimental validation. PMID:21235537

  13. Strategies for B-Cell Receptor Repertoire Analysis in Primary Immunodeficiencies: From Severe Combined Immunodeficiency to Common Variable Immunodeficiency

    PubMed Central

    IJspeert, Hanna; Wentink, Marjolein; van Zessen, David; Driessen, Gertjan J.; Dalm, Virgil A. S. H.; van Hagen, Martin P.; Pico-Knijnenburg, Ingrid; Simons, Erik J.; van Dongen, Jacques J. M.; Stubbs, Andrew P.; van der Burg, Mirjam

    2015-01-01

    The antigen receptor repertoires of B- and T-cells form the basis of the adaptive immune response. The repertoires should be sufficiently diverse to recognize all possible pathogens. However, careful selection is needed to prevent responses to self or harmless antigens. Limited antigen receptor repertoire diversity leads to immunodeficiency, whereas unselected or misdirected repertoires can result in autoimmunity. The antigen receptor repertoire harbors information about abnormalities in many immunological disorders. Recent developments in next generation sequencing allow the analysis of the antigen receptor repertoire in much greater detail than ever before. Analyzing the antigen receptor repertoire in patients with mutations in genes responsible for the generation of the antigen receptor repertoire will give new insights into repertoire formation and selection. In this perspective, we describe strategies and considerations for analysis of the naive and antigen-selected B-cell repertoires in primary immunodeficiency patients with a focus on severe combined immunodeficiency and common variable immunodeficiency. PMID:25904919

  14. Transcript origin analysis identifies antigen-presenting cells as primary targets of socially regulated gene expression in leukocytes

    PubMed Central

    Cole, Steven W.; Hawkley, Louise C.; Arevalo, Jesusa M. G.; Cacioppo, John T.

    2011-01-01

    To clarify the biological rationale for social regulation of gene expression, this study sought to identify the specific immune cell types that are transcriptionally sensitive to subjective social isolation (loneliness). Using reference distributions for the expression of each human gene in each major leukocyte subtype, we mapped the cellular origin of transcripts found to be differentially expressed in the circulating immune cells from chronically lonely individuals. Loneliness-associated genes derived primarily from plasmacytoid dendritic cells, monocytes, and, to a lesser extent, B lymphocytes. Those dynamics reflected per-cell changes in the expression of inducible genes and related more strongly to the subjective experience of loneliness than to objective social network size. Evolutionarily ancient myeloid antigen-presenting cells appear to have evolved a transcriptional sensitivity to socioenvironmental conditions that may allow them to shift basal gene expression profiles to counter the changing microbial threats associated with hostile vs. affine social conditions. PMID:21300872

  15. N-glycosylation converts non-glycoproteins into mannose receptor ligands and reveals antigen-specific T cell responses in vivo

    PubMed Central

    Kreer, Christoph; Kuepper, Janina M; Zehner, Matthias; Quast, Thomas; Kolanus, Waldemar

    2017-01-01

    N-glycosylation is generally accepted to enhance the immunogenicity of antigens because of two main reasons. First, the attachment of glycans enables recognition by endocytic receptors like the mannose receptor (MR) and hence increased uptake by dendritic cells (DCs). Second, foreign glycans are postulated to be immunostimulatory and their recognition could induce DC activation. However, a direct comparison between the immunogenicity of N-glycosylated vs. de-glycosylated proteins in vivo and a direct effect of N-glycosylated antigens on the intrinsic capacity of DCs to activate T cells have not been assessed so far. To analyze whether enforced N-glycosylation is a suited strategy to enhance the immunogenicity of non-glycosylated antigens for vaccination studies, we targeted non-glycoproteins towards the MR by introduction of artificial N-glycosylation using the methylotrophic yeast Komagataella phaffii (previously termed Pichia pastoris). We could demonstrate that the introduction of a single N-X-S/T motif was sufficient for efficient MR-binding and internalization. However, addition of N-glycosylated proteins neither influenced DC maturation nor their general capacity to activate T cells, pointing out that enforced N-glycosylation does not increase the immunogenicity of the antigen per se. Additionally, increased antigen-specific cytotoxic T cell responses in vivo after injection of N-glycosylated compared to de-glycosylated proteins were observed but this effect strongly depended on the epitope tested. A beneficial effect of N-glycosylation on antibody production could not be detected, which might be due to MR-cross-linking on DCs and to concomitant differences in IL-6 production by CD4+ T cells. These observations point out that the effect of N-glycosylation on antigen immunogenicity can vary between different antigens and therefore might have important implications for the development of vaccines using K. phaffii. PMID:28036287

  16. Inhibition of antigen-specific T lymphocyte activation by structurally related Ir gene-controlled polymers. II. Competitive inhibition of I-E- restricted, antigen-specific T cell responses

    PubMed Central

    1984-01-01

    Our previous studies have defined a highly specific competitive inhibition between a pair of structurally related antigens (GT and GAT) for antigen presentation by accessory cells. The present report investigates this phenomenon in a second antigenic system, which is controlled by a distinct Ir gene product. Two GL phi-specific, I-Ed- restricted, interleukin 2-producing T cell hybridomas were constructed. The antigenic fine specificity of these two hybrid clones was distinct. One hybrid reacted solely with GL phi while the second cross-reacted with GLleu and GLT. These latter two copolymers, as well as the antigen GL, were found to inhibit the GL phi response of the non-cross-reactive hybrid. The structurally related antigen G phi was not inhibitory for this clone's response. The cross-reactive GL phi hybrid could also be inhibited, but, in this case, G phi and not GL caused the inhibition. Reciprocal inhibitions could be demonstrated between these and other hybrids (e.g., GAT responsive), indicating a very high degree of specificity to the inhibition. The inhibition caused by the various copolymers was reversible by increasing the concentration of GL phi, This effect was localized to the antigen-presenting cell and not the T cell hybridoma. Functionally, this competition did not appear to be for antigen uptake or general antigen processing. These findings generalize the phenomenon of antigen competition to a second antigen system in the context of a second Ia molecule. The possible mechanisms accounting for the complex pattern of specificities in this system are discussed. PMID:6210339

  17. IgE-associated IGHV genes from venom and peanut allergic individuals lack mutational evidence of antigen selection.

    PubMed

    Wang, Yan; Jackson, Katherine J L; Davies, Janet; Chen, Zhiliang; Gaeta, Bruno A; Rimmer, Janet; Sewell, William A; Collins, Andrew M

    2014-01-01

    Antigen selection of B cells within the germinal center reaction generally leads to the accumulation of replacement mutations in the complementarity-determining regions (CDRs) of immunoglobulin genes. Studies of mutations in IgE-associated VDJ gene sequences have cast doubt on the role of antigen selection in the evolution of the human IgE response, and it may be that selection for high affinity antibodies is a feature of some but not all allergic diseases. The severity of IgE-mediated anaphylaxis is such that it could result from higher affinity IgE antibodies. We therefore investigated IGHV mutations in IgE-associated sequences derived from ten individuals with a history of anaphylactic reactions to bee or wasp venom or peanut allergens. IgG sequences, which more certainly experience antigen selection, served as a control dataset. A total of 6025 unique IgE and 5396 unique IgG sequences were generated using high throughput 454 pyrosequencing. The proportion of replacement mutations seen in the CDRs of the IgG dataset was significantly higher than that of the IgE dataset, and the IgE sequences showed little evidence of antigen selection. To exclude the possibility that 454 errors had compromised analysis, rigorous filtering of the datasets led to datasets of 90 core IgE sequences and 411 IgG sequences. These sequences were present as both forward and reverse reads, and so were most unlikely to include sequencing errors. The filtered datasets confirmed that antigen selection plays a greater role in the evolution of IgG sequences than of IgE sequences derived from the study participants.

  18. Molecular Cloning and Sequence Analysis of the Sta58 Major Antigen Gene of Rickettsia tsutsugamushi: Sequence homology and Antigenic Comparison of Sta58 to the 60-Kilodalton Family of Stress Proteins

    DTIC Science & Technology

    1990-05-01

    on the cell envelopes of Rickettsia 29. Messing, J. 1983. New M13 vectors for cloning. Methods prowazekii, Rickettsia rickettsii , and Rickettsia ...gene of Rickettsia tsu sugamushi:Sequence homology and antigenic comparison to the 60-kilodalton family of stresproteins. 12. PERSONAL AUTHOR(S...IuwRnuiy dy "jmber FIELD GROUP S ROUP Rickettsia tsutsugamushi, antigens, molecular cloning,. FIED_ GROU__ SUB-GROUP scrub typhus, heat-shock proteins

  19. Molecular cloning and sequence analysis of the Sta58 major antigen gene of Rickettsia tsutsugamushi: sequence homology and antigenic comparison of Sta58 to the 60-kilodalton family of stress proteins.

    PubMed Central

    Stover, C K; Marana, D P; Dasch, G A; Oaks, E V

    1990-01-01

    The scrub typhus 58-kilodalton (kDa) antigen (Sta58) of Rickettsia tsutsugamushi is a major protein antigen often recognized by humans infected with scrub typhus rickettsiae. A 2.9-kilobase HindIII fragment containing a complete sta58 gene was cloned in Escherichia coli and found to express the entire Sta58 antigen and a smaller protein with an apparent molecular mass of 11 kDa (Stp11). DNA sequence analysis of the 2.9-kilobase HindIII fragment revealed two adjacent open reading frames encoding proteins of 11 (Stp11) and 60 (Sta58) kDa. Comparisons of deduced amino acid sequences disclosed a high degree of homology between the R. tsutsugamushi proteins Stp11 and Sta58 and the E. coli proteins GroES and GroEL, respectively, and the family of primordial heat shock proteins designated Hsp10 Hsp60. Although the sequence homology between the Sta58 antigen and the Hsp60 protein family is striking, the Sta58 protein appeared to be antigenically distinct among a sample of other bacterial Hsp60 homologs, including the typhus group of rickettsiae. The antigenic uniqueness of the Sta58 antigen indicates that this protein may be a potentially protective antigen and a useful diagnostic reagent for scrub typhus fever. Images PMID:2108930

  20. Conjugation of toll-like receptor-7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects

    PubMed Central

    Wang, Xiao-Dong; Gao, Ning-Ning; Diao, Yu-Wen; Liu, Yu; Gao, Dong; Li, Wang; Wan, Yan-Yan; Zhong, Jing-Jing; Jin, Guang-Yi

    2015-01-01

    AIM: To investigate the effects of our tumor vaccines on reversing immune tolerance and generating therapeutic response. METHODS: Vaccines were synthesized by solid phase using an Fmoc strategy, where a small molecule toll-like receptor-7 agonist (T7) was conjugated to a monoclonal gastric cancer 7 antigen mono-epitope (T7-MG1) or tri-epitope (T7-MG3). Cytokines were measured in both mouse bone marrow dendritic cells and mouse spleen lymphocytes after exposed to the vaccines. BALB/c mice were intraperitoneally immunized with the vaccines every 2 wk for a total of three times, and then subcutaneously challenged with Ehrlich ascites carcinoma (EAC) cells. Three weeks later, the mice were killed, and the tumors were surgically removed and weighed. Serum samples were collected from the mice, and antibody titers were determined by ELISA using an alkaline phosphate-conjugated detection antibody for total IgG. Antibody-dependent cell-mediated cytotoxicity was detected by the lactate dehydrogenase method using natural killer cells as effectors and antibody-labeled EAC cells as targets. Cytotoxic T lymphocyte activities were also detected by the lactate dehydrogenase method using lymphocytes as effectors and EAC cells as targets. RESULTS: Vaccines were successfully synthesized and validated by analytical high performance liquid chromatography and electrospray mass spectrometry, including T7, T7-MG1, and T7-MG3. Rapid inductions of tumor necrosis factor-α and interleukin-12 in bone marrow dendritic cells and interferon γ and interleukin-12 in lymphocytes occurred in vitro after T7, T7-MG1, and T7-MG3 treatment. Immunization with T7-MG3 reduced the EAC tumor burden in BALB/c mice to 62.64% ± 5.55% compared with PBS control (P < 0.01). Six or nine weeks after the first immunization, the monoclonal gastric cancer 7 antigen antibody increased significantly in the T7-MG3 group compared with the PBS control (P < 0.01). As for antibody-dependent cell-mediated cytotoxicity

  1. The T cell antigen receptor CD3:CD4 molecular complex is diminished on the surface of pulmonary lymphocytes.

    PubMed Central

    Marathias, K.; Pinto, C.; Rodberg, G.; Preffer, F.; Wong, J.; Kradin, R.

    1994-01-01

    CD4, a 55-kd cell surface glycoprotein, binds to class II major histocompatibility complex (MHC) (Ia) antigens and functions as a coreceptor for the T cell antigen receptor (Ti alpha beta)-CD3 complex. We have observed that critical elements of the T cell antigen multireceptor complex, including Ti alpha beta, CD3, CD4, but not CD8, were diminished on CD45RO+ pulmonary T lymphocytes but not CD45RO+ peripheral blood T lymphocytes (PBL). Epitopes mapping from the first (D1) to the fourth (D4) extracytoplasmic Ig-like domains of CD4 were expressed to a lesser degree on pulmonary T cells than on PBL (P = 0.002). CD4 expression on pulmonary T cells did not increase after 72 hours of ex vivo culture in complete medium but was restored toward control levels by stimulation with phytohemagglutinin, anti-CD3, or interleukin-2. CD4 mRNA isolated from lung T cells and PBL co-migrated on Northern blots and the total levels of CD4 mRNA were comparable, suggesting that diminished CD4 expression by pulmonary T cells might reflect a posttranscriptional change. To determine whether CD4bright T cells convert with mitogen stimulation to CD4dim cells, PBLs were stimulated with immobilized anti-CD3, anti-CD4, or a molecularly engineered anti-CD3:CD4 bispecific monoclonal antibody and the ratio of the CD4:CD3 mean fluorescence staining intensities was calculated at days 3 and 13. The CD4:CD3 ratio decreased primarily for cells stimulated with anti-CD3:CD4, suggesting that co-ligation of CD3 and CD4 is required for the generation of CD4dim T cells. We conclude that diminished Ti alpha beta-CD3:CD4 expression is a characteristic of T cells in lung that is not shared by peripheral blood T cells in vivo, and speculate that this change reflects T cell activation in a millieu of limited interleukin-2 availability. Images Figure 8 Figure 9 PMID:7977652

  2. Tat engagement of p38 MAP kinase and IRF7 pathways leads to activation of interferon-stimulated genes in antigen-presenting cells.

    PubMed

    Kim, Nayoung; Kukkonen, Sami; Martinez-Viedma, Maria Del Pilar; Gupta, Sumeet; Aldovini, Anna

    2013-05-16

    As a result of its interaction with transcription factors, HIV type 1 (HIV-1) Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells Tat induces the expression of a subset of interferon (IFN)-stimulated genes (ISGs) in the absence of IFNs. We investigated the genome-wide Tat association with promoters in immature dendritic cells and in monocyte-derived macrophages. Among others, Tat associated with the MAP2K6, MAP2K3, and IRF7 promoters that are functionally part of IL-1 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The association correlated with their increased gene expression, increased activation of p38 MAPK and of phosphorylated signal transducer and activator of transcription 1 (STAT1), and consequent induction of ISGs. Probing these pathways with RNA interference, pharmacological p38 MAPK inhibition, and in cell lines lacking STAT1s or the type I IFN receptor chain confirmed the role of MAPKKs and IRF7 in Tat-mediated modulation of ISGs and excluded the involvement of IFNs in this modulation. Tat interaction with the 2 MAPKK and IRF7 promoters in HIV-1-infected cells and the resulting persistent activation of ISGs, which include inflammatory cytokines and chemokines, can contribute to the increased immune activation that characterizes HIV infection.

  3. Perilipin, a critical regulator of fat storage and breakdown, is a target gene of estrogen receptor-related receptor {alpha}

    SciTech Connect

    Akter, Mst. Hasina; Yamaguchi, Tomohiro; Hirose, Fumiko; Osumi, Takashi

    2008-04-11

    Perilipin is a protein localized on lipid droplet surfaces in adipocytes and steroidogenic cells, playing a central role in regulated lipolysis. Expression of the perilipin gene is markedly induced during adipogenesis. We found that transcription from the perilipin gene promoter is activated by an orphan nuclear receptor, estrogen receptor-related receptor (ERR){alpha}. A response element to this receptor was identified in the promoter region by a gene reporter assay, the electrophoretic-gel mobility-shift assay and the chromatin immunoprecipitation assay. Peroxisome proliferator-activated receptor {gamma} coactivator (PGC)-1{alpha} enhanced, whereas small heterodimer partner (SHP) repressed, the transactivating function of ERR{alpha} on the promoter. Thus, the perilipin gene expression is regulated by a transcriptional network controlling energy metabolism, substantiating the functional importance of perilipin in the maintenance of body energy balance.

  4. Dissecting the regulation of yeast genes by the osmotin receptor

    PubMed Central

    Kupchak, Brian R.; Villa, Nancy Y.; Kulemina, Lidia; Lyons, Thomas J.

    2008-01-01

    The Izh2p protein from Saccharomyces cerevisiae is a receptor for the plant antifungal protein, osmotin. Since Izh2p is conserved in fungi, understanding its biochemical function could inspire novel strategies for the prevention of fungal growth. However, it has been difficult to determine the exact role of Izh2p because it has pleiotropic effects on cellular biochemistry. Herein, we demonstrate that Izh2p negatively regulates functionally divergent genes through a CCCTC promoter motif. Moreover, we show that Izh2p-dependent promoters containing this motif are regulated by the Nrg1p/Nrg2p and Msn2p/Msn4p transcription factors. The fact that Izh2p can regulate gene expression through this widely dispersed element presents a reasonable explanation of its pleiotropy. The involvement of Nrg1p/Nrgp2 in Izh2p-dependent gene regulation also suggests a role for this receptor in regulating fungal differentiation in response to stimuli produced by plants. PMID:18625204

  5. The Arg160Trp allele of melanocortin-1 receptor gene might protect against vitiligo.

    PubMed

    Széll, Márta; Baltás, Eszter; Bodai, László; Bata-Csörgo, Zsuzsanna; Nagy, Nikoletta; Dallos, Attila; Pourfarzi, Reza; Simics, Eniko; Kondorosi, Ildikó; Szalai, Zsuzsanna; Tóth, Gábor K; Hunyadi, János; Dobozy, Attila; Kemény, Lajos

    2008-01-01

    Melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) play pivotal roles in the regulation of human pigmentation. We aimed to study whether single nucleotide polymorphisms (SNPs) of the MC1R and ASIP genes contribute to the pathogenesis of the polygenic pigment skin disorder, vitiligo. The PCR-amplified, full-length MC1R gene was studied with sequence analysis, and the 3' untranslated region (3' UTR) SNP of ASIP was detected using restriction fragment length polymorphism. The allele frequency of the ASIP SNP did not show any difference between the skin type, hair color and eye color-matched 97 vitiligo patients and the 59 healthy control individuals. As one of the MC1R polymorphisms showed significantly higher incidence among fair-skinned individuals (Fitzpatrick I+II, n=140) than among dark-skinned individuals (Fitzpatrick III+IV, n=90), both vitiligo patients and controls were divided into two groups and the frequency of the MC1R alleles was studied separately in fair-skinned and dark-skinned subgroups of diseased and healthy groups. C478T, one of the MC1R SNPs studied in 108 fair-skinned vitiligo patients and in 70 fair-skinned healthy control individuals, showed a significant difference (P=0.0262, odds ratio [95% confidence interval]=3.6 [0.0046-0.1003]) in allele frequency between the two groups: the allele frequency was higher in the control group, suggesting protection against vitiligo. Computer prediction of antigenicity has revealed that the Arg160Trp amino acid change caused by this SNP results in a decrease in antigenicity of the affected peptide epitope.

  6. Transcriptionally targeted in vivo gene therapy for carcinoembrionic antigen-producing adenocarcinoma.

    PubMed

    Konishi, F; Maeda, H; Yamanishi, Y; Hiyama, K; Ishioka, S; Yamakido, M

    1999-09-01

    Inoperable adenocarcinoma in colon or lung shows resistance to conventional anti-cancer therapy. For these cancers, the feasibility of transcriptionally targeted killing of carcinoembryonic antigen (CEA)-producing adenocarcinoma cells was investigated. Adenovirus vectors carrying a CEA promoter to express E. coli lacZ (AdCEALacZ) or herpes simplex thymidine kinase (AdCEATK) were made and their in vitro and in vivo tumoricidal effects on CEA-producing or non-producing colon and lung cancer cells were evaluated. In vitro infection with AdCEALacZ showed significantly higher CEA promoter-driven lacZ expression in CEA-producing adenocarcinoma cells including VMRC-LCD and LoVo than in CEA-non-producing cells. AdCEATK-infected LoVo showed higher sensitivity to ganciclovir than control vector-infected LoVo or AdCEATK-infected HeLa both in vitro and in subcutaneously implanted tumors of nude mice. Moreover, total tumor elimination in vivo was achieved by either pre-infection of as few as 30% of cells comprising tumors or by direct in vivo injection of AdCEATK to pre-established LoVo tumors. In addition, CEA promoter-driven lacZ expression in LoVo cells was enhanced by the addition of interleukin-6 (IL-6) in vitro. These results provide a rationale for CEA-promoter-driven, adenovirus-mediated gene therapy for CEA-producing adenocarcinomas in colon and lung with reduced toxicity to normal cells.

  7. Polymorphisms in prostate stem cell antigen gene rs2294008 increase gastric cancer risk in Chinese.

    PubMed

    Zeng, Zhirong; Wu, Xiaoqin; Chen, Fanggen; Yu, Jun; Xue, Ling; Hao, Yuantao; Wang, Yiming; Chen, Minhu; Sung, Joseph J Y; Hu, Pinjin

    2011-05-01

    A recent genome-wide study identified a strong association between polymorphisms in the prostate stem cell antigen (PSCA) gene and the risk of diffuse-type of gastric cancer in Japanese and Korean population. In this case-control study, we aimed to investigate the possible association between PSCA rs2294008 C/T with clinicopathological features and the prognosis of gastric cancer in a Southern Chinese population. Genotypes of 460 gastric cancer patients and 549 controls were determined by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. We found that individuals with at least one copy of the rs2294008T allele (CT or TT genotype) had an increased risk for gastric cancer compared with CC genotype (OR = 1.42, 95% CI = 1.10-1.82, P = 0.006). Further stratification analyses indicated that the effect of PSCA rs2294008T carriers was noteworthy in intestinal type (OR = 1.55, 95% CI = 1.18-2.04, P = 0.002), poorly differentiated (OR = 1.59, 95% CI = 1.19-2.13, P = 0.002), noncardia (OR = 1.55, 95% CI = 1.17-2.04, P = 0.002) subtypes of gastric cancer. Cox proportional hazards analyses demonstrated that TT genotype (HR = 2.12, 95% CI = 1.22-3.69, P = 0.008) as well as TNM staging were prognostic factors of gastric cancer patients. In conclusion, The T allele of PSCA rs2294008 is associated with increased risk of gastric cancer, especially intestinal type, poorly differentiated, early onset, and noncardia gastric cancer in Chinese population. TNM staging and TT genotype might be involved in the prognosis of gastric cancer patients.

  8. Functional characterization of ecdysone receptor gene switches in mammalian cells.

    PubMed

    Panguluri, Siva K; Kumar, Prasanna; Palli, Subba R

    2006-12-01

    Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).

  9. Nuclear Receptor Corepressor Recruitment by Unliganded Thyroid Hormone Receptor in Gene Repression during Xenopus laevis Development

    PubMed Central

    Sachs, Laurent M.; Jones, Peter L.; Havis, Emmanuelle; Rouse, Nicole; Demeneix, Barbara A.; Shi, Yun-Bo

    2002-01-01

    Thyroid hormone receptors (TR) act as activators of transcription in the presence of the thyroid hormone (T3) and as repressors in its absence. While many in vitro approaches have been used to study the molecular mechanisms of TR action, their physiological relevance has not been addressed. Here we investigate how TR regulates gene expression during vertebrate postembryonic development by using T3-dependent amphibian metamorphosis as a model. Earlier studies suggest that TR acts as a repressor during premetamorphosis when T3 is absent. We hypothesize that corepressor complexes containing the nuclear receptor corepressor (N-CoR) are key factors in this TR-dependent gene repression, which is important for premetamorphic tadpole growth. To test this hypothesis, we isolated Xenopus laevis N-CoR (xN-CoR) and showed that it was present in pre- and metamorphic tadpoles. Using a chromatin immunoprecipitation assay, we demonstrated that xN-CoR was recruited to the promoters of T3 response genes during premetamorphosis and released upon T3 treatment, accompanied by a local increase in histone acetylation. Furthermore, overexpression of a dominant-negative N-CoR in tadpole tail muscle led to increased transcription from a T3-dependent promoter. Our data indicate that N-CoR is recruited by unliganded TR to repress target gene expression during premetamorphic animal growth, an important process that prepares the tadpole for metamorphosis. PMID:12446772

  10. Endogenous antigen presentation by autoantigen-transfected Epstein-Barr virus-lymphoblastoid cells. I. Generation of human thyroid peroxidase-reactive T cells and their T cell receptor repertoire.

    PubMed Central

    Martin, A; Magnusson, R P; Kendler, D L; Concepcion, E; Ben-Nun, A; Davies, T F

    1993-01-01

    To develop a model for endogenous thyroid autoantigen presentation, we transfected EBV-transformed B lymphoblastoid cell lines (EBV-LCL), established from patients with autoimmune thyroid disease and normal controls, with cDNA for the human thyroid autoantigen thyroid peroxidase (hTPO). hTPO-antigen presentation to patient peripheral blood T cells was demonstrated after stimulation in vitro for 7 d with irradiated hTPO-transfected or untransfected autologous EBV-LCL. Anti-hTPO-reactive T cells were subsequently cloned in the presence of irradiated, autologous hTPO-transfected EBV-LCL and IL-2.10 T cell-cloned lines exhibited specific hTPO-induced proliferation (stimulation indices of 2.1-7.9) towards autologous hTPO-transfected EBV-LCL, and were subjected to human T cell receptor (hTCR) V gene analysis, using the PCR for the detection of V alpha and V beta hTcR gene families. The results indicated a preferential use of hTCR V alpha 1 and/or V alpha 3 in 9 of the 10 lines. In contrast, hTCR V beta gene family use was more variable. These data demonstrate a model for the endogenous presentation of human thyroid peroxidase in the absence of other thyroid specific antigens. The high frequency of antigen-specific T cells obtained from PBMC using this technique will facilitate further studies at both the functional and hTCR V gene level. Images PMID:7682574

  11. Structural analysis, selection, and ontogeny of the shark new antigen receptor (IgNAR): identification of a new locus preferentially expressed in early development.

    PubMed

    Diaz, Marilyn; Stanfield, Robyn L; Greenberg, Andrew S; Flajnik, Martin F

    2002-10-01

    The new antigen receptor (IgNAR) family has been detected in all elasmobranch species so far studied and has several intriguing structural and functional features. IgNAR protein, found in both transmembrane and secretory forms, is a dimer of heavy chains with no associated light chains, with each chain of the dimer having a single free and flexible V region. Four rearrangement events (among 1V, 3D, and 1J germline genes) generate an expressed NAR V gene, resulting in long and diverse CDR3 regions that contain cysteine residues. IgNAR mutation frequency is very high and "selected" mutations are found only in genes encoding the secreted form, suggesting that the primary repertoire is entirely CDR3-based. Here we further analyzed the two IgNAR types, "type 1" having one cysteine in CDR3 and "type 2" with an even number (two or four) of CDR3 cysteines, and discovered that placement of the disulfide bridges in the IgNAR V domain differentially influences the selection of mutations in CDR1 and CDR2. Ontogenetic analyses showed that IgNAR sequences from young animals were infrequently mutated, consistent with the paradigm that the shark immune system must become mature before high levels of mutation accompanied with selection can occur. Nevertheless, also in agreement with the idea that the IgNAR repertoire is entirely CDR3-based, but unlike studies in most other vertebrates, N-region diversity is present in expressed IgNAR clones at birth. During the investigation of this early IgNAR repertoire we serendipitously detected a third type of IgNAR gene that is expressed in all neonatal tissues; later in life its expression is perpetuated only in the epigonal organ, a tissue recently shown to be a (the?) primary lymphoid tissue in elasmobranchs. This "type 3" IgNAR gene still undergoes three rearrangement events (two D regions are "germline-joined"), yet CDR3 sequences were exactly of the same length and very similar sequence, suggesting that "type 3" CDR3s are selected early

  12. Fully human CD19-specific chimeric antigen receptors for T-cell therapy.

    PubMed

    Sommermeyer, D; Hill, T; Shamah, S M; Salter, A I; Chen, Y; Mohler, K M; Riddell, S R

    2017-02-16

    Impressive results have been achieved by adoptively transferring T-cells expressing CD19-specific CARs with binding domains from murine mAbs to treat B-cell malignancies. T-cell mediated immune responses specific for peptides from the murine scFv antigen-binding domain of the CAR can develop in patients and result in premature elimination of CAR-T-cells increasing the risk of tumor relapse. As fully human scFv might reduce immunogenicity, we generated CD19-specific human scFvs with similar binding characteristics as the murine FMC63-derived scFv using human Ab/DNA-libraries. CARs were constructed in various formats from several scFvs and used to transduce primary human T-cells. The resulting CD19-CAR-T-cells were specifically activated by and lysed CD19-positive tumor cell lines and primary CLL cells, and eliminated human lymphoma xenografts in immunodeficient mice. Certain fully human CAR constructs were superior to the FMC63-CAR, which is widely used in clinical trials. Imaging of cell surface distribution of the human CARs revealed no evidence of clustering without target cell engagement, and tonic signaling was not observed. To further reduce potential immunogenicity of the CARs, we also modified the fusion sites between different CAR components. The described fully human CARs for a validated clinical target may reduce immune rejection compared with murine based CARs.Leukemia accepted article preview online, 16 February 2017. doi:10.1038/leu.2017.57.

  13. Human T-cell receptor variable gene segment families

    SciTech Connect

    Arden, B.; Kabelitz, D.; Clark, S.P.; Mak, T.W.

    1995-10-01

    Multiple DNA and protein sequence alignments have been constructed for the human T-cell receptor {alpha}/{delta}, {beta}, and {gamma} (TCRA/D, B, and G) variable (V) gene segments. The traditional classification into subfamilies was confirmed using a much larger pool of sequences. For each sequence, a name was derived which complies with the standard nomenclature. The traditional numbering of V gene segments in the order of their discovery was continued and changed when in conflict with names of other segments. By discriminating between alleles at the same locus versus genes from different loci, we were able to reduce the number of more than 150 different TCRBV sequences in the database to a repertoire of only 47 functional TCRBV gene segments. An extension of this analysis to the over 100 TCRAV sequences results in a predicted repertoire of 42 functional TCRAV gene segments. Our alignment revealed two residues that distinguish between the highly homologous V{delta} and V{alpha}, one at a site that in V{sub H} contacts the constant region, the other at the interface between immunoglobulin V{sub H} and V{sub L}. This site may be responsible for restricted pairing between certain V{delta} and V{gamma} chains. On the other hand, V{beta} and V{gamma} appear to be related by the fact that their CDR2 length is increased by four residues as compared with that of V{alpha}/{delta} peptides. 150 refs., 12 figs., 5 tabs.

  14. Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived from the Envelope Glycoprotein Gene Sequence

    DTIC Science & Technology

    1990-11-27

    AD________ AD-A230 976 ARMY PROJECT NO: 89PP9961 TITLE: ANALYSIS OF DENGUE VIRUS ENHANCING EPITOPES USING PEPTIDE ANTIGENS DERIVED FROM THE ENVELOPE...INO. INO r CCESSION NO I1I TITLE (Include Security Classification) Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived From the...necessary and identify by block number) Antibody-dependent enhancement (ADE) ot dengue (DEN) virus infection in human mononuclear cells in vitro has been

  15. Cardiac gene expression data and in silico analysis provide novel insights into human and mouse taste receptor gene regulation.

    PubMed

    Foster, Simon R; Porrello, Enzo R; Stefani, Maurizio; Smith, Nicola J; Molenaar, Peter; dos Remedios, Cristobal G; Thomas, Walter G; Ramialison, Mirana

    2015-10-01

    G protein-coupled receptors are the principal mediators of the sweet, umami, bitter, and fat taste qualities in mammals. Intriguingly, the taste receptors are also expressed outside of the oral cavity, including in the gut, airways, brain, and heart, where they have additional functions and contribute to disease. However, there is little known about the mechanisms governing the transcriptional regulation of taste receptor genes. Following our recent delineation of taste receptors in the heart, we investigated the genomic loci encoding for taste receptors to gain insight into the regulatory mechanisms that drive their expression in the heart. Gene expression analyses of healthy and diseased human and mouse hearts showed coordinated expression for a subset of chromosomally clustered taste receptors. This chromosomal clustering mirrored the cardiac expression profile, suggesting that a common gene regulatory block may control the taste receptor locus. We identified unique domains with strong regulatory potential in the vicinity of taste receptor genes. We also performed de novo motif enrichment in the proximal promoter regions and found several overrepresented DNA motifs in cardiac taste receptor gene promoters corresponding to ubiquitous and cardiac-specific transcription factor binding sites. Thus, combining cardiac gene expression data with bioinformatic analyses, this study has provided insights into the noncoding regulatory landscape for taste GPCRs. These findings also have broader relevance for the study of taste GPCRs outside of the classical gustatory system, where understanding the mechanisms controlling the expression of these receptors may have implications for future therapeutic development.

  16. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strains carrying a gene that encodes Eimeria tenella antigen SO7.

    PubMed

    Konjufca, Vjollca; Jenkins, Mark; Wang, Shifeng; Juarez-Rodriguez, Maria Dolores; Curtiss, Roy

    2008-12-01

    Recombinant attenuated Salmonella vaccines against avian coccidiosis were developed to deliver Eimeria species antigens to the lymphoid tissues of chickens via the type 3 secretion system (T3SS) and the type 2 secretion system (T2SS) of Salmonella. For antigen delivery via the T3SS, the Eimeria tenella gene encoding sporozoite antigen SO7 was cloned downstream of the translocation domain of the Salmonella enterica serovar Typhimurium sopE gene in the parental pYA3868 and pYA3870 vectors to generate pYA4156 and pYA4157. Newly constructed T3SS vectors were introduced into host strain chi8879 (Delta phoP233 Delta sptP1033::xylE Delta asdA16), an attenuated derivative of the highly virulent UK-1 strain. The SopE-SO7 fusion protein was secreted by the T3SS of Salmonella. The vector pYA4184 was constructed for delivery of the SO7 antigen via the T2SS. The SO7 protein was toxic to Salmonella when larger amounts were synthesized; thus, the synthesis of this protein was placed under the control of the lacI repressor gene, whose expression in turn was dependent on the amount of available arabinose in the medium. The pYA4184 vector was introduced into host strain chi9242 (Delta phoP233 Delta asdA16 Delta araBAD23 Delta relA198::araC P(BAD) lacI TT [TT is the T4ipIII transcription terminator]). In addition to SO7, for immunization and challenge studies we used the EAMZ250 antigen of Eimeria acervulina, which was previously shown to confer partial protection against E. acervulina challenge when it was delivered via the T3SS. Immunization of chickens with a combination of the SO7 and EAMZ250 antigens delivered via the T3SS induced superior protection against challenge by E. acervulina. In contrast, chickens immunized with SO7 that was delivered via the T2SS of Salmonella were better protected from challenge by E. tenella.

  17. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  18. The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions.

    PubMed

    Gilbert, Hannah E; Asokan, Rengasamy; Holers, V Michael; Perkins, Stephen J

    2006-10-06

    Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.

  19. Lymphocyte recruitment and the kinetics of adhesion receptor expression during the pulmonary immune response to particulate antigen.

    PubMed Central

    Wolber, F. M.; Curtis, J. L.; Milik, A. M.; Fields, T.; Seitzman, G. D.; Kim, K.; Kim, S.; Sonstein, J.; Stoolman, L. M.

    1997-01-01

    The selectins and beta2 integrins participate in the recruitment of neutrophils in acute pulmonary inflammation. However, the cell adhesion receptors that mediate lymphocyte trafficking into the lung have not been defined. This study examined the relationship between cell adhesion molecules on the pulmonary vasculature and on lymphocytes recovered from the lung during a pulmonary immune response to intratracheal (I.T.) sheep red blood cells (SRBCs) in sensitized C57BL/6J mice. Silver-enhanced immunogold staining and reverse transcriptase polymerase chain reaction of lung tissues revealed sustained induction of VCAM-1, E-selectin, and P-selectin on the pulmonary vasculature for up to 7 days after I.T.-SRBC challenge. Neither the MECA 79 nor MECA 367 antigens were induced on the pulmonary vasculature during this period. In the peripheral blood, both CD4 and CD8 T-cell subsets showed an initial increase in P-selectin ligand expression after I.T.-SRBC challenge. The number of P-selectin ligand-positive T cells in the peripheral blood fell as T cells with both P-selectin and, to a lesser extent, E-selectin ligands accumulated in the bronchoalveolar lavage fluid. We conclude that I.T.-SRBC challenge in sensitized mice elicits prolonged synthesis of P-selectin, E-selectin, and VCAM-1 by the lung vasculature as well as selectin ligand synthesis by responding T cells. Furthermore, the entry of selectin-ligand-positive T cells into the circulation and their accumulation in the bronchoalveolar lavage fluid indicates that these receptors may contribute to T cell recruitment. Finally, VCAM-1 on the vasculature may also participate; however, the vascular addressins, required for homing to peripheral and mucosal lymphoid organs, are not essential for T-cell entry into the lung following I.T.-SRBC challenge. Images Figure 2 Figure 3 Figure 5 PMID:9403722

  20. Identification of the binding site in intercellular adhesion molecule 1 for its receptor, leukocyte function-associated antigen 1.

    PubMed Central

    Fisher, K L; Lu, J; Riddle, L; Kim, K J; Presta, L G; Bodary, S C

    1997-01-01

    Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1. Images PMID:9188101

  1. A reference gene set for chemosensory receptor genes of Manduca sexta.

    PubMed

    Koenig, Christopher; Hirsh, Ariana; Bucks, Sascha; Klinner, Christian; Vogel, Heiko; Shukla, Aditi; Mansfield, Jennifer H; Morton, Brian; Hansson, Bill S; Grosse-Wilde, Ewald

    2015-11-01

    The order of Lepidoptera has historically been crucial for chemosensory research, with many important advances coming from the analysis of species like Bombyx mori or the tobacco hornworm, Manduca sexta. Specifically M. sexta has long been a major model species in the field, especially regarding the importance of olfaction in an ecological context, mainly the interaction with its host plants. In recent years transcriptomic data has led to the discovery of members of all major chemosensory receptor families in the species, but the data was fragmentary and incomplete. Here we present the analysis of the newly available high-quality genome data for the species, supplemented by additional transcriptome data to generate a high quality reference gene set for the three major chemosensory receptor gene families, the gustatory (GR), olfactory (OR) and antennal ionotropic receptors (IR). Coupled with gene expression analysis our approach allows association of specific receptor types and behaviors, like pheromone and host detection. The dataset will provide valuable support for future analysis of these essential chemosensory modalities in this species and in Lepidoptera in general.

  2. Update of the androgen receptor gene mutations database.

    PubMed

    Gottlieb, B; Beitel, L K; Lumbroso, R; Pinsky, L; Trifiro, M

    1999-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 309 to 374 during the past year. We have expanded the database by adding information on AR-interacting proteins; and we have improved the database by identifying those mutation entries that have been updated. Mutations of unknown significance have now been reported in both the 5' and 3' untranslated regions of the AR gene, and in individuals who are somatic mosaics constitutionally. In addition, single nucleotide polymorphisms, including silent mutations, have been discovered in normal individuals and in individuals with male infertility. A mutation hotspot associated with prostatic cancer has been identified in exon 5. The database is available on the internet (http://www.mcgill.ca/androgendb/), from EMBL-European Bioinformatics Institute (ftp.ebi.ac.uk/pub/databases/androgen), or as a Macintosh FilemakerPro or Word file (MC33@musica.mcgill.ca).

  3. Evolution of dopamine receptor genes of the D1 class in vertebrates.

    PubMed

    Yamamoto, Kei; Mirabeau, Olivier; Bureau, Charlotte; Blin, Maryline; Michon-Coudouel, Sophie; Demarque, Michaël; Vernier, Philippe

    2013-04-01

    The receptors of the dopamine neurotransmitter belong to two unrelated classes named D1 and D2. For the D1 receptor class, only two subtypes are found in mammals, the D1A and D1B, receptors, whereas additional subtypes, named D1C, D1D, and D1X, have been found in other vertebrate species. Here, we analyzed molecular phylogeny, gene synteny, and gene expression pattern of the D1 receptor subtypes in a large range of vertebrate species, which leads us to propose a new view of the evolution of D1 dopamine receptor genes. First, we show that D1C and D1D receptor sequences are encoded by orthologous genes. Second, the previously identified Cypriniform D1X sequence is a teleost-specific paralog of the D1B sequences found in all groups of jawed vertebrates. Third, zebrafish and several sauropsid species possess an additional D1-like gene, which is likely to form another orthology group of vertebrate ancestral genes, which we propose to name D1E. Ancestral jawed vertebrates are thus likely to have possessed four classes of D1 receptor genes-D1A, D1B(X), D1C(D), and D1E-which arose from large-scale gene duplications. The D1C receptor gene would have been secondarily lost in the mammalian lineage, whereas the D1E receptor gene would have been lost independently in several lineages of modern vertebrates. The D1A receptors are well conserved throughout jawed vertebrates, whereas sauropsid D1C receptors have rapidly diverged, to the point that they were misidentified as D1D. The functional significance of the D1C receptor loss is not known. It is possible that the function may have been substituted with D1A or D1B receptors in mammals, following the disappearance of D1C receptors in these species.

  4. Domain-swapped T cell receptors improve the safety of TCR gene therapy.

    PubMed

    Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

    2016-11-08

    T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.

  5. Domain-swapped T cell receptors improve the safety of TCR gene therapy

    PubMed Central

    Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

    2016-01-01

    T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy. DOI: http://dx.doi.org/10.7554/eLife.19095.001 PMID:27823582

  6. The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters.

    PubMed

    Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

    2017-01-01

    Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains.

  7. The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters

    PubMed Central

    Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

    2017-01-01

    Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains. PMID:28224115

  8. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes

    PubMed Central

    Tamás, Peter; Hawley, Simon A.; Clarke, Rosemary G.; Mustard, Kirsty J.; Green, Kevin; Hardie, D. Grahame; Cantrell, Doreen A.

    2006-01-01

    The adenosine monophosphate (AMP)–activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKα1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca2+ with Ca2+ ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca2+ stimulation of AMPK required the activity of Ca2+–calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca2+ signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it. PMID:16818670

  9. Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase

    SciTech Connect

    Volarevic, S.; Burns, C.M.; Sussman, J.J.; Ashwell, J.D. )

    1990-09-01

    Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least first major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a trasmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and {beta}{sub 2}-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculations based upon the amounts of coprecipitated enzymatic activity or TCR {zeta} chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.

  10. Resolving protein interactions and organization downstream the T cell antigen receptor using single-molecule localization microscopy: a review

    NASA Astrophysics Data System (ADS)

    Sherman, Eilon

    2016-06-01

    Signal transduction is mediated by heterogeneous and dynamic protein complexes. Such complexes play a critical role in diverse cell functions, with the important example of T cell activation. Biochemical studies of signalling complexes and their imaging by diffraction limited microscopy have resulted in an intricate network of interactions downstream the T cell antigen receptor (TCR). However, in spite of their crucial roles in T cell activation, much remains to be learned about these signalling complexes, including their heterogeneous contents and size distribution, their complex arrangements in the PM, and the molecular requirements for their formation. Here, we review how recent advancements in single molecule localization microscopy have helped to shed new light on the organization of signalling complexes in single molecule detail in intact T cells. From these studies emerges a picture where cells extensively employ hierarchical and dynamic patterns of nano-scale organization to control the local concentration of interacting molecular species. These patterns are suggested to play a critical role in cell decision making. The combination of SMLM with more traditional techniques is expected to continue and critically contribute to our understanding of multimolecular protein complexes and their significance to cell function.

  11. Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies.

    PubMed

    Hay, Kevin A; Turtle, Cameron J

    2017-03-01

    Adoptive immunotherapy with chimeric antigen receptor-modified (CAR)-T cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T cell therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers.

  12. VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.

    PubMed Central

    Egerton, M; Ashe, O R; Chen, D; Druker, B J; Burgess, W H; Samelson, L E

    1992-01-01

    Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein. We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule. We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA. Sequence analysis has shown VCP to be a member of a family of ATP binding, homo-oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast. We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation. Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control. Images PMID:1382975

  13. Association between Duffy antigen receptor for chemokines expression and levels of inflammation markers in sickle cell anemia patients.

    PubMed

    Nebor, Danitza; Durpes, Marie Claude; Mougenel, Danielle; Mukisi-Mukaza, Martin; Elion, Jacques; Hardy-Dessources, Marie-Dominique; Romana, Marc

    2010-07-01

    Since inflammation plays a prominent role in the pathogenesis of sickle cell anemia (SCA) and Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes, we analyzed the relationship between the erythrocyte DARC phenotype and clinical expression of SCA. DARC locus was genotyped in 212 SS adult patients followed by the sickle cell center of Guadeloupe (French West Indies). After patients' stratification according to RBC DARC expression, the prevalence of renal disease, leg ulcers, priapism and osteonecrosis was compared between patient groups as well as hematological variables and plasma levels of chemokines. Duffy-positive patients exhibited higher counts of white blood cells (9.95+/-2.36 vs 8.88+/-2.32 10(9)/L, p=0.0066), polynuclear neutrophils (5.1+/-1.73 vs 4.51+/-1.71 10(9)/L, p=0.0227), higher plasma levels of IL-8 (4.46+/-1.22 vs 1.47+/-0.5 pg/mL, p=0.0202) and RANTES (27.8+/-4.3 vs 18.1+/-2.3 ng/mL, p=0.04) than Duffy-negative patients. No association was detected between RBC expression of DARC and the studied complications.

  14. P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

    PubMed Central

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8+ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8+ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8+ T cell immunity. PMID:23940597

  15. 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

  16. Sustained interactions between T cell receptors and antigens promote the differentiation of CD4⁺ memory T cells.

    PubMed

    Kim, Chulwoo; Wilson, Theodore; Fischer, Kael F; Williams, Matthew A

    2013-09-19

    During CD4⁺ T cell activation, T cell receptor (TCR) signals impact T cell fate, including recruitment, expansion, differentiation, trafficking, and survival. To determine the impact of TCR signals on the fate decision of activated CD4⁺ T cells to become end-stage effector or long-lived memory T helper 1 (Th1) cells, we devised a deep-sequencing-based approach that allowed us to track the evolution of TCR repertoires after acute infection. The transition of effector Th1 cells into the memory pool was associated with a significant decrease in repertoire diversity, and the major histocompatibility complex (MHC) class II tetramer off rate, but not tetramer avidity, was a key predictive factor in the representation of individual clonal T cell populations at the memory stage. We conclude that stable and sustained interactions with antigens during the development of Th1 responses to acute infection are a determinative factor in promoting the differentiation of Th1 memory cells.

  17. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors

    PubMed Central

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D.; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L.; Gapin, Laurent; Marrack, Philippa; Kappler, John W.

    2016-01-01

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line–encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC. PMID:27588903

  18. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

    PubMed

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L; Gapin, Laurent; Marrack, Philippa; Kappler, John W

    2016-09-20

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

  19. Kidins220/ARMS binds to the B cell antigen receptor and regulates B cell development and activation

    PubMed Central

    Fiala, Gina J.; Janowska, Iga; Prutek, Fabiola; Hobeika, Elias; Satapathy, Annyesha; Sprenger, Adrian; Plum, Thomas; Seidl, Maximilian; Dengjel, Jörn; Reth, Michael; Cesca, Fabrizia; Brummer, Tilman

    2015-01-01

    B cell antigen receptor (BCR) signaling is critical for B cell development and activation. Using mass spectrometry, we identified a protein kinase D–interacting substrate of 220 kD (Kidins220)/ankyrin repeat–rich membrane-spanning protein (ARMS) as a novel interaction partner of resting and stimulated BCR. Upon BCR stimulation, the interaction increases in a Src kinase–independent manner. By knocking down Kidins220 in a B cell line and generating a conditional B cell–specific Kidins220 knockout (B-KO) mouse strain, we show that Kidins220 couples the BCR to PLCγ2, Ca2+, and extracellular signal-regulated kinase (Erk) signaling. Consequently, BCR-mediated B cell activation was reduced in vitro and in vivo upon Kidins220 deletion. Furthermore, B cell development was impaired at stages where pre-BCR or BCR signaling is required. Most strikingly, λ light chain–positive B cells were reduced sixfold in the B-KO mice, genetically placing Kidins220 in the PLCγ2 pathway. Thus, our data indicate that Kidins220 positively regulates pre-BCR and BCR functioning. PMID:26324445

  20. Elimination of progressive mammary cancer by repeated administrations of chimeric antigen receptor-modified T cells.

    PubMed

    Globerson-Levin, Anat; Waks, Tova; Eshhar, Zelig

    2014-05-01

    Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

  1. Thyrotropin receptor gene alterations in thyroid hyperfunctioning adenomas

    SciTech Connect

    Russo, D.; Arturi, F.; Filetti, S.

    1996-04-01

    Forty-four thyroid autonomously hyperfunctioning adenomas were analyzed to assess the frequency of mutations occurring in the TSH receptor (TSHR). PCR-amplified fragments encompassing the entire exon 10 of the TSHR gene were obtained from the genomic DNA extracted from the tumors and their adjacent normal tissues and were examined by direct nucleotide sequencing. Point mutations were found in 9 of 44 adenomas examined (20%). One mutation occurred in codon 619 (Asp to Gly), four in codon 623 (three were Ala to Ser, one Ala to substitution), two in codon 632 (both Thr to Ile), and two in codon 633 (Asp to Tyr or His). All the alterations were located in a part of the gene coding for an area including the third intracellular loop and the sixth transmembrane domain of the TSH receptor. All mutations were somatic and heterozygotic, and none was simultaneous with alterations of ras or gsp oncogenes. Thus, our data show that in our series of 44 hyperfunctioning thyroid adenomas, a somatic mutation of the TSHR, responsible for the constitutive activation of the cAMP pathway, occurs in 20% of the tumors. 28 refs., 2 tabs.

  2. Variability of the Transferrin Receptor 2 Gene in AMD

    PubMed Central

    Blasiak, Janusz; Dorecka, Mariola; Kowalska, Marta; Pawlowska, Elzbieta; Szaflik, Jerzy; Szaflik, Jacek Pawel

    2014-01-01

    Oxidative stress is a major factor in the pathogenesis of age-related macular degeneration (AMD). Iron may catalyze the Fenton reaction resulting in overproduction of reactive oxygen species. Transferrin receptor 2 plays a critical role in iron homeostasis and variability in its gene may influence oxidative stress and AMD occurrence. To verify this hypothesis we assessed the association between polymorphisms of the TFR2 gene and AMD. A total of 493 AMD patients and 171 matched controls were genotyped for the two polymorphisms of the TFR2 gene: c.1892C>T (rs2075674) and c.−258+123T>C (rs4434553). We also assessed the modulation of some AMD risk factors by these polymorphisms. The CC and TT genotypes of the c.1892C>T were associated with AMD occurrence but the latter only in obese patients. The other polymorphism was not associated with AMD occurrence, but the CC genotype was correlated with an increasing AMD frequency in subjects with BMI < 26. The TT genotype and the T allele of this polymorphism decreased AMD occurrence in subjects above 72 years, whereas the TC genotype and the C allele increased occurrence of AMD in this group. The c.1892C>T and c.−258+123T>C polymorphisms of the TRF2 gene may be associated with AMD occurrence, either directly or by modulation of risk factors. PMID:24648608

  3. Variability of the transferrin receptor 2 gene in AMD.

    PubMed

    Wysokinski, Daniel; Blasiak, Janusz; Dorecka, Mariola; Kowalska, Marta; Robaszkiewicz, Jacek; Pawlowska, Elzbieta; Szaflik, Jerzy; Szaflik, Jacek Pawel

    2014-01-01

    Oxidative stress is a major factor in the pathogenesis of age-related macular degeneration (AMD). Iron may catalyze the Fenton reaction resulting in overproduction of reactive oxygen species. Transferrin receptor 2 plays a critical role in iron homeostasis and variability in its gene may influence oxidative stress and AMD occurrence. To verify this hypothesis we assessed the association between polymorphisms of the TFR2 gene and AMD. A total of 493 AMD patients and 171 matched controls were genotyped for the two polymorphisms of the TFR2 gene: c.1892C>T (rs2075674) and c.-258+123T>C (rs4434553). We also assessed the modulation of some AMD risk factors by these polymorphisms. The CC and TT genotypes of the c.1892C>T were associated with AMD occurrence but the latter only in obese patients. The other polymorphism was not associated with AMD occurrence, but the CC genotype was correlated with an increasing AMD frequency in subjects with BMI < 26. The TT genotype and the T allele of this polymorphism decreased AMD occurrence in subjects above 72 years, whereas the TC genotype and the C allele increased occurrence of AMD in this group. The c.1892C>T and c.-258+123T>C polymorphisms of the TRF2 gene may be associated with AMD occurrence, either directly or by modulation of risk factors.

  4. Tandem insertion sequence-like elements define the expression site for variable antigen genes of Borrelia hermsii.

    PubMed Central

    Barbour, A G; Carter, C J; Burman, N; Freitag, C S; Garon, C F; Bergström, S

    1991-01-01

    The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches. Images PMID:1987053

  5. Evolution of Dopamine Receptor Genes of the D1 Class in Vertebrates

    PubMed Central

    Yamamoto, Kei; Mirabeau, Olivier; Bureau, Charlotte; Blin, Maryline; Michon-Coudouel, Sophie; Demarque, Michaël; Vernier, Philippe

    2013-01-01

    The receptors of the dopamine neurotransmitter belong to two unrelated classes named D1 and D2. For the D1 receptor class, only two subtypes are found in mammals, the D1A and D1B, receptors, whereas additional subtypes, named D1C, D1D, and D1X, have been found in other vertebrate species. Here, we analyzed molecular phylogeny, gene synteny, and gene expression pattern of the D1 receptor subtypes in a large range of vertebrate species, which leads us to propose a new view of the evolution of D1 dopamine receptor genes. First, we show that D1C and D1D receptor sequences are encoded by orthologous genes. Second, the previously identified Cypriniform D1X sequence is a teleost-specific paralog of the D1B sequences found in all groups of jawed vertebrates. Third, zebrafish and several sauropsid species possess an additional D1-like gene, which is likely to form another orthology group of vertebrate ancestral genes, which we propose to name D1E. Ancestral jawed vertebrates are thus likely to have possessed four classes of D1 receptor genes—D1A, D1B(X), D1C(D), and D1E—which arose from large-scale gene duplications. The D1C receptor gene would have been secondarily lost in the mammalian lineage, whereas the D1E receptor gene would have been lost independently in several lineages of modern vertebrates. The D1A receptors are well conserved throughout jawed vertebrates, whereas sauropsid D1C receptors have rapidly diverged, to the point that they were misidentified as D1D. The functional significance of the D1C receptor loss is not known. It is possible that the function may have been substituted with D1A or D1B receptors in mammals, following the disappearance of D1C receptors in these species. PMID:23197594

  6. Anxious behavior induces elevated hippocampal Cb2 receptor gene expression.

    PubMed

    Robertson, James M; Achua, Justin K; Smith, Justin P; Prince, Melissa A; Staton, Clarissa D; Ronan, Patrick J; Summers, Tangi R; Summers, Cliff H

    2017-04-07

    Anxiety is differentially expressed across a continuum of stressful/fearful intensity, influenced endocannabinoid systems and receptors. The hippocampus plays important roles in the regulation of affective behavior, emotion, and anxiety, as well as memory. Location of Cb1/Cb2 receptor action could be important in determining emotional valence, because while the dorsal hippocampus is involved in spatial memory and cognition, the ventral hippocampus has projections to the PFC, BNST, amygdala, and HPA axis, and is important for emotional responses to stress. During repeated social defeat in a Stress-Alternatives Model arena (SAM; an oval open field with escape portals only large enough for smaller mice), smaller C57BL6/N mice are subject to fear conditioning (tone=CS), and attacked by novel larger aggressive CD1 mice (US) over four daily (5min) trials. Each SAM trial presents an opportunity for escape or submission, with stable behavioral responses established by the second day of interaction. Additional groups had access to a running wheel. Social aggression plus fear conditioning stimulates enhanced Cb2 receptor gene expression in the dorsal CA1, dorsal and ventral dentate gyrus subregions in animals displaying a submissive behavioral phenotype. Escape behavior is associated with reduced Cb2 expression in the dorsal CA1 region, with freezing and escape latency correlated with mRNA levels. Escaping and submitting animals with access to running wheels had increased Cb2 mRNA in dorsal DG/CA1. These results suggest that the Cb2 receptor system is rapidly induced during anxiogenic social interactions plus fear conditioning or exercise; with responses potentially adaptive for coping mechanisms.

  7. Analysis of gene selection in reassortant formation between canine rotavirus K9 and human rotaviruses with different antigenic specificities.

    PubMed

    Kobayashi, N; Taniguchi, K; Urasawa, T; Urasawa, S

    1993-01-01

    A number of antigenic mosaic reassortants which have neutralization proteins VP4 and VP7 derived from different parental strains were analysed in order to study gene selection in reassortant formation between animal and human rotaviruses (HRV). These reassortants were isolated from mixed infection of MA-104 cells with canine rotavirus strain K9 (subgroup I and G serotype 3) and HRV strains (with subgroup I or II antigen and G serotype 1-4, 9 or 12 antigen), through repeated selections with anti-VP4 and anti-VP7 neutralizing monoclonal antibodies directed specifically at HRV and K9, respectively. By serological and genomic analyses, all the isolated clones were found to be antigenic mosaic reassortants possessing VP4 of K9 and VP7 of HRV. In the reassortants between strain K9 and one of the six strains of subgroup II HRV, a single or a few genotypes with particular constellations of RNA segments were predominant, with only a few RNA segments including gene 4 (encoding VP4) being derived from K9. In contrast, in the reassortants between strain K9 and any one of the subgroup I HRV, more than nine different genotypes were identified and various RNA segments, except for segments 8 and 10, were derived from K9. These findings indicated that the RNA segments of K9 might be reassorted more readily with those of subgroup I HRV than with those of subgroup II HRV, suggesting the possible existence of functional mechanisms which determine the extent of diversity of genome selection depending on the pairs of parent strains in the reassortant formation.

  8. AP-1 is involved in ICOS gene expression downstream of TCR/CD28 and cytokine receptor signaling.

    PubMed

    Watanabe, Masashi; Nakajima, Shinsuke; Ohnuki, Kazunobu; Ogawa, Shuhei; Yamashita, Masakatsu; Nakayama, Toshinori; Murakami, Yasufumi; Tanabe, Kazunari; Abe, Ryo

    2012-07-01

    It has been proposed that sustained ICOS expression in chronic inflammatory immune conditions, such as autoimmunity and allergy, contributes to symptom exacerbation. Therefore modulation of ICOS gene expression could be a potential therapeutic strategy for such immune diseases. However, the precise molecular mechanisms controlling ICOS gene expression remain poorly understood. In this study, we explored transcription factors involving in ICOS gene expression and examined their roles in a physiological situation. Microarray analysis revealed that one AP-1 molecule, Fos-related antigen-2 (Fra2), was highly correlated with ICOS expression. Ectopic expression of Fra2 and other AP-1 molecules upregulated ICOS expression on T cells. We identified an AP-1-responsive site (AP1-RE) within the ICOS promoter region and demonstrated AP-1 actually binds to AP1-RE upon TCR/CD28 stimulation. Meanwhile, we found several cytokines could upregulate ICOS expression on both naïve and effector T cells in a manner independent of TCR/CD28 stimulation. These cytokine stimuli induced AP-1 binding to AP1-RE. Together, our results indicate AP-1 transcription factors are involved in ICOS gene expression downstream of both TCR/CD28 signaling and cytokine receptor signaling, and suggest AP-1 activation via cytokine receptor signaling may be one of the mechanisms maintaining high level ICOS expression in chronic inflammatory immune responses.

  9. T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-kappaB inhibitor A20.

    PubMed

    Coornaert, Beatrice; Baens, Mathijs; Heyninck, Karen; Bekaert, Tine; Haegman, Mira; Staal, Jens; Sun, Lijun; Chen, Zhijian J; Marynen, Peter; Beyaert, Rudi

    2008-03-01

    The paracaspase MALT1 mediates T cell antigen receptor-induced signaling to the transcription factor NF-kappaB and is indispensable for T cell activation and proliferation. Enhanced expression of MALT1 or aberrant expression of a fusion protein of the apoptosis inhibitor API2 and MALT1 has been linked to mucosa-associated lymphoid tissue lymphoma. Despite the presence of a caspase-like domain, MALT1 proteolytic activity has not yet been demonstrated. Here we show that T cell antigen receptor stimulation induced recruitment of the NF-kappaB inhibitor A20 into a complex of MALT1 and the adaptor protein Bcl-10, leading to MALT1-mediated processing of A20. API2-MALT1 expression likewise resulted in cleavage of A20. MALT1 cleaved human A20 after arginine 439 and impaired its NF-kappaB-inhibitory function. Our studies identify A20 as a substrate of MALT1 and emphasize the importance of MALT1 proteolytic activity in the 'fine tuning' of T cell antigen receptor signaling.

  10. Clonality Analysis of Immunoglobulin Gene Rearrangement by Next-Generation Sequencing in Endemic Burkitt Lymphoma Suggests Antigen Drive Activation of BCR as Opposed to Sporadic Burkitt Lymphoma

    PubMed Central

    Amato, Teresa; Abate, Francesco; Piccaluga, Pierpaolo; Iacono, Michele; Fallerini, Chiara; Renieri, Alessandra; De Falco, Giulia; Ambrosio, Maria Raffaella; Mourmouras, Vaselious; Ogwang, Martin; Calbi, Valeria; Rabadan, Roul; Hummel, Michael; Pileri, Stefano; Bellan, Cristiana

    2016-01-01

    Objectives: Recent studies using next-generation sequencing (NGS) analysis disclosed the importance of the intrinsic activation of the B-cell receptor (BCR) pathway in the pathogenesis of sporadic Burkitt lymphoma (sBL) due to mutations of TCF3/ID3 genes. Since no definitive data are available on the genetic landscape of endemic Burkitt (eBL), we first assessed the mutation frequency of TCF3/ID3 in eBL compared with sBL and subsequently the somatic hypermutation status of the BCR to answer whether an extrinsic activation of BCR signaling could also be demonstrated in Burkitt lymphoma. Methods: We assessed the mutations of TCF3/ID3 by RNAseq and the BCR status by NGS analysis of the immunoglobulin genes (IGs). Results: We detected mutations of TCF3/ID3 in about 30% of the eBL cases. This rate is significantly lower than that detected in sBL (64%). The NGS analysis of IGs revealed intraclonal diversity, suggesting an active targeted somatic hypermutation process in eBL compared with sBL. Conclusions: These findings support the view that the antigenic pressure plays a key role in the pathogenetic pathways of eBL, which may be partially distinct from those driving sBL development. PMID:26712879

  11. TL antigen as a transplantation antigen recognized by TL-restricted cytotoxic T cells

    PubMed Central

    1994-01-01

    In contrast to broadly expressed classical class I antigens of the major histocompatibility complex, structurally closely related TL antigens are expressed in a highly restricted fashion. Unlike classical class I antigens, TL antigens are not known to be targets of cytotoxic T cells or to mediate graft rejection. Whereas classical class I antigens function as antigen-presenting molecules to T cell receptors (TCR), the role of TL is yet to be defined. To elucidate the function of TL, we have derived transgenic mice expressing TL in most tissues including skin by introducing a TL gene, T3b of C57BL/6 mouse origin, driven by the H-2Kb promoter. By grafting the skin of transgenic mice, we demonstrate that TL can serve as a transplantation antigen and mediate a TCR-alpha/beta+ CD8+ cytotoxic T cell response. This T cell recognition of TL does not require antigen presentation by H-2 molecules. Furthermore, we show that C57BL/6 F1 mice develop CD8+ T cells that are cytotoxic for C57BL/6 TL+ leukemia cells, providing further support for the concept t