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Sample records for antigen receptor genes

  1. 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

  2. Models for antigen receptor gene rearrangement: CDR3 length.

    PubMed

    Saada, Ravit; Weinberger, Moran; Shahaf, Gitit; Mehr, Ramit

    2007-06-01

    Despite the various processing steps involved in V(D)J recombination, which could potentially introduce many biases in the length distribution of complementarity determining region 3 (CDR3) segments, the observed CDR3 length distributions for complete repertoires are very close to a normal-like distribution. This raises the question of whether this distribution is simply a result of the random steps included in the process of gene rearrangement, or has been optimized during evolution. We have addressed this issue by constructing a simulation of gene rearrangement, which takes into account the DNA modification steps included in the process, namely hairpin opening, nucleotide additions, and nucleotide deletions. We found that the near-Gaussian- shape of CDR3 length distribution can only be obtained under a relatively narrow set of parameter values, and thus our model suggests that specific biases govern the rearrangement process. In both B-cell receptor (BCR) heavy chain and T-cell receptor beta chain, we obtained a Gaussian distribution using identical parameters, despite the difference in the number and the lengths of the D segments. Hence our results suggest that these parameters most likely reflect the optimal conditions under which the rearrangement process occurs. We have subsequently used the insights gained in this study to estimate the probability of occurrence of two exactly identical BCRs over the course of a human lifetime. Whereas identical rearrangements of the heavy chain are highly unlikely to occur within one human lifetime, for the light chain we found that this probability is not negligible, and hence the light chain CDR3 alone cannot serve as an indicator of B-cell clonality. PMID:17404591

  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.Clin Cancer Res; 22(7); 1559-64. ©2016 AACR. PMID:27037253

  4. Form follows function - the three-dimensional structure of antigen receptor gene loci.

    PubMed

    Fugmann, Sebastian D

    2014-04-01

    Antigen receptor genes are assembled during lymphocyte development from individual gene segments by a somatic gene rearrangement process named V(D)J recombination. This process is tightly regulated to ensure the generation of an unbiased broad primary repertoire of immunoglobulins and T cell receptors, and to prevent aberrant recombination products that could initiate lymphomagenesis. One important mode of regulation that has recently been discovered for the immunoglobulin heavy chain (IGH) gene locus is the adoption of distinct three-dimensional structures of the locus. Changes in the spatial conformation are thought to ensure the appropriate access of the V(D)J recombinase machinery at each developmental stage, and the formation of extensive chromosome loops has been implicated in allowing equal access to widely dispersed gene elements. PMID:24549092

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

    PubMed Central

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

    2006-01-01

    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” TcRδV 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 γ/δ TcRs have for long used structural conformations recognizing free antigen. PMID:16549799

  6. Role of recombination activating genes in the generation of antigen receptor diversity and beyond.

    PubMed

    Nishana, Mayilaadumveettil; Raghavan, Sathees C

    2012-12-01

    V(D)J recombination is the process by which antibody and T-cell receptor diversity is attained. During this process, antigen receptor gene segments are cleaved and rejoined by non-homologous DNA end joining for the generation of combinatorial diversity. The major players of the initial process of cleavage are the proteins known as RAG1 (recombination activating gene 1) and RAG2. In this review, we discuss the physiological function of RAGs as a sequence-specific nuclease and its pathological role as a structure-specific nuclease. The first part of the review discusses the basic mechanism of V(D)J recombination, and the last part focuses on how the RAG complex functions as a sequence-specific and structure-specific nuclease. It also deals with the off-target cleavage of RAGs and its implications in genomic instability. PMID:23039142

  7. Gene-modified T-cell therapy using chimeric antigen receptors for pediatric hematologic malignancies.

    PubMed

    Nakazawa, Yozo

    2016-06-01

    Chimeric antigen receptor (CAR) is the generic name for synthetic T cell receptors redirected to tumor-associated antigens. Most CARs consist of an ectodomain (scFv or ligand), a hinge region, a transmembrane domain, and signaling endodomains derived from one or two co-stimulatory molecules (CD28, 4-1BB, etc) and from a CD3-ζ chain. CD19-targeted CAR T cell therapy has achieved major success in the treatment of B cell malignancies. CD19 CAR-T cells elicited complete remission in 70-90% of adult and pediatric patients with relapsed/refractory acute lymphoblastic leukemia (ALL). CD19 CAR T cell therapy from allogeneic donors including third party donors is a potential option for B-cell malignancies. CAR T cell therapies for myeloma, acute myeloid leukemia, and T-cell leukemia are still under development. Our group is currently preparing a phase I study of CD19 CAR T cell therapy in pediatric and young adult patients with ALL using a non-viral gene transfer method, the piggyBac-transposon system. PMID:27384848

  8. Adoptive Immunotherapy for Hematological Malignancies Using T Cells Gene-Modified to Express Tumor Antigen-Specific Receptors

    PubMed Central

    Fujiwara, Hiroshi

    2014-01-01

    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. PMID:25517545

  9. Epigenetic aspects of lymphocyte antigen receptor gene rearrangement or 'when stochasticity completes randomness'.

    PubMed

    Jaeger, Sébastien; Fernandez, Bastien; Ferrier, Pierre

    2013-06-01

    To perform their specific functional role, B and T lymphocytes, cells of the adaptive immune system of jawed vertebrates, need to express one (and, preferably, only one) form of antigen receptor, i.e. the immunoglobulin or T-cell receptor (TCR), respectively. This end goal depends initially on a series of DNA cis-rearrangement events between randomly chosen units from separate clusters of V, D (at some immunoglobulin and TCR loci) and J gene segments, a biomolecular process collectively referred to as V(D)J recombination. V(D)J recombination takes place in immature T and B cells and relies on the so-called RAG nuclease, a site-specific DNA cleavage apparatus that corresponds to the lymphoid-specific moiety of the VDJ recombinase. At the genome level, this recombinase's mission presents substantial biochemical challenges. These relate to the huge distance between (some of) the gene segments that it eventually rearranges and the need to achieve cell-lineage-restricted and developmentally ordered routines with at times, mono-allelic versus bi-allelic discrimination. The entire process must be completed without any recombination errors, instigators of chromosome instability, translocation and, potentially, tumorigenesis. As expected, such a precisely choreographed and yet potentially risky process demands sophisticated controls; epigenetics demonstrates what is possible when calling upon its many facets. In this vignette, we will recall the evidence that almost from the start appeared to link the two topics, V(D)J recombination and epigenetics, before reviewing the latest advances in our knowledge of this joint venture. PMID:23278765

  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. PMID:24724418

  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. HLA-DR, DQ and T cell antigen receptor constant beta genes in Japanese patients with ulcerative colitis.

    PubMed Central

    Kobayashi, K; Atoh, M; Konoeda, Y; Yagita, A; Inoko, H; Sekiguchi, S

    1990-01-01

    We studied the T cell antigen receptor (TcR) constant beta chain genes on HLA typed Japanese patients with ulcerative colitis (UC). A TcR constant beta EcoRI 6.0-kb fragment was present in all Japanese UC patients (n = 17) but completely absent in the controls (n = 35) (chi2 = 47.6, P less than 0.001). The frequency of HLA-DR2 antigen was significantly higher in UC patients (85% versus 28% in controls, P less than 0.001). Furthermore, HLA-DQw1 antigen was also increased in UC patients (96% versus 60% in controls, P less than 0.001). However, HLA-DR4 antigen was significantly decreased in UC patients (12% versus 37%, P = 0.02). HLA-DR1 antigen was not found in UC patients and was present in only 15% of the controls. These results suggest that TcR beta chain and HLA-DQw1 antigen may be important in the pathogenesis of Japanese UC. Images Fig. 1 PMID:1973647

  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. Antigen Recognition By Variable Lymphocyte Receptors

    SciTech Connect

    Han, B.W.; Herrin, B.R.; Cooper, M.D.; Wilson, I.A.

    2009-05-18

    Variable lymphocyte receptors (VLRs) rather than antibodies play the primary role in recognition of antigens in the adaptive immune system of jawless vertebrates. Combinatorial assembly of leucine-rich repeat (LRR) gene segments achieves the required repertoire for antigen recognition. We have determined a crystal structure for a VLR-antigen complex, VLR RBC36 in complex with the H-antigen trisaccharide from human blood type O erythrocytes, at 1.67 angstrom resolution. RBC36 binds the H-trisaccharide on the concave surface of the LRR modules of the solenoid structure where three key hydrophilic residues, multiple van der Waals interactions, and the highly variable insert of the carboxyl-terminal LRR module determine antigen recognition and specificity. The concave surface assembled from the most highly variable regions of the LRRs, along with diversity in the sequence and length of the highly variable insert, can account for the recognition of diverse antigens by VLRs.

  16. Expression of T cell antigen receptor genes in the thymus of irradiated mice after bone marrow transplantation

    SciTech Connect

    Matsuzaki, G.; Yoshikai, Y.; Kishihara, K.; Nomoto, K.

    1988-01-15

    Sequential appearance of the expression of T cell antigen receptor genes was investigated in the thymus of irradiated mice at the early stage after transplantation of Thy-1 congeneic H-2 compatible allogeneic bone marrow cells. The first cells to repopulate the thymus on day 7 after bone marrow transplantation were intrathymic radioresistant T cell precursors, which expanded mainly to CD4+CD8+ host-type thymocytes by day 14. A high level of gamma gene expression but a much reduced level of alpha and beta gene expression were detected in the host-type thymocytes on day 7. During regeneration of these cells, gamma-chain messages fell to low level and alpha and beta mRNA levels increased. The thymus of the recipients began to be repopulated by donor-derived T cells about 2 wk after bone marrow transplantation and was almost completely replaced by the third week. An ordered expression of gamma then beta and alpha-chain gene transcript was also observed in the donor-type thymocytes at the early stage after bone marrow transplantation. The use of thymocytes at early stage in whole-body irradiated bone marrow chimera provides a pertinent source for investigating the molecular mechanism of T cell differentiation in adult thymus.

  17. Sequential appearance of thymocyte subpopulations and T cell antigen receptor gene messages in the mouse thymus after sublethal irradiation

    SciTech Connect

    Tomooka, S.; Matsuzaki, G.; Kishihara, K.; Tanaka, K.; Yoshikai, Y.; Taniguchi, K.; Himeno, K.; Nomoto, K.

    1987-12-15

    The sequential differentiation patterns of thymocyte were observed with cell surface phenotypes and the expression of T cell antigen receptor in 800 rad irradiated adult mice. Thymus was severely reduced in size and cell number by day 5 after whole body irradiation and rapidly recovered from day 7 to day 14. Surface marker analysis on day 5 after irradiation showed thymocytes with Thy-1low L3T4+/Lyt-2- dominantly existed and suggested that these cells were radioresistant-survived cells. On the other hand, thymocytes on day 7 were composed of a large number of Thy-1high L3T4+/Lyt-2+ blast-like cells and a relatively high proportion of Thy-1high L3T4-/Lyt-2- cells which expressed a large amount of gamma-chain gene messages but scarcely any alpha- and beta-chain gene messages similar to the fetal thymocytes. On day 14, thymocytes were composed mostly of Thy-1high H-2low L3T4+/Lyt-2+ subpopulation which expressed a remarkably low level of gamma-chain gene messages, and high levels of alpha- and beta-chain transcripts analogous to those of normal adult thymus. Taken together, intrathymic radioresistent stem cells for T thymocytes seem to proliferate and differentiate after irradiation with the same pattern as was seen in a fetal thymus development.

  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. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy.

    PubMed

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. PMID:26993168

  20. Organization of the V gene segments in mouse T-cell antigen receptor [alpha]/[delta] locus

    SciTech Connect

    Wang, K.; Klotz, J.L.; Kiser, G.; Bristol, G.; Hays, E.; Lai, E.; Gese, E.; Kronenberg, M.; Hood, L. )

    1994-04-01

    The mouse T-cell receptor (TCR) [alpha]/[delta] was mapped using 17 V[alpha] and 4 V[delta] subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The [alpha]/[delta] locus spans about 1 Mb. The distance between the 3[prime]-most V gene segments (V[delta]1) and the [delta] constant gene (C[delta]) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual V[alpha] gene segments subfamilies are dispersed throughout the locus. In contrast, the V[delta] gene segments V[delta]1 to 5 are clustered at the 3[prime] end of the V gene segments cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the [alpha]/[delta] locus and on organizational features that might influence the expression of specific V gene segments in [gamma][delta] cells. 35 refs., 5 figs., 2 tabs.

  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. Identification and characterization of T-cell antigen receptor-related genes in phylogenetically diverse vertebrate species.

    PubMed

    Rast, J P; Haire, R N; Litman, R T; Pross, S; Litman, G W

    1995-01-01

    Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene superfamily in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems. PMID:7642232

  3. 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

  4. The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen.

    PubMed

    Hiyane, M I; Boscardin, S B; Rodrigues, M M

    2006-03-01

    Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vbeta genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable beta chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice. PMID:16501814

  5. Contribution of allelic variability in prostate specific antigen (PSA) & androgen receptor (AR) genes to serum PSA levels in men with prostate cancer

    PubMed Central

    Chavan, Sushant V.; Maitra, Anurupa; Roy, Nobhojit; Chavan, Padma R.

    2014-01-01

    Background & objectives: Wide variability in serum prostate specific antigen (PSA) levels exists in malignant conditions of the prostate. PSA is expressed in normal range in 20 to 25 per cent of prostate cancer cases even in presence of high grade Gleason score. This study was aimed to assess the influence of genetic variants exhibited by PSA and androgen receptor (AR) genes towards the variable expression of PSA in prostate cancer. Methods: Pre-treatment serum PSA levels from 101 prostate cancer cases were retrieved from medical record. PSA genotype analysis in promoter region and AR gene microsatellite Cytosine/Adenine/Guanine (CAG) repeat analysis in exon 1 region was performed using DNA sequencing and fragment analysis techniques. Results: A total of seven single nucleotide polymorphisms (SNPs) in the PSA promoter region were noted. Only two SNPs viz., 158G/A (P<0.001) in the proximal promoter region and -3845G/A (P<0.001) in enhancer region showed significant association with serum PSA levels. The carriers of homozygous GG genotype (P<0.001) at both of these polymorphic sites showed higher expression of PSA whereas homozygous AA genotype (P<0.001) carriers demonstrated lower PSA levels. The combination effect of PSA genotypes along with stratified AR CAG repeats lengths (long, intermediate and short) was also studied. The homozygous GG genotype along with AR long CAG repeats and homozygous AA genotype along with AR short CAG repeats at position -3845 and -158 showed strong interaction and thus influenced serum PSA levels. Interpretation & conclusions: The genetic variants exhibited by PSA gene at positions -3845G/A and -158G/A may be accountable towards wide variability of serum PSA levels in prostate cancer. Also the preferential binding of G and A alleles at these polymorphic sites along with AR long and short CAG repeats may contribute towards PSA expression. PMID:24820830

  6. Chimeric Antigen Receptor (CAR)-Engineered Lymphocytes for Cancer Therapy

    PubMed Central

    Ramos, Carlos A.; Dotti, Gianpietro

    2011-01-01

    Introduction Chimeric antigen receptors (CARs) usually combine the antigen binding site of a monoclonal antibody with the signal activating machinery of a T cell, freeing antigen recognition from major histocompatibility complex restriction and thus breaking one of the barriers to more widespread application of cellular therapy. Similar to treatment strategies employing monoclonal antibodies, T cells expressing CARs are highly targeted, but additionally offer the potential benefits of active trafficking to tumor sites, in vivo expansion and long term persistence. Furthermore, gene transfer allows the introduction of countermeasures to tumor immune evasion and of safety mechanisms. Areas covered The authors review the basic structure of so-called first and later generation CARs and their potential advantages over other immune therapy systems. It is described how these molecules can be grafted into immune cells (including retroviral and non-retroviral transduction methods) and strategies to improve the in vivo persistence and function of immune cells expressing CARs are discussed. Examples of tumor associated antigens that have been targeted in preclinical models are presented and clinical experience with these modified cells is summarized. Finally, a discussion on safety issues surrounding CAR gene transfer into T cells and potential solutions to them, are presented. Expert opinion Because of recent advances in immunology, genetics and cell processing, CAR-modified T cells will likely play an increasing role in the cellular therapy of cancer, chronic infections and autoimmune disorders. PMID:21463133

  7. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    PubMed

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  8. Designing Chimeric Antigen Receptors to Effectively and Safely Target Tumors

    PubMed Central

    Jensen, Michael C.; Riddell, Stanley R.

    2015-01-01

    The adoptive transfer of T cells engineered to express artificial chimeric antigen receptors (CARs) that target a tumor cell surface molecule has emerged as an exciting new approach for cancer immunotherapy. Clinical trials in patients with advanced B cell malignancies treated with CD19-specific CAR-modified T cells (CAR-T) have shown impressive antitumor efficacy, leading to optimism that this approach will be useful for treating common solid tumors. Because CAR-T cells recognize tumor cells independent of their expression of human leukocyte antigen (HLA) molecules, tumors that escape conventional T cells by downregulating HLA and/or mutating components of the antigen processing machinery can be eliminated. The ability to introduce or delete additional genes in T cells has the potential to provide therapeutic cell products with novel attributes that overcome impediments to immune mediated tumor elimination in immunosuppressive tumor microenvironments. This review will discuss recent concepts in the development of effective and safe synthetic CARs for adoptive T cell therapy (ACT). PMID:25621840

  9. Biologic activity of antigen receptors artificially incorporated onto B lymphocytes.

    PubMed

    Peacock, J S; Londo, T R; Roess, D A; Barisas, B G

    1986-09-15

    We describe a method for incorporating monoclonal antibody molecules onto viable murine lymphocytes and summarize the biologic activity of these artificial receptors on B cells. Mouse spleen cells incubated overnight with palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) in the presence of deoxycholic acid incorporate about 50,000 antibody molecules per cell. When concentrations of deoxycholate and palmitoyl-protein 315 are carefully controlled, this labeling procedure does not affect the viability or the normal functions of the receptor-decorated cells. The incorporated antibody specifically binds DNP-antigens, although it appears to be unable to communicate directly with internal cellular components. Yet when these receptor-decorated, unprimed cells are challenged with any one of several DNP-antigens, up to 42,000 per 10(6) B cells differentiate into Ig-secreting cells. This response is about 23-fold greater than that induced in normal cell cultures and is of the same magnitude as that induced by the polyclonal B cell activator LPS. This, in addition to the observation that only about 3.6% of receptor-decorated B cells responding to DNP-conjugated polymerized flagellin (DNP-POL) produce hapten-specific antibody, demonstrates that these antigens cause polyclonal B cell differentiation. Normal spleen cells in the presence of DNP-POL and irradiated spleen cells bearing the artificial receptors do not exhibit the polyclonal antibody response. Also, the response of receptor-decorated B cell is blocked by high but nontoxic concentrations of the nonimmunogenic hapten DNP-lysine. These observations demonstrate that the polyclonal B cell response in this system requires the binding of antigen to artificial receptors on functionally viable cells. The polyclonal B cell response to a thymus-dependent antigen DNP-conjugated bovine gamma-globulin (DNP-BGG) requires the presence of the carrier-primed T cells. On the other hand, T cell depletion by anti-Thy-1

  10. The basic principles of chimeric antigen receptor (CAR) design

    PubMed Central

    Sadelain, Michel; Brentjens, Renier; Riviere, Isabelle

    2013-01-01

    CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. A multitude of CARs has been reported over the past decade, targeting an array of cell surface tumor antigens. Their biological functions have dramatically changed following the introduction of tri-partite receptors comprising a costimulatory domain, termed second generation CARs. These have recently demonstrated clinical benefit in patients treated with CD19-targeted autologous T cells. CARs may be combined with costimulatory ligands, chimeric costimulatory receptors or cytokines to further enhance T cell potency, specificity and safety. CARs represent a new class of drugs with exciting potential for cancer immunotherapy. PMID:23550147

  11. Taste Receptor Genes

    PubMed Central

    Bachmanov, Alexander A.; Beauchamp, Gary K.

    2009-01-01

    In the past several years, tremendous progress has been achieved with the discovery and characterization of vertebrate taste receptors from the T1R and T2R families, which are involved in recognition of bitter, sweet, and umami taste stimuli. Individual differences in taste, at least in some cases, can be attributed to allelic variants of the T1R and T2R genes. Progress with understanding how T1R and T2R receptors interact with taste stimuli and with identifying their patterns of expression in taste cells sheds light on coding of taste information by the nervous system. Candidate mechanisms for detection of salts, acids, fat, complex carbohydrates, and water have also been proposed, but further studies are needed to prove their identity. PMID:17444812

  12. Targeting Antigens to Dendritic Cell Receptors for Vaccine Development

    PubMed Central

    Apostolopoulos, Vasso; Thalhammer, Theresia; Tzakos, Andreas G.

    2013-01-01

    Dendritic cells (DCs) are highly specialized antigen presenting cells of the immune system which play a key role in regulating immune responses. Depending on the method of antigen delivery, DCs stimulate immune responses or induce tolerance. As a consequence of the dual function of DCs, DCs are studied in the context of immunotherapy for both cancer and autoimmune diseases. In vaccine development, a major aim is to induce strong, specific T-cell responses. This is achieved by targeting antigen to cell surface molecules on DCs that efficiently channel the antigen into endocytic compartments for loading onto MHC molecules and stimulation of T-cell responses. The most attractive cell surface receptors, expressed on DCs used as targets for antigen delivery for cancer and other diseases, are discussed. PMID:24228179

  13. 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. PMID:26362935

  14. 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

  15. Duffy Antigen Receptor for Chemokines Regulates Post-Fracture Inflammation

    PubMed Central

    Rundle, Charles H.; Mohan, Subburaman; Edderkaoui, Bouchra

    2013-01-01

    There is now considerable experimental data to suggest that inflammatory cells collaborate in the healing of skeletal fractures. In terms of mechanisms that contribute to the recruitment of inflammatory cells to the fracture site, chemokines and their receptors have received considerable attention. Our previous findings have shown that Duffy antigen receptor for chemokines (Darc), the non-classical chemokine receptor that does not signal, but rather acts as a scavenger of chemokines that regulate cell migration, is a negative regulator of peak bone density in mice. Furthermore, because Darc is expressed by inflammatory and endothelial cells, we hypothesized that disruption of Darc action will affect post-fracture inflammation and consequently will affect fracture healing. To test this hypothesis, we evaluated fracture healing in mice with targeted disruption of Darc and corresponding wild type (WT) control mice. We found that fracture callus cartilage formation was significantly greater (33%) at 7 days post-surgery in Darc-KO compared to WT mice. The increased cartilage was associated with greater Collagen (Col) II expression at 3 days post-fracture and Col-X at 7 days post-fracture compared to WT mice, suggesting that Darc deficiency led to early fracture cartilage formation and differentiation. We then compared the expression of cytokine and chemokine genes known to be induced during inflammation. Interleukin (Il)-1β, Il-6, and monocyte chemotactic protein 1 were all down regulated in the fractures derived from Darc-KO mice at one day post-fracture, consistent with an altered inflammatory response. Furthermore, the number of macrophages was significantly reduced around the fractures in Darc-KO compared to WT mice. Based on these data, we concluded that Darc plays a role in modulating the early inflammatory response to bone fracture and subsequent cartilage formation. However, the early cartilage formation was not translated with an early bone formation at the

  16. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves' Disease.

    PubMed

    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

  17. 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

  18. [Structure and function of the T-lymphocyte antigen receptor and its role in infectious diseases].

    PubMed

    Peralta Zaragoza, O; Sánchez Magaña, T; Barrera Rodriguez, R; Madrid Marina, V

    1996-01-01

    The T lymphocytes recognize antigens through antigen receptors (TcRs) and the major histocompatibility complex (MHC) molecules: they lysate the cells that bear the antigen, or release cytokines that are mediators of the immune response. The TcRs recognize antigens in the form of short peptides bound to MHC molecules. So far, there are two isotypes of TcR: gamma/delta and alpha/beta, which appear in the sequence during T-cell ontogeny. The process of selection of TcRs during thymic ontogeny obeys to molecular mechanisms which generate intracellular events that will participate in the gene expression of the TcR. The aim of the present paper is to review the molecular, structural, and functional aspects of the TcRs, and their role in human autoimmune infectious disease. PMID:8815490

  19. How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy.

    PubMed

    Gacerez, Albert T; Arellano, Benjamine; Sentman, Charles L

    2016-12-01

    Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590-2598, 2016. © 2016 Wiley Periodicals, Inc. PMID:27163336

  20. Chimeric antigen receptors: driving immunology towards synthetic biology.

    PubMed

    Sadelain, Michel

    2016-08-01

    The advent of second generation chimeric antigen receptors and the CD19 paradigm have ushered a new therapeutic modality in oncology. In contrast to earlier forms of adoptive cell therapy, which were based on the isolation and expansion of naturally occurring T cells, CAR therapy is based on the design and manufacture of engineered T cells with optimized properties. A new armamentarium, comprising not only CARs but also chimeric costimulatory receptors, chimeric cytokine receptors, inhibitory receptors and synthetic Notch receptors, expressed in naïve, central memory or stem cell-like memory T cells, is being developed for clinical use in a wide range of cancers. Immunological principles are thus finding a new purpose thanks to advances in genetic engineering, synthetic biology and cell manufacturing sciences. PMID:27372731

  1. Antigen-specificity using chimeric antigen receptors: the future of regulatory T-cell therapy?

    PubMed

    Boardman, Dominic; Maher, John; Lechler, Robert; Smyth, Lesley; Lombardi, Giovanna

    2016-04-15

    Adoptive regulatory T-cell (Treg) therapy using autologous Tregs expandedex vivois a promising therapeutic approach which is currently being investigated clinically as a means of treating various autoimmune diseases and transplant rejection. Despite this, early results have highlighted the need for potent Tregs to yield a substantial clinical advantage. One way to achieve this is to create antigen-specific Tregs which have been shown in pre-clinical animal models to have an increased potency at suppressing undesired immune responses, compared to polyclonal Tregs. This mini review outlines where Treg therapy currently stands and discusses the approaches which may be taken to generate antigen-specific Tregs, including the potential use of chimeric antigen receptors (CARs), for future clinical trials. PMID:27068938

  2. Design of chimeric antigen receptors with integrated controllable transient functions.

    PubMed

    Juillerat, Alexandre; Marechal, Alan; Filhol, Jean-Marie; Valton, Julien; Duclert, Aymeric; Poirot, Laurent; Duchateau, Philippe

    2016-01-01

    The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug. PMID:26750734

  3. Expression and sequences of T cell antigen receptor beta-chain genes in the thymus at an early stage after sublethal irradiation

    SciTech Connect

    Yuuki, H.; Yoshikai, Y.; Kishihara, K.; Matsuzaki, G.; Ayukawa, K.; Nomoto, K.

    1989-05-15

    The sequential appearance of the thymocyte subpopulations and TCR gene messages occurred in the thymus of AKR mice (H-2k, Mlsa) from 7 to 14 days after sublethal irradiation. The thymocytes on day 7 after irradiation were composed of a large number of CD4+CD8+ blast-like cells and a relatively high proportion of CD4-CD8- cells (15 to 25%) but few CD3highCD4+CD8-/CD4-CD8+ cells. Approximately 22% of the CD4-CD8- cells were CD3high and -27% of the CD3highCD4-CD8- cells (-6% of whole CD4-CD8- cells) were F23.1+. The thymocytes on day 7 expressed a large amount of gamma- and delta-chain gene transcripts but reduced levels of alpha- and beta-chain gene transcripts. The V gene repertoire of 18 functional beta-chain cDNA derived from the thymocytes on day 7 was compared with those of 20 functional beta-chain cDNA derived from the thymocytes on day 14 which were composed of a large number of CD3lowCD4+CD8+ small-sized cells and a small number of CD3highCD4+CD8- cells. It is noteworthy that the distribution of V beta genes expressed in the thymocytes on day 7 was much the same as that in the thymocytes on day 14 but significantly different from that in normal BALB/c thymocytes as previously described. Interestingly, neither V beta 8.1 nor V beta 6 genes, which are important for recognition of the product of the Mlsa locus, was detected in these two cDNA libraries. These results suggest that clonal selection of TCR V beta repertoire, irrespective of positive or negative selection, appears to occur at the early stage of T cell differentiation, i.e., on the blast-like CD4+CD8+ thymocytes.

  4. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

    PubMed

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A

    2016-04-01

    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell

  5. The structure, rearrangement, and ontogenic expression of DB and JB gene segments of the Mexican axolotl T-cell antigen receptor beta chain (TCRB).

    PubMed

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

    1996-01-01

    We sequenced a total of 189 independent rearrangements in which the VB7.1 element is associated with CB1 (99 clones) or CB2 (90 clones) isotypes of the T-cell receptor (TCR) beta chain in the Mexican axolotl. Three stages of development were analyzed: 2.5 months, 10 months, and 25 months. Three JB1 segments were associated with the VB-CB1 rearrangements and six JB2 segments with VB-CB2. As in other vertebrates, some amino acid positions were conserved in all Jbetas (e. g., Phe-108, Gly-109, Gly-111, Thr-112, and Val-116). Two 11 nucleotides DB-like sequences, differed by one (A or T) central residue and could be productively read in the three putative reading frames. Most of the DB1 and JB1 segments were in the VB-CB1 clones, and most of the DB2 and JB2 segments were in the VB-CB2 clones, suggesting that the TCRB locus is organized into independent DB-JB-CB clusters that used the same collection of VB segments. About 40% of the beta-chain VDJ junctions in 2.5-month-old larvae had N nucleotides, compared with about 73% in 10 - 25-month old animals. The beta-chain VDJ junctions had about 30% of defective rearrangements at all stages of development, which could be due to the slow rate of cell division in the axolotl lymphoid organs, and the large genome in this urodele. Many of the axolotl CDRbeta3 sequences deduced for in frame VDJ rearrangements are the same in animals of different origins. Such redundancy could be a statistical effect due to the small number of thymocytes in the developing axolotl, rather than to some bias due to junctional preferences. PMID:8753858

  6. Identification of genes encoding Schistosoma mansoni antigens using an antigenic sequence tag strategy.

    PubMed

    Zouain, C S; Azevedo, V A; Franco, G R; Pena, S D; Goes, A M

    1998-12-01

    Another approach for the identification of genes that code for antigenic products is described using an antigenic sequence tag (AST) strategy. A Schistosoma mansoni adult worm cDNA library was screened with affinity chromatography-purified immunoglobulins from infected human sera and a mild oxidation treatment with sodium periodate. From 1 or both ends of 30 cDNA clones, 30 ASTs were obtained. Of these, 22 were previously known Sm antigens. One clone had matches with entries for other organisms in the databases and 6 had homology with Sm-expressed sequence tags (EST) entries. These clones, together with another 1 that had no significant database matches, were considered new antigenic genes in S. mansoni. The strategy proved to be efficient for the identification of genes that could be used for immunological studies and evaluation as vaccine candidates. PMID:9920341

  7. Functional Development of the T Cell Receptor for Antigen

    PubMed Central

    Ebert, Peter J.R.; Li, Qi-Jing; Huppa, Johannes B.; Davis, Mark M.

    2016-01-01

    For over three decades now, the T cell receptor (TCR) for antigen has not ceased to challenge the imaginations of cellular and molecular immunologists alike. T cell antigen recognition transcends every aspect of adaptive immunity: it shapes the T cell repertoire in the thymus and directs T cell-mediated effector functions in the periphery, where it is also central to the induction of peripheral tolerance. Yet, despite its central position, there remain many questions unresolved: how can one TCR be specific for one particular peptide-major histocompatibility complex (pMHC) ligand while also binding other pMHC ligands with an immunologically relevant affinity? And how can a T cell’s extreme specificity (alterations of single methyl groups in their ligand can abrogate a response) and sensitivity (single agonist ligands on a cell surface are sufficient to trigger a measurable response) emerge from TCR–ligand interactions that are so low in affinity? Solving these questions is intimately tied to a fundamental understanding of molecular recognition dynamics within the many different contexts of various T cell–antigen presenting cell (APC) contacts: from the thymic APCs that shape the TCR repertoire and guide functional differentiation of developing T cells to the peripheral APCs that support homeostasis and provoke antigen responses in naïve, effector, memory, and regulatory T cells. Here, we discuss our recent findings relating to T cell antigen recognition and how this leads to the thymic development of foreign-antigen-responsive αβT cells. PMID:20800817

  8. A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

    PubMed

    Miller, A

    1977-01-01

    The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented. PMID:68706

  9. The mutation G298A-->Ala100Thr on the coding sequence of the Duffy antigen/chemokine receptor gene in non-caucasian Brazilians.

    PubMed

    Estalote, Angela Cristina; Proto-Siqueira, Rodrigo; Silva, Wilson Araújo Da; Zago, Marco Antonio; Palatnik, Marcos

    2005-01-01

    Ala100Thr has been suggested to be a Caucasian genetic marker on the FY*B allele. As the Brazilian population has arisen from miscegenation among Portuguese, Africans, and Indians, this mutation could possibly be found in Euro- and Afro-Brazilians, or in Brazilian Indians. Fifty-three related individuals and a random sample of 100 subjects from the Brazilian population were investigated using the polymerase chain reaction and four restriction fragment length polymorphisms. Confirming the working hypothesis, among the related individuals three Afro-Brazilians (two of them a mother and daughter) and a woman of Amerindian descent had the Ala100Thr mutation on the FY*B allele. Five non-related Euro-Brazilians also carried the mutation. All nine individuals presented the Fy(a-b+) phenotype. We conclude that the Ala100Thr mutation can occur in populations other than Caucasians and that this mutation does not affect Duffy expression on red blood cells. Gene frequencies for this allele in the non-related individuals were in agreement with those of other populations. The Duffy frequencies of two Amerindian tribes were also investigated. PMID:16110438

  10. 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

  11. 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. PMID:26819347

  12. 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.

  13. Advances in chimeric antigen receptor immunotherapy for neuroblastoma.

    PubMed

    Heczey, Andras; Louis, Chrystal U

    2013-12-01

    Neuroblastoma (NBL) is the most common extracranial pediatric solid tumor and has heterogeneous biology and behavior. Patients with high-risk disease have poor prognosis despite complex multimodal therapy; therefore, novel curative approaches are needed. Immunotherapy is a novel therapeutic approach that harnesses the inherent activity of the immune system to control and eliminate malignant cells. One form of immunotherapy uses chimeric antigen receptors (CAR) to target tumor-associated antigens. CARs are derived from the antigen-binding domain of a monoclonal antibody (MAb) coupled with the intracellular signaling portion of the T cell receptor. CARs can combine the specificity and effectiveness of MAbs with the active bio-distribution, direct cytotoxicity, and long-term persistence of T cells. NBL provides an attractive target for CAR immunotherapy as many of its tumor-associated antigens are not expressed at significant levels on normal tissues, thus decreasing potential treatment related toxicity. Two previous clinical trials utilizing L1-cell adhesion molecule (L1-CAM) and disialoganglioside (GD2) specific CARs (GD2-CAR) have demonstrated safety and anti-tumor efficacy in heavily pretreated relapsed/refractory neuroblastoma patients. Based on these promising results and on improved techniques that can further potentiate CAR therapies, two clinical trials are currently investigating the use of GD2-CARs in children with NBL. Several approaches may further enhance anti-tumor activity and persistence of CAR modified cells, and if these can be safely translated into the clinic, CAR-based immunotherapy could become a viable adjunct or potential alternative to conventional treatment options for patients with NBL. PMID:24333408

  14. The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

    PubMed Central

    LIU, Chaohong; FALLEN, Margaret K.; MILLER, Heather; UPADHYAYA, Arpita; SONG, Wenxia

    2014-01-01

    The B cell antigen receptor (BCR) is the sensor on the B cell surface that surveys foreign molecules (antigen) in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR-mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling. PMID:24999354

  15. Chimeric Antigen Receptor T Cell Therapy in Hematology

    PubMed Central

    Ataca, Pınar; Arslan, Önder

    2015-01-01

    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. PMID:26377367

  16. 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. PMID:26377367

  17. Utilizing Chimeric Antigen Receptors to Direct Natural Killer Cell Activity

    PubMed Central

    Hermanson, David L.; Kaufman, Dan S.

    2015-01-01

    Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains. PMID:25972867

  18. Toxicities of chimeric antigen receptor T cells: recognition and management.

    PubMed

    Brudno, Jennifer N; Kochenderfer, James N

    2016-06-30

    Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy. PMID:27207799

  19. Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics.

    PubMed

    Kim, Mi-Gyeong; Kim, Dongyoon; Suh, Soo-Kyung; Park, Zewon; Choi, Min Joung; Oh, Yu-Kyoung

    2016-04-01

    Chimeric antigen receptor-modified T cells (CAR-T) have emerged as a new modality for cancer immunotherapy due to their potent efficacy against terminal cancers. CAR-Ts are reported to exert higher efficacy than monoclonal antibodies and antibody-drug conjugates, and act via mechanisms distinct from T cell receptor-engineered T cells. These cells are constructed by transducing genes encoding fusion proteins of cancer antigen-recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. CAR-Ts are classified as first-, second- and third-generation, depending on the intracellular signaling domain number of T cell receptors. This review covers the current status of CAR-T research, including basic proof-of-concept investigations at the cell and animal levels. Currently ongoing clinical trials of CAR-T worldwide are additionally discussed. Owing to the lack of existing approved products, several unresolved concerns remain with regard to safety, efficacy and manufacturing of CAR-T, as well as quality control issues. In particular, the cytokine release syndrome is the major side-effect impeding the successful development of CAR-T in clinical trials. Here, we have addressed the challenges and regulatory perspectives of CAR-T therapy. PMID:26895243

  20. 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

  1. Rearrangements of T-cell antigen receptor gamma and delta chain genes are detected in the long-term cultured bone marrow cells of athymic nude mice but not in those of euthymic mice.

    PubMed Central

    Yoshikai, Y; Takeda, Y; Ohga, S; Kishihara, K; Matsuzaki, G; Nomoto, K

    1989-01-01

    We have previously shown that extrathymic rearrangements of T-cell receptor (TcR) gamma and delta chain genes occur in the peripheral lymphoid tissues of athymic nude mice. To further determine where the TcR gene rearrangements occur in nude mice, we investigated the rearrangement and expression of the TcR genes in the long-term cultured bone marrow (LTBM) cells which were homogenous in developments without mature T cells as assessed by FACS analysis. The LTBM derived from euthymic mice contained TcR gamma and delta chain genes in germline configuration, while gene rearrangements of both locus were detected in the LTBM cells from nude mice. These results suggested that gamma and delta gene rearrangements do occur in the bone marrow cells of nude mice and that the T-cell precursors in bone marrow may be increased in frequency in such animals. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2541071

  2. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

    PubMed

    Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

    2016-05-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. PMID:26858358

  3. 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. PMID:26129802

  4. Monomeric and oligomeric complexes of the B cell antigen receptor.

    PubMed

    Schamel, W W; Reth, M

    2000-07-01

    The current structural model of the B cell antigen receptor (BCR) describes it as a symmetric protein complex in which one membrane-bound immunoglobulin molecule (mIg) is noncovalently bound on each side by an Ig-alpha/Ig-beta heterodimer. Using peptide-tagged Ig-alpha proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE), and biosynthetical labeling of B cells, we find that the mIg:Ig-alpha/Ig-beta complex has a stoichiometry of 1:1 and not 1:2. An anti-Flag stimulation of B cells coexpressing Flag-tagged and wild-type Ig-alpha proteins results in the phosphorylation of both Ig-alpha proteins, suggesting that on the surface of living B cells, several BCR monomers are in contact with each other. A BN-PAGE analysis after limited detergent lysis provides further evidence for an oligomeric BCR structure. PMID:10933390

  5. Chimeric antigen receptor-modified T cells strike back.

    PubMed

    Frigault, Matthew J; Maus, Marcela V

    2016-07-01

    Chimeric antigen receptors (CARs) are engineered molecules designed to endow a polyclonal T-cell population with the ability to recognize tumor-associated surface antigens. In their simplest form, CARs comprise a targeting moiety in the form of a single-chain variable fragment from an antibody connected to various intracellular signaling domains allowing for T-cell activation. This powerful approach combines the specificity of an antibody with the cytotoxic ability of a T cell. There has been much excitement since early phase trials of CAR-T cells targeting CD19 expressed on B-cell malignancies demonstrated remarkable efficacy in inducing long-term, stable remissions in otherwise relapsed/refractory disease. Despite these successes, we have just begun to understand the intricacies of CAR biology with efforts underway to utilize this platform in the treatment of other, previously refractory malignancies. Challenges currently include identification of viable cancer targets, management strategies for potentially severe and irreversible toxicities and overcoming the immunosuppressive nature of the tumor microenvironment. This review will focus on basic CAR structure and function, previous success and new approaches aimed at the broader application of CAR-T-cell therapy. PMID:27021308

  6. Development of chimeric antigen receptors for multiple myeloma.

    PubMed

    Martínez-Cingolani, Carolina; Bories, Jean Christophe

    2016-04-15

    Multiple myeloma (MM) is a haematologic malignancy characterized by the expansion of monoclonal plasma cells in the bone marrow. It is associated with serum or urine monoclonal protein and organ damage including renal failure, anaemia, hypercalcaemia and bone lesions. Despite recent improvements MM still remains an incurable disease. Previous studies have shown that the adoptive transfer of autologous T-cells modified to express chimeric antigen receptors (CAR) is effective in cases of acute and chronic lymphoid leukaemia. However, the adjustment of CAR-T-cell therapy to MM is hindered by the scarcity of antigens specific to the tumour plasma cells. Most candidate targets are shared by healthy tissues, and entail high risks of toxicity. Therefore several strategies have been proposed to regulate CAR-T-cell function as well as to enhance CAR-T-cell specificity against tumour cells. In this article we summarize the surface markers that have been investigated as targets to eliminate MM plasma cells and the MM-specific CARs that have been developed to date. Then we describe the different CAR-T-cell designs that could be applied in the case of MM to circumvent current problems of toxicity. PMID:27068946

  7. 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

  8. Recognition of Antigen-Specific B Cell Receptors From Chronic Lymphocytic Leukemia Patients By Synthetic Antigen Surrogates

    PubMed Central

    Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas

    2014-01-01

    In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe discovery of non-peptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used for discovery of other classes of antigen surrogates. PMID:25467125

  9. Current status of chimeric antigen receptor therapy for haematological malignancies.

    PubMed

    Maude, Shannon; Barrett, David M

    2016-01-01

    The field of adoptive cell transfer includes chimeric antigen receptor (CAR) engineered T cells, constructs that emerged from basic research into principles of immunology and have transformed into clinically effective therapies for haematological malignancies. T cells engineered to express these artificial receptors hold great promise, but also carry significant risk. While permanent genetic modification of mature T cells appears safe, modulating their in vivo function is difficult, partly because the robust response can trigger other arms of the immune system. Suicide systems and toxicity management with cytokine blockade or signal transduction modulators have emerged as a new frontier in this field, a far cry from early problems getting CAR T cells to work at all. Currently, clinical trials in patients with relapsed or refractory B cell malignancies treated with CD19-specific CAR T cells have induced durable remissions in adults and children. Results from these trials indicate that more work needs to be done to understand biomarkers of efficacy, the role of T cell persistence and how to integrate this care into standard practice. Cell therapy will not be a 'one size fits all' class of medicine, and here we will discuss the development of this therapy and important questions for its future. PMID:26560054

  10. The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor

    PubMed Central

    Schmitt, Thomas M.; Baskar, Sivasubramanian; Lupo-Stanghellini, Maria Teresa; Nishida, Tetsuya; Yamamoto, Tori N.; Bleakley, Marie; Turtle, Cameron J.; Chang, Wen-Chung; Greisman, Harvey A.; Wood, Brent; Maloney, David G.; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2010-01-01

    Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells. PMID:20702778

  11. IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo

    PubMed Central

    Koneru, Mythili; Purdon, Terence J.; Spriggs, David; Koneru, Susmith; Brentjens, Renier J.

    2015-01-01

    A novel approach for the treatment of ovarian cancer includes immunotherapy with genetically engineered T cells targeted to ovarian cancer cell antigens. Using retroviral transduction, T cells can be created that express an artificial T cell receptor (TCR) termed a chimeric antigen receptor (CAR). We have generated a CAR, 4H11-28z, specific to MUC-16ecto antigen, which is the over-expressed on a majority of ovarian tumor cells and is the retained portion of MUC-16 after cleavage of CA-125. We previously demonstrated that T cells modified to express the 4H11-28z CAR eradicate orthotopic human ovarian cancer xenografts in SCID-Beige mice. However, despite the ability of CAR T cells to localize to tumors, their activation in the clinical setting can be inhibited by the tumor microenvironment, as is commonly seen for endogenous antitumor immune response. To potentially overcome this limitation, we have recently developed a construct that co-expresses both MUC16ecto CAR and IL-12 (4H11-28z/IL-12). In vitro, 4H11-28z/IL-12 CAR T cells show enhanced proliferation and robust IFNγ secretion compared to 4H11-28z CAR T cells. In SCID-Beige mice with human ovarian cancer xenografts, IL-12 secreting CAR T cells exhibit enhanced antitumor efficacy as determined by increased survival, prolonged persistence of T cells, and higher systemic IFNγ. Furthermore, in anticipation of translating these results into a phase I clinical trial which will be the first to study IL-12 secreting CAR T cells in ovarian cancer, an elimination gene has been included to allow for deletion of CAR T cells in the context of unforeseen or off-tumor on-target toxicity. PMID:25949921

  12. T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells.

    PubMed

    Zah, Eugenia; Lin, Meng-Yin; Silva-Benedict, Anne; Jensen, Michael C; Chen, Yvonne Y

    2016-06-01

    The adoptive transfer of T cells expressing anti-CD19 chimeric antigen receptors (CARs) has shown remarkable curative potential against advanced B-cell malignancies, but multiple trials have also reported patient relapses due to the emergence of CD19-negative leukemic cells. Here, we report the design and optimization of single-chain, bispecific CARs that trigger robust cytotoxicity against target cells expressing either CD19 or CD20, two clinically validated targets for B-cell malignancies. We determined the structural parameters required for efficient dual-antigen recognition, and we demonstrate that optimized bispecific CARs can control both wild-type B-cell lymphoma and CD19(-) mutants with equal efficiency in vivo To our knowledge, this is the first bispecific CAR capable of preventing antigen escape by performing true OR-gate signal computation on a clinically relevant pair of tumor-associated antigens. The CD19-OR-CD20 CAR is fully compatible with existing T-cell manufacturing procedures and implementable by current clinical protocols. These results present an effective solution to the challenge of antigen escape in CD19 CAR T-cell therapy, and they highlight the utility of structure-based rational design in the development of receptors with higher-level complexity. Cancer Immunol Res; 4(6); 498-508. ©2016 AACRSee related Spotlight by Sadelain, p. 473. PMID:27059623

  13. Candidate vaccine antigens and genes in Pasteurella multocida.

    PubMed

    Adler, B; Bulach, D; Chung, J; Doughty, S; Hunt, M; Rajakumar, K; Serrano, M; van Zanden, A; Zhang, Y; Ruffolo, C

    1999-08-20

    Pasteurella multocida is the causative agent of fowl cholera and other diseases of production animals. Isolates are classified into five groups based on capsular antigens and into 16 serotypes based on LPS antigens. Strains causing fowl cholera are most frequently designated A:1, A:3 or A:4. Whole cell bacterins can provide some degree of protection, but only against the homologous LPS serotype. There is good evidence that cross-protective antigens are expressed only under in vivo conditions. Empirically derived, live, attenuated vaccines can protect against heterologous serotypes, but because the basis for attenuation is undefined, reversion to virulence is not uncommon. Work in our laboratory is aimed at using a variety of approaches to identify potential protective antigens or virulence genes to be used as candidates for attenuating mutations or as the basis for vaccine antigen delivery systems. The gene encoding an outer membrane protein, Oma87, which is a homologue of the D15 protective antigen of Haemophilus influenzae, was cloned and sequenced. Rabbit antiserum prepared against recombinant Oma87 could passively protect mice against infection. Type 4 fimbriae form the basis of vaccines against ovine footrot and bovine keratoconjunctivitis. We have identified type 4 fimbriae on the surface of P. multocida, purified the fimbrial subunit protein, PtfA, and determined its N-terminal amino acid sequence. Subsequent cloning of the ptfA gene and its inactivation will now be used to assess the importance of type 4 fimbriae in virulence. There has long been anecdotal evidence for the importance of capsule in virulence, but unequivocal genetic evidence for such a role is lacking. We have cloned and characterised the capsule biosynthetic locus in P. multocida A:1 and identified four bex genes involved in capsule transport and genes encoding enzymes involved in the biosynthesis and transfer of the N-acetyl glucosamine and glucuronic acid components of the capsule. It has

  14. Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage K8 and Its Genomic Analysis

    PubMed Central

    Pan, Xuewei; Cui, Xiaoli; Zhang, Fenjiao; He, Yang; Li, Lingyan; Yang, Hongjiang

    2016-01-01

    Phage therapy requires the comprehensive understanding of the mechanisms underlying the host-phage interactions. In this work, to identify the genes related to Pseudomonas aeruginosa phage K8 receptor synthesis, 16 phage-resistant mutants were selected from a Tn5G transposon mutant library of strain PAK. The disrupted genetic loci were identified and they were related to O-specific antigen (OSA) synthesis, including gene wbpR, ssg, wbpV, wbpO, and Y880_RS05480, which encoded a putative O-antigen polymerase Wzy. The Lipopolysaccharide profile of the Y880_RS05480 mutant was analyzed and shown to lack the O-antigen. Therefore, the data from characterization of Y880_RS05480 by TMHMM and SDS-PAGE silver staining analysis suggest that this locus might encode Wzy. The complete phage K8 genome was characterized as 93879 bp in length and contained identical 1188-bp terminal direct repeats. Comparative genomic analysis showed that phage K8 was highly homologous to members of the genus PaP1-like phages. On the basis of our genetic findings, OSA of P. aeruginosa PAK is proven to be the receptor of phage K8. The highly conserved structural proteins among the genetic closely related phages suggest that they may recognize the same receptor. PMID:26973628

  15. Evidence of antigen receptor-influenced oligoclonal B lymphocyte expansion in the synovium of a patient with longstanding rheumatoid arthritis.

    PubMed Central

    Lee, S K; Bridges, S L; Kirkham, P M; Koopman, W J; Schroeder, H W

    1994-01-01

    Plasma cell infiltration of synovium is common in longstanding rheumatoid arthritis (RA). The mechanism(s) underlying synovial B cell proliferation remains unclear. One theory invokes nonspecific polyclonal stimuli; another implicates antigen as the driving force. Antigen-driven repertoires are characteristically enriched for related sets of V gene segments containing similar sequence in the antigen binding site (complementarity-determining regions; CDRs). To study the forces shaping B cell proliferation, we analyzed V kappa transcripts expressed in the synovium of an RA patient. We found Humkv325, a developmentally regulated V kappa III gene segment associated with autoantibody reactivity, in > 10% of randomly-chosen synovial C kappa cDNAs. Two sets of sequences contained identical charged amino acid residues at the V kappa-J kappa join, apparently due to N region addition. We generated "signature" oligonucleotides from these CDR3s and probed PCR amplified V kappa products from the synovium and PBLs of the same patient, and from PBLs and spleen of individuals without rheumatic disease. Significant expression of transcripts containing these unique CDR3 sequences occurred only in the patient's synovium. Thus, in this synovium there is expansion of a limited set of B cell clones expressing antigen receptors that bear evidence of antigen selection. Images PMID:8282807

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

    PubMed

    Morris, Emma C; Stauss, Hans J

    2016-06-30

    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

  17. Chimeric antigen receptors and bispecific antibodies to retarget T cells in pediatric oncology

    PubMed Central

    Suzuki, Maya; Curran, Kevin J.; Cheung, Nai-Kong V.

    2016-01-01

    Cancer immunotherapy using antigen-specific T cells has broad therapeutic potential. Chimeric antigen receptors and bispecific antibodies can redirect T cells to kill tumors without human leukocyte antigens (HLA) restriction. Key determinants of clinical potential include the choice of target antigen, antibody specificity, antibody affinity, tumor accessibility, T cell persistence, and tumor immune evasion. For pediatric cancers, additional constraints include their propensity for bulky metastatic disease and the concern for late toxicities from treatment. Nonetheless, the recent preclinical and clinical developments of these T cell based therapies are highly encouraging. PMID:25832831

  18. Mutually Exclusive Expression of Virulence Genes by Malaria Parasites Is Regulated Independently of Antigen Production

    PubMed Central

    Dzikowski, Ron; Frank, Matthias; Deitsch, Kirk

    2006-01-01

    The primary virulence determinant of Plasmodium falciparum malaria parasite–infected cells is a family of heterogeneous surface receptors collectively referred to as PfEMP1. These proteins are encoded by a large, polymorphic gene family called var. The family contains approximately 60 individual genes, which are subject to strict, mutually exclusive expression, with the single expressed var gene determining the antigenic, cytoadherent, and virulence phenotype of the infected cell. The mutually exclusive expression pattern of var genes is imperative for the parasite's ability to evade the host's immune response and is similar to the process of “allelic exclusion” described for mammalian Ig and odorant receptor genes. In mammalian systems, mutually exclusive expression is ensured by negative feedback inhibition mediated by production of a functional protein. To investigate how expression of the var gene family is regulated, we have created transgenic lines of parasites in which expression of individual var loci can be manipulated. Here we show that no such negative feedback system exists in P. falciparum and that this process is dependent solely on the transcriptional regulatory elements immediately adjacent to each gene. Transgenic parasites that are selected to express a var gene in which the PfEMP1 coding region has been replaced by a drug-selectable marker silence all other var genes in the genome, thus effectively knocking out all PfEMP1 expression and indicating that the modified gene is still recognized as a member of the var gene family. Mutually exclusive expression in P. falciparum is therefore regulated exclusively at the level of transcription, and a functional PfEMP1 protein is not necessary for viability or for proper gene regulation in cultured parasites. PMID:16518466

  19. The In Vivo Pattern of Binding of RAG1 and RAG2 to Antigen Receptor Loci

    PubMed Central

    Ji, Yanhong; Resch, Wolfgang; Corbett, Elizabeth; Yamane, Arito; Casellas, Rafael; Schatz, David G.

    2010-01-01

    Summary The critical initial step in V(D)J recombination, binding of RAG1 and RAG2 to recombination signal sequences flanking antigen receptor V, D, and J gene segments, has not previously been characterized in vivo. Here we demonstrate that RAG protein binding occurs in a highly focal manner to a small region of active chromatin encompassing Igκ and Tcrα J gene segments and Igh and Tcrβ J and J-proximal D gene segments. Formation of these small RAG-bound regions, which we refer to as recombination centers, occurs in a developmental stage- and lineage-specific manner. Each RAG protein is independently capable of specific binding within recombination centers. While RAG1 binding was detected only at regions containing recombination signal sequences, RAG2 binds at thousands of sites in the genome containing histone 3 trimethylated at lysine 4. We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination. PMID:20398922

  20. Evolution of an Expanded Mannose Receptor Gene Family

    PubMed Central

    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. PMID:25390371

  1. Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes.

    PubMed

    Hammill, Joanne A; Afsahi, Arya; Bramson, Jonathan L; Helsen, Christopher W

    2016-01-01

    The adoptive transfer of a bolus of tumor-specific T lymphocytes into cancer patients is a promising therapeutic strategy. In one approach, tumor specificity is conferred upon T cells via engineering expression of exogenous receptors, such as chimeric antigen receptors (CARs). Here, we describe the generation and production of both murine and human CAR-engineered T lymphocytes using retroviruses. PMID:27581020

  2. A complement receptor locus: genes encoding C3b/C4b receptor and C3d/Epstein-Barr virus receptor map to 1q32.

    PubMed

    Weis, J H; Morton, C C; Bruns, G A; Weis, J J; Klickstein, L B; Wong, W W; Fearon, D T

    1987-01-01

    The alternative or classical pathways for complement system component C3 may be triggered by microorganisms and antigen-antibody complexes. In particular, an activated fragment of C3, C3b, covalently attaches to microorganisms or antigen-antibody complexes, which in turn bind to the C3b receptor, also known as complement receptor 1. The genes encoding the proteins that constitute the C3-activating enzymes have been cloned and mapped to a "complement activation" locus in the major histocompatibility complex, and we demonstrate in this study such a locus on the long arm of chromosome 1 at band 1q32. PMID:3782802

  3. Antigen

    MedlinePlus

    An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune ... and is trying to fight it off. An antigen may be a substance from the environment, such ...

  4. 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.

  5. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.

    PubMed

    Levine, B L

    2015-03-01

    Performance enhancement of the immune system can now be generated through ex vivo gene modification of T cells in order to redirect native specificity to target tumor antigens. This approach combines the specificity of antibody therapy, the expanded response of cellular therapy and the memory activity of vaccine therapy. Recent clinical trials of chimeric antigen receptor (CAR) T cells directed toward CD19 as a stand-alone therapy have shown sustained complete responses in patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. As these drug products are individually derived from a patient's own cells, a different manufacturing approach is required for this kind of personalized therapy compared with conventional drugs. Key steps in the CAR T-cell manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of modified T cells and cryopreservation in infusible media. In this review, the steps involved in isolating, genetically modifying and scaling-out the CAR T cells for use in a clinical setting are described in the context of in-process and release testing and regulatory standards. PMID:25675873

  6. Insulin receptor substrate 1 translocation to the nucleus by the human JC virus T-antigen.

    PubMed

    Lassak, Adam; Del Valle, Luis; Peruzzi, Francesca; Wang, Jin Ying; Enam, Sahnila; Croul, Sidney; Khalili, Kamel; Reiss, Krzysztof

    2002-05-10

    Insulin receptor substrate 1 (IRS-1) is the major signaling molecule for the insulin and insulin-like growth factor I receptors, which transduces both metabolic and growth-promoting signals, and has transforming properties when overexpressed in the cells. Here we show that IRS-1 is translocated to the nucleus in the presence of the early viral protein-T-antigen of the human polyomavirus JC. Nuclear IRS-1 was detected in T-antigen-positive cell lines and in T-antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JC virus T-antigen binding was localized within the N-terminal portion of IRS-1 molecule, and the binding was independent from IRS-1 tyrosine phosphorylation and was strongly inhibited by IRS-1 serine phosphorylation. In addition, competition for the IRS-1-T-antigen binding by a dominant negative mutant of IRS-1 inhibited growth and survival of JC virus T-antigen-transformed cells in anchorage-independent culture conditions. Based on these findings, we propose a novel role for the IRS-1-T-antigen complex in controlling cellular equilibrium during viral infection. It may involve uncoupling of IRS-1 from its surface receptor and translocation of its function to the nucleus. PMID:11877394

  7. Genetic location of genes encoding enterobacterial common antigen.

    PubMed Central

    Meier, U; Mayer, H

    1985-01-01

    A new rff mutation (rff-726) of Escherichia coli is described which affects the biosynthesis of the enterobacterial common antigen. This mutation was detected in an rfe-defective strain. A Tn10 insertion near the rfe locus was isolated to facilitate further mapping. Both mutations rfe and rff were mapped by transduction with bacteriophage P1, giving the gene order ilv rfe rff uvrD metE. The F' factor F14 was able to complement both mutations rfe and rff, whereas the F' factor F16 could complement the rfe but not the rff mutation. The rff mutation did not affect the biosynthesis of N-acetyl-D-mannosaminuronic acid, as the previously described rff mutations in Salmonella typhimurium do (H. C. Lew, H. Nikaido, and P. H. Mäkelä, J. Bacteriol. 136:227-233, 1978), and also did not affect the biosynthesis of other enterobacterial common antigen components; however, the biosynthesis of the complete enterobacterial common antigen molecule was blocked. PMID:3894334

  8. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

    PubMed

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

  9. Comparison of O-Antigen Gene Clusters of Escherichia coli (Shigella) Sonnei and Plesiomonas shigelloides O17: Sonnei Gained Its Current Plasmid-Borne O-Antigen Genes from P. shigelloides in a Recent Event

    PubMed Central

    Shepherd, James G.; Wang, Lei; Reeves, Peter R.

    2000-01-01

    Escherichia coli Sonnei has an O antigen identical to that of Plesiomonas shigelloides O17, and its O-antigen gene cluster is located on a plasmid. By sequencing the chromosomal O-antigen gene cluster of P. shigelloides O17 and comparing it with that of Sonnei, we showed that Sonnei gained its O-antigen genes recently. PMID:10992522

  10. Human platelet antigen gene frequencies in the Austrian population.

    PubMed

    Holensteiner, A; Walchshofer, S; Adler, A; Kittl, E M; Mayr, W R; Panzer, S

    1995-01-01

    Gene frequencies for the human platelet antigen systems HPA-1, -2, -3, and -5 were determined directly from DNA isolated from cord blood of more than 900 randomly selected Caucasoid newborns in Vienna, Austria. Genotyping was performed by specific amplification of the respective regions coding for platelet glycoproteins GP Ib, IIb, IIIa, and Ia by PCR. These PCR products were analyzed after restriction enzyme digestion and electrophoresis. The observed gene frequencies were: HPA-1a: 0.852, HPA-1b: 0.148; HPA-2a: 0.918, HPA-2b: 0.082; HPA-3a: 0.612, HPA-3b: 0.388; HPA-5a: 0.892, HPA-5b: 0.108. There was a good fit with the Hardy-Weinberg equilibrium. Results from serological determinations and genotyping showed no discrepancies. PMID:7607581

  11. 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

  12. 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

  13. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    PubMed

    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

  14. DNA sequence of a gene encoding a BALB/c mouse Ld transplantation antigen.

    PubMed

    Moore, K W; Sher, B T; Sun, Y H; Eakle, K A; Hood, L

    1982-02-01

    The sequence of a gene, denoted 27.5, encoding a transplantation antigen for the BALB/c mouse has been determined. Gene transfer studies and comparison of the translated sequence with the partial amino acid sequence of the Ld transplantation antigen establish that gene 27.5 encodes an Ld polypeptide. A comparison of the gene 27.5 sequence with several complementary DNA sequences suggests that the BALB/c mouse may contain a number of closely related L-like genes. Gene 27.5 has eight exons that correlate with the structural domains of the transplantation antigen. PMID:7058332

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

    PubMed Central

    Chen, Yamei

    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.

  16. 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...

  17. Topoisomerase inhibitors modulate gene expression of B-cell translocation gene 2 and prostate specific antigen in prostate carcinoma cells.

    PubMed

    Chiang, Kun-Chun; Tsui, Ke-Hung; Chung, Li-Chuan; Yeh, Chun-Nan; Chang, Phei-Lang; Chen, Wen-Tsung; Juang, Horng-Heng

    2014-01-01

    Camptothecin (CPT) and doxorubicin (DOX) have been demonstrated to have potent anti-tumor activity. The B-cell translocation gene 2 (BTG2) is involved in the regulation of cell cycle progression. We evaluated the molecular mechanisms of CPT and DOX on cell proliferation and the expressions of BTG2 and prostate specific antigen (PSA) in prostate carcinoma cells. Our results indicated that CPT or DOX treatments induced Go/G1 cell cycle arrest in LNCaP cells and apoptosis at higher dosage. Immunoblot and transient gene expression assay indicated that CPT or DOX treatments induced p53 and BTG2 gene expression, with the later effect dependent on the p53 response element within BTG2 promoter area since mutation of the p53 response element from GGGAAAGTCC to GGAGTCC or from GGCAGAGCCC to GGCACC by site-directed mutagenesis abolished the stimulation of CPT or DOX on the BTG2 promoter activity, which is also supported by our results that cotreatments of pifithrin-α, an inhibitor of p53 dependent transcriptional activation, blocked the induction of CPT or DOX on BTG2 gene expression. CPT or DOX also downregulated the protein expressions of androgen receptor (AR) and PSA. Transient gene expression assays suggested that CPT or DOX's attenuation of PSA promoter activity is dependent on both the androgen and p53 response elements within of the PSA promoter. Our results indicated that CPT and DOX attenuate cell proliferation via upregulation of BTG2 gene expression through the p53-dependent pathway. The CPT and DOX block the PSA gene expression by upregulation of p53 activity and downregulation of androgen receptor activity. PMID:24586533

  18. Topoisomerase Inhibitors Modulate Gene Expression of B-Cell Translocation Gene 2 and Prostate Specific Antigen in Prostate Carcinoma Cells

    PubMed Central

    Chung, Li-Chuan; Yeh, Chun-Nan; Chang, Phei-Lang; Chen, Wen-Tsung; Juang, Horng-Heng

    2014-01-01

    Camptothecin (CPT) and doxorubicin (DOX) have been demonstrated to have potent anti-tumor activity. The B-cell translocation gene 2 (BTG2) is involved in the regulation of cell cycle progression. We evaluated the molecular mechanisms of CPT and DOX on cell proliferation and the expressions of BTG2 and prostate specific antigen (PSA) in prostate carcinoma cells. Our results indicated that CPT or DOX treatments induced Go/G1 cell cycle arrest in LNCaP cells and apoptosis at higher dosage. Immunoblot and transient gene expression assay indicated that CPT or DOX treatments induced p53 and BTG2 gene expression, with the later effect dependent on the p53 response element within BTG2 promoter area since mutation of the p53 response element from GGGAAAGTCC to GGAGTCC or from GGCAGAGCCC to GGCACC by site-directed mutagenesis abolished the stimulation of CPT or DOX on the BTG2 promoter activity, which is also supported by our results that cotreatments of pifithrin-α, an inhibitor of p53 dependent transcriptional activation, blocked the induction of CPT or DOX on BTG2 gene expression. CPT or DOX also downregulated the protein expressions of androgen receptor (AR) and PSA. Transient gene expression assays suggested that CPT or DOX’s attenuation of PSA promoter activity is dependent on both the androgen and p53 response elements within of the PSA promoter. Our results indicated that CPT and DOX attenuate cell proliferation via upregulation of BTG2 gene expression through the p53-dependent pathway. The CPT and DOX block the PSA gene expression by upregulation of p53 activity and downregulation of androgen receptor activity. PMID:24586533

  19. Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy.

    PubMed

    Maus, Marcela V; June, Carl H

    2016-04-15

    Chimeric antigen receptors (CAR) are engineered fusion proteins constructed from antigen recognition, signaling, and costimulatory domains that can be expressed in cytotoxic T cells with the purpose of reprograming the T cells to specifically target tumor cells. CAR T-cell therapy uses gene transfer technology to reprogram a patient's own T cells to stably express CARs, thereby combining the specificity of an antibody with the potent cytotoxic and memory functions of a T cell. In early-phase clinical trials, CAR T cells targeting CD19 have resulted in sustained complete responses within a population of otherwise refractory patients with B-cell malignancies and, more specifically, have shown complete response rates of approximately 90% in patients with relapsed or refractory acute lymphoblastic leukemia. Given this clinical efficacy, preclinical development of CAR T-cell therapy for a number of cancer indications has been actively investigated, and the future of the CAR T-cell field is extensive and dynamic. Several approaches to increase the feasibility and safety of CAR T cells are currently being explored, including investigation into the mechanisms regulating the persistence of CAR T cells. In addition, numerous early-phase clinical trials are now investigating CAR T-cell therapy beyond targeting CD19, especially in solid tumors. Trials investigating combinations of CAR T cells with immune checkpoint blockade therapies are now beginning and results are eagerly awaited. This review evaluates several of the ongoing and future directions of CAR T-cell therapy.Clin Cancer Res; 22(8); 1875-84. ©2016 AACR SEE ALL ARTICLES IN THIS CCR FOCUS SECTION, "OPPORTUNITIES AND CHALLENGES IN CANCER IMMUNOTHERAPY". PMID:27084741

  20. 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

  1. Increasing the Antigenicity of Synthetic Tumor-Associated Carbohydrate Antigens by Targeting Toll-Like Receptors

    PubMed Central

    Ingale, Sampat; Wolfert, Margreet A.; Buskas, Therese; Boons, Geert-Jan

    2009-01-01

    Epithelial cancer cells often over express mucins that are aberrantly glycosylated. Although it has been realized that these compounds offer exciting opportunities for the development of immunotherapy for cancer, their use is hampered by the low antigenicity of classical immunogens composed of a glycopeptide derived from a mucin conjugated to a foreign carrier protein. We have designed, chemically synthesized, and immunologically evaluated a number of fully synthetic vaccine candidates to establish a strategy to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. The compounds were also designed to study in detail the importance of TLR engagement for these antigenic responses. We have found that covalent attachment of a TLR2 agonist, a promiscuous peptide T-helper epitope, and a tumor-associated glycopeptide, gives a compound (1) that elicit in mice exceptionally high titers of IgG antibodies which recognize MCF7 cancer cells expressing the tumor-associated carbohydrate. Immunizations with glycolipopeptide (2), which contains lipidated amino acids instead of a TLR2 ligand, gave significantly lower titers of IgG antibodies demonstrating that TLR engagement is critical for optimum antigenic responses. Although mixtures of compound 2 with Pam3CysSK4 (3) or monophosphoryl lipid A (4) elicited similar titers of IgG antibodies compared to 1, the resulting antisera had an impaired ability to recognize cancer cells. It was also found that it is essential to covalently link the helper T-epitope to B-epitope probably because internalization of the helper T-epitope by B-cells requires assistance of the B-epitope. The results presented here show that synthetic vaccine development is amenable to structure activity relationship studies for successful optimization of carbohydrate-based cancer vaccines. PMID:19145607

  2. Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors

    PubMed Central

    Harding, Clifford V.; Boom, W. Henry

    2011-01-01

    Mycobacterium tuberculosis survives in antigen-presenting cells (APCs) such as macrophages and dendritic cells. APCs present antigens in association with major histocompatibility complex (MHC) class II molecules to stimulate CD4+ T cells, and this process is essential to contain M. tuberculosis infection. Immune evasion allows M. tuberculosis to establish persistent or latent infection in macrophages and results in Toll-like receptor 2 (TLR2)-dependent inhibition of MHC class II transactivator expression, MHC class II molecule expression and antigen presentation. This reduction of antigen presentation might reflect a general mechanism of negative-feedback regulation that prevents excessive T cell-mediated inflammation and that M. tuberculosis has subverted to create a niche for survival in infected macrophages and evasion of recognition by CD4+ T cells. PMID:20234378

  3. Structure of the human progesterone receptor gene.

    PubMed

    Misrahi, M; Venencie, P Y; Saugier-Veber, P; Sar, S; Dessen, P; Milgrom, E

    1993-11-16

    The complete organization of the human progesterone receptor (hPR) gene has been determined. It spans over 90 kbp and contains eight exons. The first exon encodes the N-terminal part of the receptor. The DNA binding domain is encoded by two exons, each exon corresponding to one zinc finger. The steroid binding domain is encoded by five exons. The nucleotide sequence of 1144 bp of the 5' flanking region has been determined. PMID:8241270

  4. The role of antigenically different virus neuraminidases as structures implicated in receptor-binding processes.

    PubMed

    Coimbra, M V; Luiz, M O; Cabral, M C; Couceiro, J N

    1995-06-01

    Influenza A viruses exhibit segmented nucleic acid coding for eight different proteins, two of them as glycoproteins exposed on their lipoprotein envelopes, hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin exhibits receptor-binding activity while neuraminidase develops sialidase cleavage activity which acts on cell receptors. Influenza A strains responsible for human, avian, equine and porcine respiratory infections all over the world present antigenically different hemagglutinin (H1 to H14) and neuraminidase (N1 to N9) structures on their surface. The objective of the present investigation was to study the role of N2, N8, and N9, antigenically diverse neuraminidase structures of human (N2) and animal (N8 and N9) influenza viruses, in the receptor-binding process. Receptor-binding activity of N2 and N8 was analyzed by crossed tests using H3N2 and H3N8 antisera and the hemagglutination inhibition test as a model. Hemagglutinating activity of antigenically different N2 and N8 structures was demonstrable and was inhibited by homologous antisera (N2-H3N2, N8-H3N8) but not by heterologous antisera (N2-H3N8,N8-H3N2). This previously demonstrated N9 hemagglutinating activity was analyzed for receptor-binding specificity using hemagglutination tests and NeuAc alpha2,3Gal and NeuAc alpha2,6Gal derivatized erythrocytes. This highly purified N9 strain was obtained from a virus strain isolated from terns by Dr. Peter Colman (CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia). It exhibited receptor-binding specificity for NeuAc alpha2,3Gal sequences, a property similar to that observed in hemagglutinins from avian strains. These results indicate the importance of antigenically different neuraminidase structures as alternative agents for developing receptor-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8547843

  5. Cell-type specific regulation of gene expression by simian virus 40 T antigens

    SciTech Connect

    Cantalupo, Paul G.; Saenz-Robles, Maria Teresa; Rathi, Abhilasha V.; Beerman, Rebecca W.; Patterson, William H.; Whitehead, Robert H.; Pipas, James M.

    2009-03-30

    SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25741594

  7. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

    PubMed

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies. PMID:23840834

  8. Comparison of Lentiviral and Sleeping Beauty Mediated αβ T Cell Receptor Gene Transfer

    PubMed Central

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm’s tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies. PMID:23840834

  9. 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

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

    PubMed

    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

  11. Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic

    SciTech Connect

    Ayeb, M.E.; Bahraoui, E.M.; Granier, C.; Beress, L.; Rochat, H.

    1986-11-04

    Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp=9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and ..cap alpha..-NH/sub 2/ of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.

  12. On the logic of restrictive recognition of peptide by the T-cell antigen receptor

    PubMed Central

    2011-01-01

    This essay provides an analysis of the inadequacy of the current view of restrictive recognition of peptide by the T-cell antigen receptor. A competing model is developed, and the experimental evidence for the prevailing model is reinterpreted in the new framework. The goal is to contrast the two models with respect to their consistency, coverage of the data, explanatory power, and predictability. PMID:20931295

  13. Augmentation of Antigen Receptor–mediated Responses by Histamine H1 Receptor Signaling

    PubMed Central

    Banu, Yasmin; Watanabe, Takeshi

    1999-01-01

    Histamine is considered one of the important mediators of immediate hypersensitivity and inflammation, and acts via G protein–coupled receptors. Here, we report that histamine may affect antigen receptor–mediated immune responses of T and B cells via a signal(s) from histamine H1 receptors (H1Rs). Histamine exhibited enhancing effects on the in vitro proliferative responses of anti-CD3ε– or anti-IgM–stimulated spleen T and B cells, respectively, at the culture condition that the fetal calf serum was dialyzed before culture and c-kit–positive cells were depleted from the spleen cells. In studies of histamine H1R knockout mice, H1R-deficient T cells had low proliferative responses to anti-CD3ε cross-linking or antigen stimulation in vitro. B cells from H1R-deficient mice were also affected, demonstrating low proliferative responses to B cell receptor cross-linking. Antibody production against trinitrophenyl-Ficoll was reduced in H1R-deficient mice. Other aspects of T and B cell function were normal in the H1R knockout mice. H1R-deficient T and B cells showed normal responses upon stimulation with interleukin (IL)-2, IL-4, CD40 ligand, CD40 ligand plus IL-4, and lipopolysaccharide. Collectively, these results imply that the signal generated by histamine through H1R augments antigen receptor–mediated immune responses, suggesting cross-talk between G protein–coupled receptors and antigen receptor–mediated signaling. PMID:9989982

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

    PubMed Central

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

    2016-01-01

    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. PMID:26999600

  15. Killer cell immunoglobulin-like receptor gene association with cryptorchidism.

    PubMed

    Niepiekło-Miniewska, Wanda; Kuśnierczyk, Piotr; Havrylyuk, Anna; Kamieniczna, Marzena; Nakonechnyy, Andrij; Chopyak, Valentyna; Kurpisz, Maciej

    2015-12-01

    Cryptorchidism is a condition where a testis persists in the abdominal cavity. Thus, due to elevated temperature we may expect induction of aberrant immune reactions depending on genetic constitution of individual. This may be reflected by development of anti-sperm antibodies (ASA) in cryptorchid males. Also, natural killer (NK) cells which belong to innate immunity may control adaptive immunity. Therefore, the gene system encoding polymorphic NK cell immunoglobulin receptors (KIRs) has been studied. 109 prepubertal boys with cryptorchidism and 136 ethnically matched young male donors were selected to study NK cell KIRs. DNA was isolated using automatic Maxwell(®) system from the peripheral venous blood drawn onto anticoagulant. Olerup SSP KIR Genotyping kit including Taq polymerase was used for detection of KIR genes. Human leukocyte antigen-C (HLA-C) groups, C1 and C2 were established using a Olerup SSP KIR HLA Ligand kit. KIR2DL2 (killer immunoglobulin-like receptor two-domain long 2) and KIR2DS2 (killer immunoglobulin-like receptor two-domain short 2) genes were less frequent in patients than in control individuals (corrected p values: 0.0110 and 0.0383, respectively). However, no significant differences were observed between ASA-positive and ASA-negative patients, or between bilateral or unilateral cryptorchidism. No association between KIR ligands C1 and C2, alone or together with KIR2DL2, was found. However, the results suggest that KIR2DL2+/KIR2DS2+ genotype may be, to some extent, protective against cryptorchidism. PMID:26679162

  16. Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

    SciTech Connect

    Abbas, A.K.; Haber, S.; Rock, K.L.

    1985-09-01

    Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes.

  17. A BTP1 prophage gene present in invasive non-typhoidal Salmonella determines composition and length of the O-antigen of the lipopolysaccharide

    PubMed Central

    Kintz, Erica; Davies, Mark R; Hammarlöf, Disa L; Canals, Rocío; Hinton, Jay C D; van der Woude, Marjan W

    2015-01-01

    Salmonella Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non-typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non-iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O-antigen modification. GtrCBTP1 is essential for maintaining O-antigen length in isolate D23580, since a gtrBTP1 mutant yields a short O-antigen. This phenotype can be complemented by gtrCBTP1 or very closely related gtrC genes. The short O-antigen of the gtrBTP1 mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O-antigen cleavage. Expression of the gtrCBTP1 gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrCBTP1 expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrCBTP1 mediates modification of the BTP1 phage O-antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that uses the same O-antigen co-receptor. PMID:25586744

  18. A BTP1 prophage gene present in invasive non-typhoidal Salmonella determines composition and length of the O-antigen of the lipopolysaccharide.

    PubMed

    Kintz, Erica; Davies, Mark R; Hammarlöf, Disa L; Canals, Rocío; Hinton, Jay C D; van der Woude, Marjan W

    2015-04-01

    Salmonella Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non-typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non-iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O-antigen modification. GtrC(BTP) (1) is essential for maintaining O-antigen length in isolate D23580, since a gtr(BTP) (1) mutant yields a short O-antigen. This phenotype can be complemented by gtrC(BTP) (1) or very closely related gtrC genes. The short O-antigen of the gtr(BTP) (1) mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O-antigen cleavage. Expression of the gtrC(BTP) (1) gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrC(BTP) (1) expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrC(BTP) (1) mediates modification of the BTP1 phage O-antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that uses the same O-antigen co-receptor. PMID:25586744

  19. Regulation of T cell receptor beta gene rearrangements and allelic exclusion by the helix-loop-helix protein, E47.

    PubMed

    Agata, Yasutoshi; Tamaki, Nobuyuki; Sakamoto, Shuji; Ikawa, Tomokatsu; Masuda, Kyoko; Kawamoto, Hiroshi; Murre, Cornelis

    2007-12-01

    Allelic exclusion of antigen-receptor genes is ensured primarily by monoallelic locus activation upon rearrangement and subsequently by feedback inhibition of continued rearrangement. Here, we demonstrated that the basic helix-loop-helix protein, E47, promoted T cell receptor beta (TCRbeta) gene rearrangement by directly binding to target gene segments to increase chromatin accessibility in a dosage-sensitive manner. Feedback signaling abrogated E47 binding, leading to a decline in accessibility. Conversely, enforced expression of E47 induced TCRbeta gene rearrangement by antagonizing feedback inhibition. Thus, the abundance of E47 is rate limiting in locus activation, and feedback signaling downregulates E47 activity to ensure allelic exclusion. PMID:18093539

  20. Identification of antigen-specific B cell receptor sequences using public repertoire analysis

    PubMed Central

    Galson, Jacob D.; Rance, Richard; Parkhill, Julian; Lunter, Gerton; Pollard, Andrew J.; Kelly, Dominic F.

    2014-01-01

    High-throughput sequencing allows detailed study of the B cell receptor (BCR) repertoire post-immunization but it remains unclear to what extent the de novo identification of antigen-specific sequences from the total BCR repertoire is possible. A Hib-MenC-TT conjugate vaccine containing H. influenzae type b (Hib) and group C meningococcal (MenC) polysaccharides as well as tetanus toxoid (TT) was used to investigate the BCR repertoire of adult humans following immunization and test the hypothesis that public or convergent repertoire analysis could identify antigen specific sequences. A number of antigen-specific BCR sequences have previously been reported for Hib and TT which made a vaccine containing these 2 antigens an ideal immunological stimulus. Analysis of identical complementarity determining region (CDR)3 amino acid (AA) sequences that were shared by individuals in the post-vaccine repertoire identified a number of known Hib-specific sequences but only one previously described TT sequence. The extension of this analysis to non-identical but highly similar CDR3 AA sequences revealed a number of other TT-related sequences. The anti-Hib avidity index post-vaccination was strongly correlated with the relative frequency of Hib-specific sequences, indicating that the post-vaccination public BCR repertoire may be related to more conventional measures of immunogenicity correlating with disease protection. Analysis of public BCR repertoire provided evidence of convergent BCR evolution in individuals exposed to the same antigens. If this finding is confirmed, the public repertoire could be used for rapid and direct identification of protective antigen-specific BCR sequences from peripheral blood. PMID:25392534

  1. Nonviral RNA transfection to transiently modify T cells with chimeric antigen receptors for adoptive therapy.

    PubMed

    Riet, Tobias; Holzinger, Astrid; Dörrie, Jan; Schaft, Niels; Schuler, Gerold; Abken, Hinrich

    2013-01-01

    Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy. PMID:23296935

  2. Examining Variable Domain Orientations in Antigen Receptors Gives Insight into TCR-Like Antibody Design

    PubMed Central

    Dunbar, James; Knapp, Bernhard; Fuchs, Angelika; Shi, Jiye; Deane, Charlotte M.

    2014-01-01

    The variable domains of antibodies and T-Cell receptors (TCRs) share similar structures. Both molecules act as sensors for the immune system but recognise their respective antigens in different ways. Antibodies bind to a diverse set of antigenic shapes whilst TCRs only recognise linear peptides presented by a major histocompatibility complex (MHC). The antigen specificity and affinity of both receptors is determined primarily by the sequence and structure of their complementarity determining regions (CDRs). In antibodies the binding site is also known to be affected by the relative orientation of the variable domains, VH and VL. Here, the corresponding property for TCRs, the Vβ-Vα orientation, is investigated and compared with that of antibodies. We find that TCR and antibody orientations are distinct. General antibody orientations are found to be incompatible with binding to the MHC in a canonical TCR-like mode. Finally, factors that cause the orientation of TCRs and antibodies to be different are investigated. Packing of the long Vα CDR3 in the domain-domain interface is found to be influential. In antibodies, a similar packing affect can be achieved using a bulky residue at IMGT position 50 on the VH domain. Along with IMGT VH 50, other positions are identified that may help to promote a TCR-like orientation in antibodies. These positions should provide useful considerations in the engineering of therapeutic TCR-like antibodies. PMID:25233457

  3. Pre-Clustering of the B Cell Antigen Receptor Demonstrated by Mathematically Extended Electron Microscopy

    PubMed Central

    Fiala, Gina J.; Kaschek, Daniel; Blumenthal, Britta; Reth, Michael; Timmer, Jens; Schamel, Wolfgang W. A.

    2013-01-01

    The B cell antigen receptor (BCR) plays a crucial role in adaptive immunity, since antigen-induced signaling by the BCR leads to the activation of the B cell and production of antibodies during an immune response. However, the spatial nano-scale organization of the BCR on the cell surface prior to antigen encounter is still controversial. Here, we fixed murine B cells, stained the BCRs on the cell surface with immuno-gold and visualized the distribution of the gold particles by transmission electron microscopy. Approximately 30% of the gold particles were clustered. However the low staining efficiency of 15% precluded a quantitative conclusion concerning the oligomerization state of the BCRs. To overcome this limitation, we used Monte-Carlo simulations to include or to exclude possible distributions of the BCRs. Our combined experimental-modeling approach assuming the lowest number of different BCR sizes to explain the observed gold distribution suggests that 40% of the surface IgD-BCR was present in dimers and 60% formed large laminar clusters of about 18 receptors. In contrast, a transmembrane mutant of the mIgD molecule only formed IgD-BCR dimers. Our approach complements high resolution fluorescence imaging and clearly demonstrates the existence of pre-formed BCR clusters on resting B cells, questioning the classical cross-linking model of BCR activation. PMID:24367367

  4. Mycoplasma Superantigen Is a CDR3-dependent Ligand for the T Cell Antigen Receptor

    PubMed Central

    Hodtsev, Andrew S.; Choi, Yongwon; Spanopoulou, Eugenia; Posnett, David N.

    1998-01-01

    Superantigens are defined as proteins that activate a large number of T cells through interaction with the Vβ region of the T cell antigen receptor (TCR). Here we demonstrate that the superantigen produced by Mycoplasma arthritidis (MAM), unlike six bacterial superantigens tested, interacts not only with the Vβ region but also with the CDR3 (third complementarity-determining region) of TCR-β. Although MAM shares typical features with other superantigens, direct interaction with CDR3-β is a feature of nominal peptide antigens situated in the antigen groove of major histocompatibility complex (MHC) molecules rather than superantigens. During peptide recognition, Vβ and Vα domains of the TCR form contacts with MHC and the complex is stabilized by CDR3–peptide interactions. Similarly, recognition of MAM is Vβ-dependent and is apparently stabilized by direct contacts with the CDR3-β region. Thus, MAM represents a new type of ligand for TCR, distinct from both conventional peptide antigens and other known superantigens. PMID:9449712

  5. 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

  6. 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. PMID:27262111

  7. 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. PMID:27006117

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

    PubMed

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

    2010-06-01

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

  9. Adoptive immunotherapy for acute leukemia: New insights in chimeric antigen receptors.

    PubMed

    Heiblig, Maël; Elhamri, Mohamed; Michallet, Mauricette; Thomas, Xavier

    2015-08-26

    Relapses remain a major concern in acute leukemia. It is well known that leukemia stem cells (LSCs) hide in hematopoietic niches and escape to the immune system surveillance through the outgrowth of poorly immunogenic tumor-cell variants and the suppression of the active immune response. Despite the introduction of new reagents and new therapeutic approaches, no treatment strategies have been able to definitively eradicate LSCs. However, recent adoptive immunotherapy in cancer is expected to revolutionize our way to fight against this disease, by redirecting the immune system in order to eliminate relapse issues. Initially described at the onset of the 90's, chimeric antigen receptors (CARs) are recombinant receptors transferred in various T cell subsets, providing specific antigens binding in a non-major histocompatibility complex restricted manner, and effective on a large variety of human leukocyte antigen-divers cell populations. Once transferred, engineered T cells act like an expanding "living drug" specifically targeting the tumor-associated antigen, and ensure long-term anti-tumor memory. Over the last decades, substantial improvements have been made in CARs design. CAR T cells have finally reached the clinical practice and first clinical trials have shown promising results. In acute lymphoblastic leukemia, high rate of complete and prolonged clinical responses have been observed after anti-CD19 CAR T cell therapy, with specific but manageable adverse events. In this review, our goal was to describe CAR structures and functions, and to summarize recent data regarding pre-clinical studies and clinical trials in acute leukemia. PMID:26328018

  10. Adoptive immunotherapy for acute leukemia: New insights in chimeric antigen receptors

    PubMed Central

    Heiblig, Maël; Elhamri, Mohamed; Michallet, Mauricette; Thomas, Xavier

    2015-01-01

    Relapses remain a major concern in acute leukemia. It is well known that leukemia stem cells (LSCs) hide in hematopoietic niches and escape to the immune system surveillance through the outgrowth of poorly immunogenic tumor-cell variants and the suppression of the active immune response. Despite the introduction of new reagents and new therapeutic approaches, no treatment strategies have been able to definitively eradicate LSCs. However, recent adoptive immunotherapy in cancer is expected to revolutionize our way to fight against this disease, by redirecting the immune system in order to eliminate relapse issues. Initially described at the onset of the 90’s, chimeric antigen receptors (CARs) are recombinant receptors transferred in various T cell subsets, providing specific antigens binding in a non-major histocompatibility complex restricted manner, and effective on a large variety of human leukocyte antigen-divers cell populations. Once transferred, engineered T cells act like an expanding “living drug” specifically targeting the tumor-associated antigen, and ensure long-term anti-tumor memory. Over the last decades, substantial improvements have been made in CARs design. CAR T cells have finally reached the clinical practice and first clinical trials have shown promising results. In acute lymphoblastic leukemia, high rate of complete and prolonged clinical responses have been observed after anti-CD19 CAR T cell therapy, with specific but manageable adverse events. In this review, our goal was to describe CAR structures and functions, and to summarize recent data regarding pre-clinical studies and clinical trials in acute leukemia. PMID:26328018

  11. Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells.

    PubMed

    Lynn, Rachel C; Poussin, Mathilde; Kalota, Anna; Feng, Yang; Low, Philip S; Dimitrov, Dimiter S; Powell, Daniel J

    2015-05-28

    T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ(+) AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ(+) THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34(+) HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA. PMID:25887778

  12. Individual heritable differences result in unique cell lymphocyte receptor repertoires of naïve and antigen-experienced cells.

    PubMed

    Rubelt, Florian; Bolen, Christopher R; McGuire, Helen M; Vander Heiden, Jason A; Gadala-Maria, Daniel; Levin, Mikhail; Euskirchen, Ghia M; Mamedov, Murad R; Swan, Gary E; Dekker, Cornelia L; Cowell, Lindsay G; Kleinstein, Steven H; Davis, Mark M

    2016-01-01

    The adaptive immune system's capability to protect the body requires a highly diverse lymphocyte antigen receptor repertoire. However, the influence of individual genetic and epigenetic differences on these repertoires is not typically measured. By leveraging the unique characteristics of B, CD4(+) T and CD8(+) T-lymphocyte subsets from monozygotic twins, we quantify the impact of heritable factors on both the V(D)J recombination process and on thymic selection. We show that the resulting biases in both V(D)J usage and N/P addition lengths, which are found in naïve and antigen experienced cells, contribute to significant variation in the CDR3 region. Moreover, we show that the relative usage of V and J gene segments is chromosomally biased, with ∼1.5 times as many rearrangements originating from a single chromosome. These data refine our understanding of the heritable mechanisms affecting the repertoire, and show that biases are evident on a chromosome-wide level. PMID:27005435

  13. 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...

  14. 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 ...

  15. Widespread ectopic expression of olfactory receptor genes

    PubMed Central

    Feldmesser, Ester; Olender, Tsviya; Khen, Miriam; Yanai, Itai; Ophir, Ron; Lancet, Doron

    2006-01-01

    Background Olfactory receptors (ORs) are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information. PMID:16716209

  16. Targeting HER2-positive cancer cells with receptor-redirected anthrax protective antigen

    PubMed Central

    McCluskey, Andrew J.; Olive, Andrew J.; Starnbach, Michael N.; Collier, R. John

    2012-01-01

    Targeted therapeutics have emerged in recent years as an attractive approach to treating various types of cancer. One approach is to modify a cytocidal protein toxin to direct its action to a specific population of cancer cells. We created a targeted toxin in which the receptor-binding and pore-forming moiety of anthrax toxin, termed Protective Antigen (PA), was modified to redirect its receptor specificity to HER2, a marker expressed at the surface of a significant fraction of breast and ovarian tumors. The resulting fusion protein (mPA-ZHER2) delivered cytocidal effectors specifically into HER2-positive tumor cells, including a trastuzumab-resistant line, causing death of the cells. No off-target killing of HER2-negative cells was observed, either with homogeneous populations or with mixtures of HER2-positive and HER2-negative cells. A mixture of mPA variants targeting different receptors mediated killing of cells bearing either receptor, without affecting cells devoid of these receptors. Anthrax toxin may serve as an effective platform for developing therapeutics to ablate cells bearing HER2 or other tumor-specific cell-surface markers. PMID:23290417

  17. Cytokine release syndrome in cancer immunotherapy with chimeric antigen receptor engineered T cells.

    PubMed

    Xu, Xiao-Jun; Tang, Yong-Min

    2014-02-28

    Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells is a promising therapy for cancers. However, the safety of this approach is concerned. Cytokine release syndrome (CRS) is a common but lethal complication of CAR-T cell therapy. The development of CRS correlates with CAR structures, tumor type and burden, and patients' genetic polymorphisms. CRS related adverse events may be reduced by designing safer CARs and CAR-T cells and following strict dose-escalation scheme. Timely and effective cytokine-directed treatment with corticosteroid and various cytokine antagonists is important to avoid CRS associated death. PMID:24141191

  18. Adoptive Therapy with Chimeric Antigen Receptor Modified T Cells of Defined Subset Composition

    PubMed Central

    Riddell, Stanley R.; Sommermeyer, Daniel; Berger, Carolina; Liu, Lingfeng (Steven); Balakrishnan, Ashwini; Salter, Alex; Hudecek, Michael; Maloney, David G.; Turtle, Cameron J.

    2014-01-01

    The ability to engineer T cells to recognize tumor cells through genetic modification with a synthetic chimeric antigen receptor has ushered in a new era in cancer immunotherapy. The most advanced clinical applications are in in targeting CD19 on B cell malignancies. The clinical trials of CD19 CAR therapy have thus far not attempted to select defined subsets prior to transduction or imposed uniformity of the CD4 and CD8 cell composition of the cell products. This review will discuss the rationale for and challenges to utilizing adoptive therapy with genetically modified T cells of defined subset and phenotypic composition. PMID:24667960

  19. 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.

  20. Chimeric antigen receptor T cell therapy: 25years in the making.

    PubMed

    Gill, Saar; Maus, Marcela V; Porter, David L

    2016-05-01

    Chimeric antigen receptor (CAR) T cell therapy of cancer is generating enormous enthusiasm. Twenty-five years after the concept was first proposed, major advances in molecular biology, virology, and good manufacturing practices (GMP)-grade cell production have transformed antibody-T cell chimeras from a scientific curiosity to a fact of life for academic cellular immunotherapy researchers and, increasingly, for patients. In this review, we explain the preclinical concept, outline how it has been translated to the clinic, and draw lessons from the first years of CAR T cell therapy for the practicing clinician. PMID:26574053

  1. 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

  2. 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. PMID:26860370

  3. Efficacy of an immunotoxin to folate receptor beta in the intra-articular treatment of antigen-induced arthritis

    PubMed Central

    2012-01-01

    Introduction We previously demonstrated that synovial sublining macrophages express folate receptor beta (FRβ). The aim of this study was to evaluate the efficacy of intra-articular administration of a recombinant immunotoxin to FRβ for treating rat antigen-induced arthritis. Methods A monoclonal antibody (mAb) to rat FRβ was produced by immunizing mice with B300-19 cells (murine pre-B cells) transfected with the rat FRβ gene. Recombinant immunotoxin was prepared by conjugating the Fv portion of the anti-rat FRβ mAb heavy chain with a truncated Pseudomonas exotoxin A and the Fv portion of the anti-rat FRβ mAb light chain. Antigen-induced arthritis was induced through intra-articular injection of methylated bovine serum albumin (mBSA) after two subcutaneous injections of mBSA and complete Freund's adjuvant. Immunotoxin was intra-articularly injected into the arthritis joint every other day for seven days after arthritis onset. Joint swelling was measured and histological scores of inflammation, synovial thickness, cartilage, and bone destruction were determined. Immunohistochemistry was performed to detect osteoclast and osteoclast precursor FRβ-expressing macrophages and cathepsin K-positive cells on day 21. Results Intra-articular administration of the immunotoxin attenuated joint swelling (61% suppression; P < 0.01 compared to the control on day 21) and improved histological findings, particularly cartilage and bone destruction (scores of rats treated with control versus the immunotoxin: 2.2 versus 0.5; P < 0.01), by reducing the number of FRβ-expressing macrophages and cathepsin K-positive cells. Conclusions Intra-articular administration of an immunotoxin to FRβ is effective for improving rat antigen-induced arthritis. PMID:22551402

  4. 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...

  5. Cellular gene expression induced by parasite antigens and allergens in neonates from parasite-infected mothers.

    PubMed

    Soboslay, Peter T; Orlikowsky, Thorsten; Huang, Xiangsheng; Gille, Christian; Spring, Bärbel; Kocherscheidt, Lars; Agossou, Abram; Banla, Meba; Bonin, Michael; Köhler, Carsten

    2016-05-01

    Prenatal exposure to parasite antigens or allergens will influence the profile and strength of postnatal immune responses, such contact may tolerize and increase susceptibility to future infections or sensitize to environmental allergens. Exposure in utero to parasite antigens will distinctly alter cellular gene expression in newborns. Gene microarrays were applied to study gene expression in umbilical cord blood cell (UCBC) from parasite-exposed (Para-POS) and non-exposed (Para-NEG) neonates. UCBC were activated with antigens of helminth (Onchocerca volvulus), amoeba (Entamoeba histolytica) or allergens of mite (Dermatophagoides farinae). When UCBC from Para-POS and Para-NEG newborns were exposed to helminth antigens or allergens consistent differences occurred in the expression of genes encoding for MHC class I and II alleles, signal transducers of activation and transcription (STATs), cytokines, chemokines, immunoglobulin heavy and light chains, and molecules associated with immune regulation (SOCS, TLR, TGF), inflammation (TNF, CCR) and apoptosis (CASP). Expression of genes associated with innate immune responses were enhanced in Para-NEG, while in Para-POS, the expression of MHC class II and STAT genes was reduced. Within functional gene networks for cellular growth, proliferation and immune responses, Para-NEG neonates presented with significantly higher expression values than Para-POS. In Para-NEG newborns, the gene cluster and pathway analyses suggested that gene expression profiles may predispose for the development of immunological, hematological and dermatological disorders upon postnatal helminth parasite infection or allergen exposure. Thus, prenatal parasite contact will sensitize without generating aberrant inflammatory immune responses, and increased pro-inflammatory but decreased regulatory gene expression profiles will be present in those neonates lacking prenatal parasite antigen encounter. PMID:27062712

  6. 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

  7. Antigen-induced regulation of T-cell motility, interaction with antigen-presenting cells and activation through endogenous thrombospondin-1 and its receptors

    PubMed Central

    Bergström, Sten-Erik; Uzunel, Mehmet; Talme, Toomas; Bergdahl, Eva; Sundqvist, Karl-Gösta

    2015-01-01

    Antigen recognition reduces T-cell motility, and induces prolonged contact with antigen-presenting cells and activation through mechanisms that remain unclear. Here we show that the T-cell receptor (TCR) and CD28 regulate T-cell motility, contact with antigen-presenting cells and activation through endogenous thrombospondin-1 (TSP-1) and its receptors low-density lipoprotein receptor-related protein 1 (LRP1), calreticulin and CD47. Antigen stimulation induced a prominent up-regulation of TSP-1 expression, and transiently increased and subsequently decreased LRP1 expression whereas calreticulin was unaffected. This antigen-induced TSP-1/LRP1 response down-regulated a motogenic mechanism directed by LRP1-mediated processing of TSP-1 in cis within the same plasma membrane while promoting contact with antigen-presenting cells and activation through cis interaction of the C-terminal domain of TSP-1 with CD47 in response to N-terminal TSP-1 triggering by calreticulin. The antigen-induced TSP-1/LRP1 response maintained a reduced but significant motility level in activated cells. Blocking CD28 co-stimulation abrogated LRP1 and TSP-1 expression and motility. TCR/CD3 ligation alone enhanced TSP-1 expression whereas CD28 ligation alone enhanced LRP1 expression. Silencing of TSP-1 inhibited T-cell conjugation to antigen-presenting cells and T helper type 1 (Th1) and Th2 cytokine responses. The Th1 response enhanced motility and increased TSP-1 expression through interleukin-2, whereas the Th2 response weakened motility and reduced LRP1 expression through interleukin-4. Ligation of the TCR and CD28 therefore elicits a TSP-1/LRP1 response that stimulates prolonged contact with antigen-presenting cells and, although down-regulating motility, maintains a significant motility level to allow serial contacts and activation. Th1 and Th2 cytokine responses differentially regulate T-cell expression of TSP-1 and LRP1 and motility. PMID:25393517

  8. IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression.

    PubMed

    Chmielewski, Markus; Kopecky, Caroline; Hombach, Andreas A; Abken, Hinrich

    2011-09-01

    During malignant progression cancer cells tend to lose cell surface expression of MHC and other immune antigens, making them invisible to cytotoxic T cells and therefore inaccessible to tumor antigen-directed immunotherapy. Moreover, cancer cell variants that have lost antigen expression frequently contribute to deadly tumor relapses that occur following treatments that had been initially effective. In an effort to destroy antigen-loss cancer cells in tumors, we created a strategy that combines a chimeric antigen receptor (CAR)-redirected T-cell attack with an engineered local release of the cytokine interleukin 12 (IL-12), which recruits and reinforces macrophage function. Cytotoxic T cells were engineered to release inducible IL-12 upon CAR engagement in the tumor lesion, resulting in destruction of antigen-loss cancer cells that would normally escape. Importantly, elimination of the antigen-loss cancer cells was accompanied by an accumulation of activated macrophages that was critical to the antitumor response, because removing the macrophages abolished the response and restoring them reengaged it. Neutralizing TNF-α also abrogated the elimination of antigen-loss cancer cells, implying this proinflammatory factor in the process. Taken together, our results show how IL-12 supplementation by CAR T cells can target otherwise inaccessible tumor lesions, in a manner associated with reduced systemic toxicity, by recruiting and activating innate immune cells for a proinflammatory response. PMID:21742772

  9. Immunotherapy of malignant disease using chimeric antigen receptor engrafted T cells.

    PubMed

    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

  10. 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

  11. Chimeric antigen receptor-redirected T cells return to the bench.

    PubMed

    Geldres, Claudia; Savoldo, Barbara; Dotti, Gianpietro

    2016-02-01

    While the clinical progress of chimeric antigen receptor T cell (CAR-T) immunotherapy has garnered attention to the field, our understanding of the biology of these chimeric molecules is still emerging. Our aim within this review is to bring to light the mechanistic understanding of these multi-modular receptors and how these individual components confer particular properties to CAR-Ts. In addition, we will discuss extrinsic factors that can be manipulated to influence CAR-T performance such as choice of cellular population, culturing conditions and additional modifications that enhance their activity particularly in solid tumors. Finally, we will also consider the emerging toxicity associated with CAR-Ts. By breaking apart the CAR and examining the role of each piece, we can build a better functioning cellular vehicle for optimized treatment of cancer patients. PMID:26797495

  12. Molecular characteristics of an immobilization antigen gene of the fish-parasitic protozoan Ichthyophthirius multifiliis strain ARS-6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ichthyophthirius multifiliis, a ciliated protozoan parasite of fish, expresses surface antigens (i-antigens), which react with host antibodies that render them immobile. The nucleotide sequence of an i-antigen gene of Ichthyophthirius multifiliis strain ARS-6 was deduced. The predicted protein of 47...

  13. Enhancement of the in vivo persistence and antitumor efficacy of CD19 chimeric antigen receptor T cells through the delivery of modified TERT mRNA

    PubMed Central

    Bai, Yun; Kan, Shifeng; Zhou, Shixin; Wang, Yuting; Xu, Jun; Cooke, John P; Wen, Jinhua; Deng, Hongkui

    2015-01-01

    Chimeric antigen receptor T cell immunotherapy is a promising therapeutic strategy for treating tumors, demonstrating its efficiency in eliminating several hematological malignancies in recent years. However, a major obstacle associated with current chimeric antigen receptor T cell immunotherapy is that the limited replicative lifespan of chimeric antigen receptor T cells prohibits the long-term persistence and expansion of these cells in vivo, potentially hindering the long-term therapeutic effects of chimeric antigen receptor T cell immunotherapy. Here we showed that the transient delivery of modified mRNA encoding telomerase reverse transcriptase to human chimeric antigen receptor T cells targeting the CD19 antigen (CD19 chimeric antigen receptor T cells) would transiently elevate the telomerase activity in these cells, leading to increased proliferation and delayed replicative senescence without risk of insertion mutagenesis or immortalization. Importantly, compared to conventional CD19 chimeric antigen receptor T cells, after the transient delivery of telomerase reverse transcriptase mRNA, these CD19 chimeric antigen receptor T cells showed improved persistence and proliferation in mouse xenograft tumor models of human B-cell malignancies. Furthermore, the transfer of CD19 chimeric antigen receptor T cells after the transient delivery of telomerase reverse transcriptase mRNA enhanced long-term antitumor effects in mouse xenograft tumor models compared with conventional CD19 chimeric antigen receptor T cell transfer. The results of the present study provide an effective and safe method to improve the therapeutic potential of chimeric antigen receptor T cells, which might be beneficial for treating other types of cancer, particularly solid tumors. PMID:27462436

  14. 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

  15. 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

  16. Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum

    PubMed Central

    Artzy-Randrup, Yael; Rorick, Mary M; Day, Karen; Chen, Donald; Dobson, Andrew P; Pascual, Mercedes

    2012-01-01

    The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier ‘strain theory’ by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity. DOI: http://dx.doi.org/10.7554/eLife.00093.001 PMID:23251784

  17. In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses.

    PubMed Central

    Ballay, A; Levrero, M; Buendia, M A; Tiollais, P; Perricaudet, M

    1985-01-01

    We have developed an adenovirus vector to express foreign proteins under the control of the adenovirus E1a promoter. Two recombinant plasmids, harbouring either the S gene or the pre-S2 region and the S gene of hepatitis B virus under the control of the E1a promoter, were used to construct two recombinant adenoviruses. These two viruses direct the synthesis of hepatitis B virus surface antigen (HBsAg) particles during the time course of an infectious cycle. When the pre-S2 region is present in the constructed virus, the synthesis of particles carrying the receptor for polymerized human serum albumin (pHSA) is observed. Moreover, the inoculation of rabbits with this latter purified recombinant adenovirus elicits the production of antibodies that react with both HBsAg and pHSA receptor. Images Fig. 4. PMID:3004975

  18. The Novelty of Human Cancer/Testis Antigen Encoding Genes in Evolution

    PubMed Central

    Dobrynin, Pavel; Matyunina, Ekaterina; Malov, S. V.; Kozlov, A. P.

    2013-01-01

    In order to be inherited in progeny generations, novel genes should originate in germ cells. Here, we suggest that the testes may play a special “catalyst” role in the birth and evolution of new genes. Cancer/testis antigen encoding genes (CT genes) are predominantly expressed both in testes and in a variety of tumors. By the criteria of evolutionary novelty, the CT genes are, indeed, novel genes. We performed homology searches for sequences similar to human CT in various animals and established that most of the CT genes are either found in humans only or are relatively recent in their origin. A majority of all human CT genes originated during or after the origin of Eutheria. These results suggest relatively recent origin of human CT genes and align with the hypothesis of the special role of the testes in the evolution of the gene families. PMID:23691492

  19. Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

    PubMed Central

    McConnell, I; Hurd, C M

    1976-01-01

    Immunoglobulin (Ig) bearing, antigen-binding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibody-secreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibody-secreting cells. These receptors are either lost from the B cell surface as they differentiate into antibody-secreting cells or are blocked in vivo by immune complexes or C3b. PMID:800392

  20. 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

  1. 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. PMID:27203746

  2. Phase I study utilizing a novel antigen-presenting cell-targeted vaccine with Toll-like receptor stimulation to induce immunity to self antigens in cancer patients

    PubMed Central

    Morse, Michael A.; Chapman, Robert; Powderly, John; Blackwell, Kimberly; Keler, Tibor; Green, Jennifer; Riggs, Renee; He, Li-Zhen; Ramakrishna, Venky; Vitale, Laura; Zhao, Biwei; Butler, Stephen A.; Hobeika, Amy; Osada, Takuya; Davis, Thomas; Clay, Timothy; Lyerly, H. Kim

    2011-01-01

    Purpose The use of tumor-derived proteins as cancer vaccines is complicated by tolerance to these self antigens. Tolerance may be broken by immunization with activated, autologous, ex vivo generated and antigen-loaded, antigen-presenting cells (APC); however, targeting tumor antigen directly to APC in vivo would be a less complicated strategy. We wished to test whether targeted delivery of an otherwise poorly immunogenic, soluble antigen to APC through their mannose receptors (MR) would induce clinically relevant immunity. Experimental Design Two phase I studies were performed with CDX-1307, a vaccine composed of human chorionic gonadotropin beta chain (hCG-β) fused to a MR-specific monoclonal antibody, administered either locally (intradermally) or systemically (intravenously) in patients with advanced epithelial malignancies. An initial dose-escalation of single agent CDX-1307 was followed by additional cohorts of CDX-1307 combined with GM-CSF and the Toll-like receptor (TLR)-3 agonist poly-ICLC and TLR7/8 agonist resiquimod to activate the APC. Results CDX-1307 induced consistent humoral and T cell responses to hCG-β when co-administered with TLR agonists. Greater immune responses and clinical benefit, including the longest duration of stable disease, were observed with immunization combined with local TLR agonists. Immune responses were induced equally efficiently in patients with elevated and non-elevated levels of serum hCG-β. Antibodies within the serum of vaccinated participants had tumor suppressive function in vitro. Toxicity consisted chiefly of mild injection site reactions. Conclusions APC targeting and activation induce adaptive immunity against poorly immunogenic self antigens which has implications for enhancing the efficacy of cancer immunotherapy. PMID:21632857

  3. Diacylglycerol kinase ζ limits B cell antigen receptor-dependent activation of ERK signaling to inhibit early antibody responses.

    PubMed

    Wheeler, Matthew L; Dong, Matthew B; Brink, Robert; Zhong, Xiao-Ping; DeFranco, Anthony L

    2013-10-15

    Signaling downstream of the B cell antigen receptor (BCR) is tightly regulated to enable cells to gauge the strength and duration of antigen-receptor interactions and to respond appropriately. We investigated whether metabolism of the second messenger diacylglycerol (DAG) by members of the family of DAG kinases (DGKs) played a role in modulating the magnitude of signaling by DAG downstream of the BCR. In the absence of DGKζ, the threshold for BCR signaling, measured as activation of the Ras-extracellular signal-regulated kinase (ERK) pathway, was markedly reduced in mature follicular B cells, which resulted in enhanced responses to antigen in vitro and in vivo. Inhibition of DAG signaling by DGKζ limited the number of antibody-secreting cells that were generated early in response to T cell-independent type 2 antigens, as well as to T cell-dependent antigens. Furthermore, the effect of loss of DGKζ closely resembled the effect of increasing the affinity of the BCR for antigen during the T cell-dependent antibody response. These results suggest that the magnitude of DAG signaling is important for translating the affinity of the BCR for antigen into the amount of antibody produced during the early stages of an immune response. PMID:24129701

  4. Relationship between Fc receptors, antigen-binding sites on T and B cells, and H-2 complex-associated determinants.

    PubMed

    Basten, A; Miller, J F; Abraham, R

    1975-03-01

    The relationship between H-2 complex-associated determinants, Fc receptors, and specific antigen-recognition sites on T and B cells was examined by binding and functional assays. The Fc receptor was detected by radiolabeled immune complexes or aggregated human IgG. Both these reagents selectively bound to B cells, not to T cells. When spleen cells, from mice primed to several antigens, were exposed to highly substituted radioactive aggregates, their capacity to transfer both a direct and indirect plaque-forming cell response to these antigens was abrogated. Addition of B cells, but not of T cells, restored responsiveness. Complexed Ig binding to Fc receptors was prevented by pretreatment of mixed lymphoid cell populations with antisera directed against membrane components on the same cell (e.g., H-2) and on other cells (e.g., theta). The lack of specificity of inhibition was thought to be due to the formation on cell surfaces of antigen-antibody complexes which would then attach to the Fc receptor during the incubation precedure. Specific blockade of the Fc receptor during the incubation procedure. Specific blockade of the Fc receptor however occurred when B cells were pretreated with the Fab fragments of anti-H-2 antibody. This was demonstrated autoradiographically and by inhibition of aggregate-induced suicide. The blocking activity of ante-H-2 Fab was removed by absorption with spleen cells from thymectomized irradiated mice but not with thymus cells of appropriate specificity. This suggested that the antibodies involved had specificity for determinants on the B-cell membrane distinct from those coded by the K or D end of the H-2 complex, and either absent from, or poorly represented on, thymus cells. Specific antigen-induced suicide of B cells was achieved simply by incubating the cells with radioactive antigen in the cold. T-cell suicide on the other hand required that the 125I-labeled antigen be presented to the T cells at 37 degrees-C on the surface of

  5. Synthetic Peptide Ligands of the Antigen Binding Receptor Induce Programmed Cell Death in a Human B-Cell Lymphoma

    NASA Astrophysics Data System (ADS)

    Renschler, Markus F.; Bhatt, Ramesh R.; Dower, William J.; Levy, Ronald

    1994-04-01

    Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

  6. 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

  7. Androgen receptor gene polymorphism in zebra species

    PubMed Central

    Ito, Hideyuki; Langenhorst, Tanya; Ogden, Rob; Inoue-Murayama, Miho

    2015-01-01

    Androgen receptor genes (AR) have been found to have associations with reproductive development, behavioral traits, and disorders in humans. However, the influence of similar genetic effects on the behavior of other animals is scarce. We examined the loci AR glutamine repeat (ARQ) in 44 Grevy's zebras, 23 plains zebras, and three mountain zebras, and compared them with those of domesticated horses. We observed polymorphism among zebra species and between zebra and horse. As androgens such as testosterone influence aggressiveness, AR polymorphism among equid species may be associated with differences in levels of aggression and tameness. Our findings indicate that it would be useful to conduct further studies focusing on the potential association between AR and personality traits, and to understand domestication of equid species. PMID:26236645

  8. Hypervariable antigen genes in malaria have ancient roots

    PubMed Central

    2013-01-01

    Background The var genes of the human malaria parasite Plasmodium falciparum are highly polymorphic loci coding for the erythrocyte membrane proteins 1 (PfEMP1), which are responsible for the cytoaherence of P. falciparum infected red blood cells to the human vasculature. Cytoadhesion, coupled with differential expression of var genes, contributes to virulence and allows the parasite to establish chronic infections by evading detection from the host’s immune system. Although studying genetic diversity is a major focus of recent work on the var genes, little is known about the gene family's origin and evolutionary history. Results Using a novel hidden Markov model-based approach and var sequences assembled from additional isolates and species, we are able to reveal elements of both the early evolution of the var genes as well as recent diversifying events. We compare sequences of the var gene DBLα domains from divergent isolates of P. falciparum (3D7 and HB3), and a closely-related species, Plasmodium reichenowi. We find that the gene family is equally large in P. reichenowi and P. falciparum -- with a minimum of 51 var genes in the P. reichenowi genome (compared to 61 in 3D7 and a minimum of 48 in HB3). In addition, we are able to define large, continuous blocks of homologous sequence among P. falciparum and P. reichenowi var gene DBLα domains. These results reveal that the contemporary structure of the var gene family was present before the divergence of P. falciparum and P. reichenowi, estimated to be between 2.5 to 6 million years ago. We also reveal that recombination has played an important and traceable role in both the establishment, and the maintenance, of diversity in the sequences. Conclusions Despite the remarkable diversity and rapid evolution found in these loci within and among P. falciparum populations, the basic structure of these domains and the gene family is surprisingly old and stable. Revealing a common structure as well as conserved

  9. Antigen uptake and expression of antigen presentation-related immune genes in flounder (Paralichthys olivaceus) after vaccination with an inactivated Edwardsiella tarda immersion vaccine, following hyperosmotic treatment.

    PubMed

    Gao, Yingli; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

    2016-08-01

    Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders (Paralichthys olivaceus) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30 min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) (P < 0.05), and the highest amount of antigen was detected in flounders immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group (P > 0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine (P < 0.05) compared with the spleen, kidney and liver. Antigen uptake in the gill and skin both peaked at 30 min post immersion, which was significantly higher than the levels of uptake measured in the other tissues (P < 0.05), and then quickly declined. In contrast, antigen uptake in the spleen, kidney and liver gradually increased 3 h post immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group (P < 0.05). In the mucosal-associated tissues, the expression of MHC Iα and CD8α genes peaked at 24 hpi, while the expression of MHC IIα and CD4-1 genes showed up-regulation in the gill and skin

  10. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    PubMed Central

    Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

    2014-01-01

    Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

  11. Understanding the structural specificity of Tn antigen for its receptor: an NMR solution study.

    PubMed

    D'Amelio, Nicola; Coslovi, Anna; Rossi, Marco; Uggeri, Fulvio; Paoletti, Sergio

    2012-04-01

    The present work aims at understanding the structural basis of the biological recognition of Tn antigen (GalNAc-α-O-L-Ser), a specific epitope expressed by tumor cells, and the role of its amino acidic moiety in the interaction with its receptor (the isolectin B4 extracted from Vicia villosa). An NMR structural characterization of the α and β anomers, based on J couplings and molecular modeling revealed a structure in very good agreement with data reported in literature for variants of the same molecules. In order to demonstrate the involvement of the amino acid in the ligand-receptor recognition, also GalNAc-α-O-D-Ser was studied; the change in the stereochemistry is in fact expected to impact on the interaction only in case the serine is part of the epitope. Relaxation properties in the presence of the receptor clearly indicated a selective recognition of the natural L form, probably due to the formation of a water-mediated hydrogen bond with Asn 129 of the protein. PMID:22341503

  12. Structure and gene cluster of the O-antigen of Escherichia coli O137.

    PubMed

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

    2016-03-01

    The O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of Escherichia coli O137 and studied by sugar analysis and NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit was established: Formula: see text] Both structure and gene cluster of the E. coli O137 polysaccharide are related to those of the E. coli K40 polysaccharide (Amor et al., 1999), which lacks the side-chain glucosylation but contains serine that is amide-linked to GlcA. Functions of genes in the O137-antigen gene cluster were assigned by a comparison with those in K40 and sequences in the available databases. Particularly, predicted glycosyltransferases encoded in the gene cluster were assigned to the formation of three glycosidic linkages in the O-polysaccharide repeating unit. PMID:26845703

  13. PiggyBac-mediated Cancer Immunotherapy Using EBV-specific Cytotoxic T-cells Expressing HER2-specific Chimeric Antigen Receptor

    PubMed Central

    Nakazawa, Yozo; Huye, Leslie E; Salsman, Vita S; Leen, Ann M; Ahmed, Nabil; Rollins, Lisa; Dotti, Gianpietro; Gottschalk, Stephen M; Wilson, Matthew H; Rooney, Cliona M

    2011-01-01

    Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy. PMID:21772253

  14. Evolutionary origin and human-specific expansion of a cancer/testis antigen gene family.

    PubMed

    Zhang, Qu; Su, Bing

    2014-09-01

    Cancer/testis (CT) antigens are encoded by germline genes and are aberrantly expressed in a number of human cancers. Interestingly, CT antigens are frequently involved in gene families that are highly expressed in germ cells. Here, we presented an evolutionary analysis of the CTAGE (cutaneous T-cell-lymphoma-associated antigen) gene family to delineate its molecular history and functional significance during primate evolution. Comparisons among human, chimpanzee, gorilla, orangutan, macaque, marmoset, and other mammals show a rapid and primate specific expansion of CTAGE family, which starts with an ancestral retroposition in the haplorhini ancestor. Subsequent DNA-based duplications lead to the prosperity of single-exon CTAGE copies in catarrhines, especially in humans. Positive selection was identified on the single-exon copies in comparison with functional constraint on the multiexon copies. Further sequence analysis suggests that the newly derived CTAGE genes may obtain regulatory elements from long terminal repeats. Our result indicates the dynamic evolution of primate genomes, and the recent expansion of this CT antigen family in humans may confer advantageous phenotypic traits during early human evolution. PMID:24916032

  15. 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...

  16. A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells.

    PubMed

    Hamana, Hiroshi; Shitaoka, Kiyomi; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Muraguchi, Atsushi

    2016-06-10

    T-cell receptor (TCR) gene therapy is a promising approach for the treatment of infectious diseases and cancers. However, the paired cloning and functional assays of antigen-specific TCRα and TCRβ is time-consuming and laborious. In this study, we developed a novel, rapid and efficient antigen-specific TCR-cloning system by combining three technologies: multiplex one-step RT-PCR, transcriptionally active PCR (TAP) and luciferase reporter assays. Multiplex one-step RT-PCR with leader primers designed from leader peptide sequences of TCRs enabled us to amplify cDNAs of TCRα and β pairs from single T-cells with remarkably high efficiency. The combination of TAP fragments and HEK293T-based NFAT-luciferase reporter cells allowed for a rapid functional assay without the need to construct expression vectors. Using this system, we cloned human TCRs specific for Epstein-Barr virus BRLF-1-derived peptide as well as mouse TCRs specific for melanoma-associated antigen tyrosinase-related protein 2 (TRP-2) within four days. These results suggest that our system provides rapid and efficient cloning of functional antigen-specific human and mouse TCRs and contributes to TCR-based immunotherapy for cancers and infectious diseases. PMID:27155153

  17. Improving therapy of chronic lymphocytic leukemia with chimeric antigen receptor T cells.

    PubMed

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

    2016-04-01

    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 redirecting 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

  18. Structure of a lamprey variable lymphocyte receptor in complex with a protein antigen

    PubMed Central

    Velikovsky, C Alejandro; Deng, Lu; Tasumi, Satoshi; Iyer, Lakshminarayan M; Kerzic, Melissa C; Aravind, L; Pancer, Zeev; Mariuzza, Roy A

    2009-01-01

    Variable lymphocyte receptors (VLRs) are leucine-rich repeat (LRR) proteins that mediate adaptive immunity in jawless vertebrates. VLRs are fundamentally different from the antibodies of jawed vertebrates, which consist of immunoglobulin (Ig) domains. We determined the structure of an anti-hen egg white lysozyme (HEL) VLRB, isolated by yeast display, bound to HEL. The VLR, whose affinity resembles that of IgM antibodies, uses nearly all its concave surface to bind the protein, in addition to a loop that penetrates into the enzyme active site. The VLR-HEL structure, combined with sequence analysis, revealed an almost perfect match between ligand-contacting positions and positions with highest sequence diversity. Thus, we have defined the generalized antigen-binding site of VLRs. We further demonstrated that VLRs can be affinity-matured to affinities as high as those of IgG antibodies, making VLRs potential alternatives to antibodies for biotechnology applications. PMID:19543291

  19. Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges.

    PubMed

    Han, Ethan Q; Li, Xiu-ling; Wang, Chun-rong; Li, Tian-fang; Han, Shuang-yin

    2013-01-01

    Recent years have witnessed much progress in both basic research and clinical trials regarding cancer immunotherapy with chimeric antigen receptor (CAR)-engineered T cells. The unique structure of CAR endows T cell tumor specific cytotoxicity and resistance to immunosuppressive microenvironment in cancers, which helps patients to better tackle the issue of immunological tolerance. Adoptive immunotherapy (AIT) using this supernatural T cell have gained momentum after decades of intense debates because of the promising results obtained from preclinical models and clinical trials. However, it is very important for us to evaluate thoroughly the challenges/obstacles before widespread clinical application, which clearly warrants more studies to improve our understanding of the mechanism underlying AIT. In this review, we focus on the critical issues related to the clinical outcomes of CAR-based adoptive immunotherapy and discuss the rationales to refine this new cancer therapeutic modality. PMID:23829929

  20. 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. PMID:24986688

  1. 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

  2. Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor

    PubMed Central

    Jena, Bipulendu; Dotti, Gianpietro

    2010-01-01

    Infusions of antigen-specific T cells have yielded therapeutic responses in patients with pathogens and tumors. To broaden the clinical application of adoptive immunotherapy against malignancies, investigators have developed robust systems for the genetic modification and characterization of T cells expressing introduced chimeric antigen receptors (CARs) to redirect specificity. Human trials are under way in patients with aggressive malignancies to test the hypothesis that manipulating the recipient and reprogramming T cells before adoptive transfer may improve their therapeutic effect. These examples of personalized medicine infuse T cells designed to meet patients' needs by redirecting their specificity to target molecular determinants on the underlying malignancy. The generation of clinical grade CAR+ T cells is an example of bench-to-bedside translational science that has been accomplished using investigator-initiated trials operating largely without industry support. The next-generation trials will deliver designer T cells with improved homing, CAR-mediated signaling, and replicative potential, as investigators move from the bedside to the bench and back again. PMID:20439624

  3. Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates.

    PubMed

    Sarkar, Mohosin; Liu, Yun; Qi, Junpeng; Peng, Haiyong; Morimoto, Jumpei; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2016-04-01

    Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset. PMID:26851280

  4. CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia

    PubMed Central

    Maude, Shannon L.; Teachey, David T.; Porter, David L.

    2015-01-01

    Relapsed and refractory acute lymphoblastic leukemia (ALL) remains difficult to treat, with minimal improvement in outcomes seen in more than 2 decades despite advances in upfront therapy and improved survival for de novo ALL. Adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a powerful targeted immunotherapy, showing striking responses in highly refractory populations. Complete remission (CR) rates as high as 90% have been reported in children and adults with relapsed and refractory ALL treated with CAR-modified T cells targeting the B-cell–specific antigen CD19. Distinct CAR designs across several studies have produced similar promising CR rates, an encouraging finding. Even more encouraging are durable remissions observed in some patients without additional therapy. Duration of remission and CAR-modified T-cell persistence require further study and more mature follow-up, but emerging data suggest these factors may distinguish CAR designs. Supraphysiologic T-cell proliferation, a hallmark of this therapy, contributes to both efficacy and the most notable toxicity, cytokine release syndrome (CRS), posing a unique challenge for toxicity management. This review will discuss the current landscape of CD19 CAR clinical trials, CRS pathophysiology and management, and remaining challenges. PMID:25999455

  5. 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. PMID:23632026

  6. 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

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

    PubMed

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

    2016-08-23

    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

  8. Identification of cancer/testis-antigen genes by massively parallel signature sequencing

    PubMed Central

    Chen, Yao-Tseng; Scanlan, Matthew J.; Venditti, Charis A.; Chua, Ramon; Theiler, Gregory; Stevenson, Brian J.; Iseli, Christian; Gure, Ali O.; Vasicek, Tom; Strausberg, Robert L.; Jongeneel, C. Victor; Old, Lloyd J.; Simpson, Andrew J. G.

    2005-01-01

    Massively parallel signature sequencing (MPSS) generates millions of short sequence tags corresponding to transcripts from a single RNA preparation. Most MPSS tags can be unambiguously assigned to genes, thereby generating a comprehensive expression profile of the tissue of origin. From the comparison of MPSS data from 32 normal human tissues, we identified 1,056 genes that are predominantly expressed in the testis. Further evaluation by using MPSS tags from cancer cell lines and EST data from a wide variety of tumors identified 202 of these genes as candidates for encoding cancer/testis (CT) antigens. Of these genes, the expression in normal tissues was assessed by RT-PCR in a subset of 166 intron-containing genes, and those with confirmed testis-predominant expression were further evaluated for their expression in 21 cancer cell lines. Thus, 20 CT or CT-like genes were identified, with several exhibiting expression in five or more of the cancer cell lines examined. One of these genes is a member of a CT gene family that we designated as CT45. The CT45 family comprises six highly similar (>98% cDNA identity) genes that are clustered in tandem within a 125-kb region on Xq26.3. CT45 was found to be frequently expressed in both cancer cell lines and lung cancer specimens. Thus, MPSS analysis has resulted in a significant extension of our knowledge of CT antigens, leading to the discovery of a distinctive X-linked CT-antigen gene family. PMID:15905330

  9. 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.

  10. Distribution of killer cell immunoglobulin-like receptor genes in Poles.

    PubMed

    Majorczyk, E; Łuszczek, W; Nowak, I; Pawlik, A; Wiśniewski, A; Jasek, M; Kuśnierczyk, P

    2008-08-01

    Killer cell immunoglobulin-like receptors (KIRs) present on natural killer cells and minor subpopulations of T cells recognize class I human leucocyte antigen (HLA) molecules on the surface of target cells. Humans differ by the presence or absence of some KIR genes on their chromosomes. As KIRs are important for the outcome of tissue transplantation (particularly for haematopoietic stem cell transplantation) and possibly for pregnancy and autoimmune diseases, knowledge of the KIR gene distribution in a given human population is of practical value. Therefore, we tested 363 healthy individuals from Western Poland for the presence or absence of KIR genes. Results are compared with those published for other human populations. KIR gene frequencies in Poles are close to these in other Caucasoids but different from those in Asian and African populations, and particularly distant from those in Australian Aborigines. PMID:18976447

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

    PubMed

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

    2008-01-01

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

  12. Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells.

    PubMed

    Betting, David J; Mu, Xi Y; Kafi, Kamran; McDonnel, Desmond; Rosas, Francisco; Gold, Daniel P; Timmerman, John M

    2009-01-01

    Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources. PMID:19000731

  13. Enhanced immune stimulation by a therapeutic lymphoma tumor antigen vaccine produced in insect cells involves mannose receptor targeting to antigen presenting cells

    PubMed Central

    Betting, David J.; Mu, Xi Y.; Kafi, Kamran; McDonnel, Desmond; Rosas, Francisco; Gold, Daniel P.; Timmerman, John M.

    2009-01-01

    Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) coupled to the carrier protein keyhole limpet hemocyanin (Id-KLH) is undergoing clinical investigation, and methods to improve the immunogenicity of these and other protein tumor antigen vaccines are being sought. Id proteins can be produced via tumor-myeloma hybridomas or recombinant methods in mammalian, bacteria, or insect cells. We now demonstrate that terminal mannose residues, characteristic of recombinant proteins produced in insect cells, yield Id proteins with significantly enhanced immunostimulatory properties compared to Id proteins derived from mammalian cells. Recombinant baculovirus-infected insect cell-derived Id showed higher binding to and activation of human dendritic cells mediated by mannose receptors. In vivo, insect cell-derived Id elicited higher levels of tumor-specific CD8+ CTL and improved eradication of pre-established murine lymphoma. Insect cell and mammalian Id generated similar levels of tumor-specific antibodies, showing no impairment in antibody responses to native tumor antigen despite the glycoslylation differences in the immunogen. Combining insect cell production and maleimide-based KLH conjugation offered the highest levels of anti-tumor immunity. Our data comparing sources of recombinant Id protein tumor antigens used in therapeutic cancer vaccines demonstrate that insect cell-derived antigens can offer several immunologic advantages over proteins derived from mammalian sources. PMID:19000731

  14. DNA SEQUENCING, ANALYSIS, AND IDENTIFICATION OF SEROGROUP-SPECIFIC GENES IN THE ESCHERICHIA COLI O28AC AND O118 O ANTIGEN GENE CLUSTERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The DNA sequence of the O antigen gene clusters of Escherichia coli serogroups O28ac and O118 was determined, and 7 and 13 ORFs were identified, respectively, encoding genes required for O antigen sugar biosynthesis, transfer, and processing. Analysis of the DNA sequence revealed that the wzx (O ...

  15. 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. PMID:19033646

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

    PubMed

    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

    2015-04-01

    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

  17. 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

  18. 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

  19. Dopamine receptor-mediated regulation of neuronal "clock" gene expression.

    PubMed

    Imbesi, M; Yildiz, S; Dirim Arslan, A; Sharma, R; Manev, H; Uz, T

    2009-01-23

    Using a transgenic mice model (i.e. "clock" knockouts), clock transcription factors have been suggested as critical regulators of dopaminergic behaviors induced by drugs of abuse. Moreover, it has been shown that systemic administration of psychostimulants, such as cocaine and methamphetamine regulates the striatal expression of clock genes. However, it is not known whether dopamine receptors mediate these regulatory effects of psychostimulants at the cellular level. Primary striatal neurons in culture express dopamine receptors as well as clock genes and have been successfully used in studying dopamine receptor functioning. Therefore, we investigated the role of dopamine receptors on neuronal clock gene expression in this model using specific receptor agonists. We found an inhibitory effect on the expression of mClock and mPer1 genes with the D2-class (i.e. D2/D3) receptor agonist quinpirole. We also found a generalized stimulatory effect on the expression of clock genes mPer1, mClock, mNPAS2 (neuronal PAS domain protein 2), and mBmal1 with the D1-class (i.e. D1) receptor agonist SKF38393. Further, we tested whether systemic administration of dopamine receptor agonists causes similar changes in striatal clock gene expression in vivo. We found quinpirole-induced alterations in mPER1 protein levels in the mouse striatum (i.e. rhythm shift). Collectively, our results indicate that the dopamine receptor system may mediate psychostimulant-induced changes in clock gene expression. Using striatal neurons in culture as a model, further research is needed to better understand how dopamine signaling modulates the expression dynamics of clock genes (i.e. intracellular signaling pathways) and thereby influences neuronal gene expression, neuronal transmission, and brain functioning. PMID:19017537

  20. Identification of chemosensory receptor genes from vertebrate genomes.

    PubMed

    Niimura, Yoshihito

    2013-01-01

    Chemical senses are essential for the survival of animals. In vertebrates, mainly three different types of receptors, olfactory receptors (ORs), vomeronasal receptors type 1 (V1Rs), and vomeronasal receptors type 2 (V2Rs), are responsible for the detection of chemicals in the environment. Mouse or rat genomes contain >1,000 OR genes, forming the largest multigene family in vertebrates, and have >100 V1R and V2R genes as well. Recent advancement in genome sequencing enabled us to computationally identify nearly complete repertories of OR, V1R, and V2R genes from various organisms, revealing that the numbers of these genes are highly variable among different organisms depending on each species' living environment. Here I would explain bioinformatic methods to identify the entire repertoires of OR, V1R, and V2R genes from vertebrate genome sequences. PMID:24014356

  1. Modification of Expanded NK Cells with Chimeric Antigen Receptor mRNA for Adoptive Cellular Therapy.

    PubMed

    Chu, Yaya; Flower, Allyson; Cairo, Mitchell S

    2016-01-01

    NK cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors and cells infected by viruses. The regulation of NK activation vs inhibition is regulated by the expression of a variety of NK receptors (NKRs) and specific NKRs' ligands expressed on their targets. However, factors limiting NK therapy include small numbers of active NK cells in unexpanded peripheral blood and lack of specific tumor targeting. Chimeric antigen receptors (CAR) usually include a single-chain Fv variable fragment from a monoclonal antibody, a transmembrane hinge region, and a signaling domain such as CD28, CD3-zeta, 4-1BB (CD137), or 2B4 (CD244) endodimers. Redirecting NK cells with a CAR will circumvent the limitations of the lack of NK targeting specificity. This chapter focuses on the methods to expand human NK cells from peripheral blood by co-culturing with feeder cells and to modify the expanded NK cells efficiently with the in vitro transcribed CAR mRNA by electroporation and to test the functionality of the CAR-modified expanded NK cells for use in adoptive cellular immunotherapy. PMID:27177669

  2. Aptamer-MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen.

    PubMed

    Jolly, Pawan; Tamboli, Vibha; Harniman, Robert L; Estrela, Pedro; Allender, Chris J; Bowen, Jenna L

    2016-01-15

    This study reports the design and evaluation of a new synthetic receptor sensor based on the amalgamation of biomolecular recognition elements and molecular imprinting to overcome some of the challenges faced by conventional protein imprinting. A thiolated DNA aptamer with established affinity for prostate specific antigen (PSA) was complexed with PSA prior to being immobilised on the surface of a gold electrode. Controlled electropolymerisation of dopamine around the complex served to both entrap the complex, holding the aptamer in, or near to, it's binding conformation, and to localise the PSA binding sites at the sensor surface. Following removal of PSA, it was proposed that the molecularly imprinted polymer (MIP) cavity would act synergistically with the embedded aptamer to form a hybrid receptor (apta-MIP), displaying recognition properties superior to that of aptamer alone. Electrochemical impedance spectroscopy (EIS) was used to evaluate subsequent rebinding of PSA to the apta-MIP surface. The apta-MIP sensor showed high sensitivity with a linear response from 100pg/ml to 100ng/ml of PSA and a limit of detection of 1pg/ml, which was three-fold higher than aptamer alone sensor for PSA. Furthermore, the sensor demonstrated low cross-reactivity with a homologous protein (human Kallikrein 2) and low response to human serum albumin (HSA), suggesting possible resilience to the non-specific binding of serum proteins. PMID:26318788

  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. 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

  5. Single-round of antigen receptor signaling programs naïve B cells to receive T cell help

    PubMed Central

    Damdinsuren, Bazarragchaa; Zhang, Yongqing; Khalil, Ashraf; Wood, William H.; Becker, Kevin G.; Shlomchik, Mark J.; Sen, Ranjan

    2010-01-01

    SUMMARY To simulate transient B cell activation that is the likely initiator of T-dependent responses, we examined the molecular and functional consequences of a single-round of immunoglobulin M (IgM) signaling. This form of activation triggered early cytosolic signaling and the transcription factor NF-κB activation indistinguishably from conventional continuous IgM cross-linking, but did not induce G1 progression. However, single-round IgM signaling changed the expression of chemokine and chemokine receptor genes implicated in initiating T-dependent responses, as well as accentuated responsiveness to CD40 signaling. Several features of single-round IgM signaling in vitro were recapitulated in B cells after short-term exposure to antigen in vivo. We propose that transient BCR signals prime B cells to receive T cell help by increasing the probability of B-T encounter and creating a cellular environment that is hyper-responsive to CD40 signaling. PMID:20226693

  6. Cloning of T-cell antigen receptor beta chain cDNAs from Atlantic salmon (Salmo salar).

    PubMed

    Hordvik, I; Jacob, A L; Charlemagne, J; Endresen, C

    1996-01-01

    Atlantic salmon (Salmo salar) cDNAs encoding the T-cell antigen receptor beta chain (TCRB) were isolated from leukocyte RNA by reverse transcription - polymerase chain reaction (RT-PCR). Twenty-five distinct cDNA fragments covering the variable (V) - diversity (D) - joining (J) junction and part of the constant (C) region were characterized; the sequences of which indicate interchangeable V/D/J usage and expression in the context of one TCRBC gene. Full-length TCRBC sequence information was derived from a leukocyte cDNA library. Key residues of the salmon TCRBC region are in good agreement with those of other species. One distinct exception is the absence of the hinge region cysteine residue which is involved in covalent bonding between the alpha and beta chain in mammalian TCRs. As in amphibian and avian species, the salmon TCRBC membrane proximal region is considerably shorter than the mammalian. An octamer sequence (GGACAGGG) very similar to amphibian, avian, and mammalian D sequences could be recognized in the VDJ junctions from salmon. The pattern of VDJ variability also indicates that mechanisms like trimming and addition occur in fish as in higher vertebrates. Compared with mammals, a relatively high frequency (32%) of the VDJ junctions in salmon were out of frame. PMID:8881032

  7. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia.

    PubMed

    Kenderian, S S; Ruella, M; Shestova, O; Klichinsky, M; Aikawa, V; Morrissette, J J D; Scholler, J; Song, D; Porter, D L; Carroll, M; June, C H; Gill, S

    2015-08-01

    Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML. PMID:25721896

  8. A Highly Focused Antigen Receptor Repertoire Characterizes γδ T Cells That are Poised to Make IL-17 Rapidly in Naive Animals

    PubMed Central

    Wei, Yu-Ling; Han, Arnold; Glanville, Jacob; Fang, Fengqin; Zuniga, Luis Alejandro; Lee, Jacob S.; Cua, Daniel J.; Chien, Yueh-hsiu

    2015-01-01

    Interleukin (IL)-17 plays a key role in immunity. In acute infections, a rapid IL-17 response must be induced without prior antigen exposure, and γδ T cells are the major initial IL-17 producers. In fact, some γδ T cells make IL-17 within hours after an immune challenge. These cells appear to acquire the ability to respond to IL-1 and IL-23 and to make IL-17 naturally in naïve animals. They are known as the natural Tγδ17 (nTγδ17) cells. The rapidity of the nTγδ17 response, and the apparent lack of explicit T cell receptor (TCR) engagement for its induction have led to the view that this is a cytokine (IL-1, IL-23)-mediated response. However, pharmacological inhibition or genetic defects in TCR signaling drastically reduce the nTγδ17 response and/or their presence. To better understand antigen recognition in this rapid IL-17 response, we analyzed the antigen receptor repertoire of IL-1R+/IL-23R+ γδ T cells, a proxy for nTγδ17 cells in naïve animals directly ex vivo, using a barcode-enabled high throughput single-cell TCR sequence analysis. We found that regardless of their anatomical origin, these cells have a highly focused TCR repertoire. In particular, the TCR sequences have limited V gene combinations, little or no junctional diversity and much reduced or no N region diversity. In contrast, IL-23R− cells at mucosal sites similar to most of the splenic γδ T cells and small intestine epithelial γδ lymphocytes expressed diverse TCRs. This remarkable commonality and restricted repertoire of IL-1R+/IL-23R+ γδ T cells underscores the importance of antigen recognition in their establishment/function. PMID:25852688

  9. Network-based gene expression analysis of intracranial aneurysm tissue reveals role of antigen presenting cells.

    PubMed

    Krischek, B; Kasuya, H; Tajima, A; Akagawa, H; Sasaki, T; Yoneyama, T; Ujiie, H; Kubo, O; Bonin, M; Takakura, K; Hori, T; Inoue, I

    2008-07-17

    Little is known about the pathology and pathogenesis of the rupture of intracranial aneurysms. For a better understanding of the molecular processes involved in intracranial aneurysm (IA) formation we performed a gene expression analysis comparing ruptured and unruptured aneurysm tissue to a control artery. Tissue samples of six ruptured and four unruptured aneurysms, and four cerebral arteries serving as controls, were profiled using oligonucleotide microarrays. Gene ontology classification of the differentially expressed genes was analyzed and regulatory functional networks and canonical pathways were identified with a network-based computational pathway analysis tool. Real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed as confirmation. Analysis of aneurysmal and control tissue revealed 521 differentially expressed genes. The most significantly associated gene ontology term was antigen processing (P=1.64E-16). Further network-based analysis showed the top scoring regulatory functional network to be built around overexpressed major histocompatibility class (MHC) I and II complex related genes and confirmed the canonical pathway "Antigen Presentation" to have the highest upregulation in IA tissue (P=7.3E-10). Real time RT-PCR showed significant overexpression of MHC class II genes. Immunohistochemical staining showed strong positivity for MHC II molecule specific antibody (HLA II), for CD68 (macrophages, monocytes), for CD45RO (T-cells) and HLA I antibody. Our results offer strong evidence for MHC class II gene overexpression in human IA tissue and that antigen presenting cells (macrophages, monocytes) play a key role in IA formation. PMID:18538937

  10. 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. PMID:16595374

  11. Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes.

    PubMed

    Jeyaseelan, S; Hsuan, S L; Kannan, M S; Walcheck, B; Wang, J F; Kehrli, M E; Lally, E T; Sieck, G C; Maheswaran, S K

    2000-01-01

    Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all beta2 integrins. We designed experiments with the following objectives: to identify which member of the beta2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced beta2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis. PMID:10603370

  12. 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

  13. Genetic diversity and population structure of genes encoding vaccine candidate antigens of Plasmodium vivax

    PubMed Central

    2012-01-01

    Background A major concern in malaria vaccine development is genetic polymorphisms typically observed among Plasmodium isolates in different geographical areas across the world. Highly polymorphic regions have been observed in Plasmodium falciparum and Plasmodium vivax antigenic surface proteins such as Circumsporozoite protein (CSP), Duffy-binding protein (DBP), Merozoite surface protein-1 (MSP-1), Apical membrane antigen-1 (AMA-1) and Thrombospondin related anonymous protein (TRAP). Methods Genetic variability was assessed in important polymorphic regions of various vaccine candidate antigens in P. vivax among 106 isolates from the Amazon Region of Loreto, Peru. In addition, genetic diversity determined in Peruvian isolates was compared to population studies from various geographical locations worldwide. Results The structured diversity found in P. vivax populations did not show a geographic pattern and haplotypes from all gene candidates were distributed worldwide. In addition, evidence of balancing selection was found in polymorphic regions of the trap, dbp and ama-1 genes. Conclusions It is important to have a good representation of the haplotypes circulating worldwide when implementing a vaccine, regardless of the geographic region of deployment since selective pressure plays an important role in structuring antigen diversity. PMID:22417572

  14. Comparison of Z and R3 antigen expression and of genes encoding other antigenic markers in invasive human and bovine Streptococcus agalactiae strains from Norway.

    PubMed

    Maeland, Johan A; Radtke, Andreas

    2013-12-27

    Streptococcus agalactiae (GBS) may cause a variety of infectious diseases in humans caused by human GBS and mastitis in cattle caused by bovine GBS. Over the last few years molecular testing has provided evidence that human and bovine GBS have evolved along diverse phylogenetic lines. In the present study 173 invasive human GBS strains and 52 invasive bovine strains were tested for altogether 18 strain-variable and surface-localized antigenic markers including all 10 capsular polysaccharides (CPS) and proteins including Cβ, the alpha-like proteins, R3 and the recently described Z1 and Z2 antigens. PCR was used to detect encoding genes and antibody-based methods to detect expression of antigens. Thirteen of the 18 markers were detected in isolates of both strain categories. Seven of the ten CPS antigens were detected in both groups with types III and V predominating in the human GBS strains, types IV and V in the bovine isolates. Z1, Z2 and/or R3 expression and the genes encoding Cβ, Cα, Alp1, Alp2/3 or R4 (Rib) were detected in both groups. Protein antigen-CPS associations well known for human strains were essentially the same in the bovine isolates. The results show that in spite of evolution along different lines, human and bovine GBS share a variety of surface-exposed antigenic markers, substantiating close relationship between the two GBS subpopulations. PMID:24120184

  15. 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. PMID:10680014

  16. 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

  17. 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. PMID:27378344

  18. Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice.

    PubMed

    Yong, Carmen S M; Westwood, Jennifer A; Schröder, Jan; Papenfuss, Anthony T; von Scheidt, Bianca; Moeller, Maria; Devaud, Christel; Darcy, Phillip K; Kershaw, Michael H

    2015-01-01

    Genetically modified CD8+ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer. PMID:26505904

  19. Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice

    PubMed Central

    Yong, Carmen S. M.; Westwood, Jennifer A.; Schröder, Jan; Papenfuss, Anthony T.; von Scheidt, Bianca; Moeller, Maria; Devaud, Christel

    2015-01-01

    Genetically modified CD8+ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer. PMID:26505904

  20. Non random usage of T cell receptor alpha gene expression in atopy using anchored PCR.

    PubMed

    Mansur, A H; Gelder, C M; Holland, D; Campell, D A; Griffin, A; Cunliffe, W; Markham, A F; Morrison, J F

    1996-01-01

    The T cell receptor (TCR) alpha beta heterodimer recognises antigenic peptide fragments presented by Class II MHC. This interaction initiates T cell activation and cytokine release with subsequent recruitment of inflammatory cells. Previous work from our group suggests a qualitative difference in variable alpha gene expression in atopy as compared to non atopic controls. In this study we examine TCR alpha repertoire using anchored PCR to provide a quantitative assessment of the V alpha and J alpha repertoire. One atopic (DRB1*0701,DRB1*15: DRB4*0101, DRB5*01: DQB1* 0303, DQB1*601/2) and one non-atopic (DRB1*0701,DRB1*03011/2: DRB4*01, DRB3*0x: DQB1* 0303, DQB1*0201/2) control were studied. Variable gene usage was markedly limited in the atopic individual. V alpha 1, 3, 8 accounted for 60% and J alpha 12, 31 30% of the gene usage. There was evidence of preferential V alpha-J alpha gene pairing and clonal expansion. We conclude that there is a marked non random TCR alpha gene distribution in atopy using both V alpha family and anchored PCR. This may be due in part to antigen driven clonal expansion. PMID:9095269

  1. Dexamethasone modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human T lymphocytes.

    PubMed

    Nambiar, M P; Enyedy, E J; Fisher, C U; Warke, V G; Juang, Y T; Tsokos, G C

    2001-02-25

    Dexamethasone is a potent anti-inflammatory and immunosupressive agent that has complex, yet incompletely defined, effects on the immune response. Here, we explored the effect of dexamethasone on the expression of TCR zeta chain and TCR/CD3-induced early signaling events in human T lymphocytes. Immunoblotting studies using TCR zeta chain specific mAb showed a dose-dependent biphasic effect of dexamethasone on TCR zeta chain expression, that is, it was increased when cells were incubated with 10 nM, whereas the expression was decreased when incubated with 100 nM dexamethasone. The dose-dependent biphasic effect of dexamethsone on the TCR zeta chain expression was also revealed by FACS analysis of permeabilized cells. Time course studies showed that upregulation of the TCR zeta chain at 10 nM dexamethasone reached maximum levels at 24 h and remained elevated up to 48 h. Other subunits of the TCR/CD3 complex were minimally affected under these conditions. The increased expression of the TCR zeta chain following treatment with 10 nM dexamethasone correlated with increased anti-CD3 antibody-induced tyrosine phosphorylation of the TCR zeta chain and downstream signaling intermediate ZAP-70 and PLC gamma with faster kinetics. Similarly, the induction of TCR zeta chain expression at 10 nM dexamethasone correlated with increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response. Reporter gene assays using TCR zeta chain promoter-driven luciferase gene constructs in Jurkat cells showed that treatment with 10 nM dexamethasone increased TCR zeta chain promoter activity and that the region between -160 and +58 was responsible for the observed effect. These results suggest that dexamethasone primarily acts at the transcriptional level and differentially modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human peripheral T lymphocytes. PMID:11277620

  2. Vitamin A and immune function: retinoic acid modulates population dynamics in antigen receptor and CD38-stimulated splenic B cells.

    PubMed

    Chen, Qiuyan; Ross, A Catharine

    2005-10-01

    Vitamin A and its active metabolite, all-trans retinoic acid (RA), regulate the antibody response in vivo, although the underlying mechanisms are not well understood. We have investigated the regulation by RA of B cell population dynamics and Ig gene expression in purified splenic mouse B cells stimulated through the B cell antigen receptor (BCR) and/or CD38, a BCR coreceptor. After ligation of the BCR and/or CD38, B cells became more heterogeneous in size. RA substantially restrained this change, concomitant with inhibition of cell proliferation. To examine B cell heterogeneity more closely, we categorized stimulated B cells by size (forward angle light scatter) and determined cell division dynamics, germ-line Ig heavy chain gene transcription and surface IgG1 (sIgG1) expression. Flow cytometric analysis of carboxyfluorescein diacetate succinimidyl ester-labeled B cells costained for sIgG1 showed that the more proliferative groups of B cells were smaller, whereas cells expressing more sIgG1 were larger. RA enriched the latter population, whereas cell division frequency in general and the number of smaller B cells that had undergone division cycles were reduced. Although RA significantly inhibited Ig germ-line transcript levels in the total B cell population, CD19(-)IgG1(+) B cells, which represent a more differentiated phenotype, were enriched. Furthermore, pax-5 mRNA was decreased and activation-induced cytidine deaminase mRNA was increased in RA-treated stimulated B cells. Thus, RA regulated factors known to be required for Ig class switch recombination and modulated the population dynamics of ligation-stimulated B cells, while promoting the progression of a fraction of B cells into differentiated sIgG-expressing cells. PMID:16093312

  3. A Tet-On Inducible System for Controlling CD19-Chimeric Antigen Receptor Expression upon Drug Administration.

    PubMed

    Sakemura, Reona; Terakura, Seitaro; Watanabe, Keisuke; Julamanee, Jakrawadee; Takagi, Erina; Miyao, Kotaro; Koyama, Daisuke; Goto, Tatsunori; Hanajiri, Ryo; Nishida, Tetsuya; Murata, Makoto; Kiyoi, Hitoshi

    2016-08-01

    T cells genetically modified with a CD19 chimeric antigen receptor (CD19CAR) are remarkably effective against B-cell malignancies in clinical trials. However, major concerns remain regarding toxicities, such as hypogammaglobulinemia, due to B-cell aplasia or severe cytokine release syndrome after overactivation of CAR T cells. To resolve these adverse events, we aimed to develop an inducible CAR system by using a tetracycline regulation system that would be activated only in the presence of doxycycline (Dox). In this study, the second-generation CD19CAR was fused into the third-generation Tet-On vector (Tet-CD19CAR) and was retrovirally transduced into primary CD8(+) T cells. Tet-CD19CAR T cells were successfully generated and had minimal background CD19CAR expression without Dox. Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD19(+) cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells. The Dox(+) Tet-CD19CAR T cells also had significant antitumor activity in a xenograft model. However, without Dox, Tet-CD19CAR T cells lost CAR expression and CAR T-cell functions in vitro and in vivo, clearly segregating the "On" and "Off" status of Tet-CD19CAR cells by Dox administration. In addition to suicide-gene technology, controlling the expression and the functions of CAR with an inducible vector is a potential solution for CAR T-cell therapy-related toxicities, and may improve the safety profile of CAR T-cell therapy. This strategy might also open the way to treat other malignancies in combination with other CAR or TCR gene-modified T cells. Cancer Immunol Res; 4(8); 658-68. ©2016 AACRSee related Spotlight by June, p. 643. PMID:27329987

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

    PubMed

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

    1992-12-01

    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

  5. Multiple human D sub 5 dopamine receptor genes: A functional receptor and two pseudogenes

    SciTech Connect

    Grandy, D.K.; Yuan Zhang; Bouvier, C.; Qunyong Zhou; Johnson, R.A.; Allen, L.; Buck, K.; Bunzow, J.R.; Salon, J.; Civelli, O. )

    1991-10-15

    Three genes closely related to the D{sub 1} dopamine receptor were identified in the human genome. One of the genes lacks introns and encodes a functional human dopamine receptor, D{sub 5}, whose deduced amino acid sequence is 49% identical to that of the human D{sub 1} receptor. Compared with the human D{sub 1} dopamine receptor, the D{sub 5} receptor displayed a higher affinity for dopamine and was able to stimulate a biphasic rather than a monophasic intracellular accumulation of cAMP. Neither of the other two genes was able to direct the synthesis of a receptor. nucleotide sequence analysis revealed that these two genes are 98% identical to each other and 95% identical to the D{sub 5} sequence. Relative to the D{sub 5} sequence, both contain insertions and deletions that result in several in-frame termination codons. Premature termination of translation is the most likely explanation for the failure of these genes to produce receptors in COS-7 and 293 cells even though their messages are transcribed. The authors conclude that the two are pseudogenes. Blot hybridization experiments performed on rat genomic DNA suggest that there is one D{sub 5} gene in this species and that the pseudogenes may be the result of a relatively recent evolutionary event.

  6. 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.

  7. Transgenic Mice Harboring SV40 T-Antigen Genes Develop Characteristic Brain Tumors

    PubMed Central

    Brinster, Ralph L.; Chen, Howard Y.; Messing, Albee; van Dyke, Terry; Levine, Arnold J.; Palmiter, Richard D.

    2016-01-01

    Summary A high percentage of transgenic mice developing from eggs microinjected with plasmids containing the SV40 early region genes and a metallothionein fusion gene develop tumors within the choroid plexus. A line of mice has been established in which nearly every affected animal succumbs to this brain tumor. Thymic hypertrophy and kidney pathology are also observed in some mice. SV40 T-antigen mRNA and protein are readily detected in affected tissues; however, SV40 T-antigen gene expression is barely detectable in unaffected tissues or in susceptible tissues prior to overt pathology, suggesting that tumorigenesis depends upon activation of the SV40 genes. Comparison of DNA from tumor tissue (or cell lines derived from tumors) with DNA from unaffected tissues reveals structural rearrangements as well as changes in DNA methylation of the foreign DNA. The SV40 genes are frequently amplified in tumor tissue, which further indicates that their expression is intimately involved in tumorigenesis in transgenic mice. PMID:6327063

  8. 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.

  9. Chimeric Antigen Receptor-Modified T Cells for Solid Tumors: Challenges and Prospects

    PubMed Central

    Guo, Yelei; Wang, Yao; Han, Weidong

    2016-01-01

    Recent studies have highlighted the successes of chimeric antigen receptor-modified T- (CART-) cell-based therapy for B-cell malignancies, and early phase clinical trials have been launched in recent years. The few published clinical studies of CART cells in solid tumors have addressed safety and feasibility, but the clinical outcome data are limited. Although antitumor effects were confirmed in vitro and in animal models, CART-cell-based therapy still faces several challenges when directed towards solid tumors, and it has been difficult to achieve the desired outcomes in clinical practice. Many studies have struggled to improve the clinical responses to and benefits of CART-cell treatment of solid tumors. In this review, the status quo of CART cells and their clinical applications for solid tumors will be summarized first. Importantly, we will suggest improvements that could increase the therapeutic effectiveness of CART cells for solid tumors and their future clinical applications. These interventions will make treatment with CART cells an effective and routine therapy for solid tumors. PMID:26998495

  10. Expression of the duffy antigen/receptor for chemokines (DARC) by the inflamed synovial endothelium.

    PubMed

    Patterson, Angela M; Siddall, Hilary; Chamberlain, Giselle; Gardner, Lucy; Middleton, Jim

    2002-05-01

    The expression of chemokine binding sites on the endothelial cells of venules in inflamed synovia was examined and whether the Duffy antigen/receptor for chemokines (DARC) was involved. In situ binding assays were performed to determine the expression of chemokine binding sites from rheumatoid (n = 10) and non-rheumatoid (n = 10) synovia. The expression of DARC protein and mRNA was examined by immunohistochemistry and northern blotting. The involvement of DARC in chemokine binding was studied by incubating sections with blocking antibodies to DARC (Fy3 and 6), to find out if these reduced 125I-IL-8 binding. Binding of radiolabelled chemokines IL-8, RANTES, MCP-1, but not MIP-1alpha, was found on venular endothelial cells in inflamed synovia from both rheumatoid and non-rheumatoid patients. Excess homologous unlabelled chemokine displaced binding and excess unlabelled RANTES could displace radiolabelled IL-8 binding. DARC protein expression was demonstrated on venular endothelial cells in all samples and DARC mRNA could be detected in extracts from synovia. There was downregulation of DARC protein and mRNA in rheumatoid samples. Binding of IL-8 to both rheumatoid and non-rheumatoid synovia was significantly reduced in the presence of anti-DARC Fy3 and Fy6 monoclonal antibodies. These findings show the expression of a multispecific chemokine binding site on the inflamed synovial endothelium, with evidence for involvement of DARC. This suggests a potential role for DARC in the inflammatory processes involved in synovitis. PMID:12081195

  11. Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer.

    PubMed

    Cao, Yu; Rodgers, David T; Du, Juanjuan; Ahmad, Insha; Hampton, Eric N; Ma, Jennifer S Y; Mazagova, Magdalena; Choi, Sei-Hyun; Yun, Hwa Young; Xiao, Han; Yang, Pengyu; Luo, Xiaozhou; Lim, Reyna K V; Pugh, Holly M; Wang, Feng; Kazane, Stephanie A; Wright, Timothy M; Kim, Chan Hyuk; Schultz, Peter G; Young, Travis S

    2016-06-20

    Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR-T cells (specific for the corresponding FITC or PNE) to Her2-expressing tumor cells, and afford dose-titratable activation of CAR-T cells ex vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch-based control of the CAR-T response. PMID:27145250

  12. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells

    PubMed Central

    Hudecek, Michael; Lupo-Stanghellini, Maria-Teresa; Kosasih, Paula L.; Sommermeyer, Daniel; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2013-01-01

    Purpose The adoptive transfer of T-cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module has activity in advanced malignancies. ROR1 is a tumor-associated molecule expressed on prevalent B-lymphoid and epithelial cancers, and is absent on normal mature B-cells and vital tissues, making it a candidate for CAR T-cell therapy. Experimental Design We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T-cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma engrafted into immunodeficient mice. Results ROR1-CARs containing a short ‘Hinge-only’ extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared to CARs with long ‘Hinge-CH2-CH3’ spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation induced T-cell death. T-cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR modified T-cells in mediating regression of JeKo-1 mantle cell lymphoma in immunodeficient mice. Conclusions Our results demonstrate that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T-cells modified with an optimized ROR1-CAR have significant anti-tumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. PMID:23620405

  13. Transcriptional activation by EBV nuclear antigen 1 is essential for the expression of EBV's transforming genes

    PubMed Central

    Altmann, Markus; Pich, Dagmar; Ruiss, Romana; Wang, Jindong; Sugden, Bill; Hammerschmidt, Wolfgang

    2006-01-01

    EBV is a paradigm for human tumor viruses because, although it infects most people benignly, it also can cause a variety of cancers. Both in vivo and in vitro, EBV infects B lymphocytes in G0, induces them to become blasts, and can maintain their proliferation in cell culture or in vivo as tumors. How EBV succeeds in these contrasting cellular environments in expressing its genes that control the host has not been explained. We have genetically dissected the EBV nuclear antigen 1 (EBNA1) gene that is required for replication of the viral genome, to elucidate its possible role in the transcription of viral genes. Strikingly, EBNA1 is essential to drive transcription of EBV's transforming genes after infection of primary B lymphocytes. PMID:16966603

  14. 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.

  15. 77 FR 62520 - Prospective Grant of Exclusive License: The Development of Anti-CD22 Chimeric Antigen Receptors...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-15

    ...This is notice, in accordance with 35 U.S.C. 209(c)(1) and 37 CFR part 404.7(a)(1)(i), that the National Institutes of Health, Department of Health and Human Services, is contemplating the grant of an exclusive license to practice the inventions embodied in (a) U.S. Patent Application 61/549,516 entitled ``Anti-CD22 Chimeric Antigen Receptors'' [HHS Ref. E-265-2011/0-US-01], and (b) U.S.......

  16. Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

    PubMed Central

    Tyagi, Kriti; Gupta, Deepali; Saini, Ekta; Choudhary, Shilpa; Jamwal, Abhishek; Alam, Mohd. Shoeb; Zeeshan, Mohammad; Tyagi, Rupesh K.; Sharma, Yagya D.

    2015-01-01

    Background The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites. Methods Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively. Results Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite. Conclusions Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host. PMID:26393350

  17. B7H6-specific chimeric antigen receptors lead to tumor elimination and host anti-tumor immunity

    PubMed Central

    Wu, Ming-Ru; Zhang, Tong; DeMars, Leslie R.; Sentman, Charles L.

    2015-01-01

    Chimeric antigen receptor (CAR) T cell therapies have demonstrated durable and potentially curative therapeutic efficacy against B cell leukemia in clinical trials. A CAR strategy can target any tumor surface antigens as long as an antigen-binding receptor can be generated. New CARs which target solid tumors and have the potential to target multiple tumor types are needed. In this study, B7H6, a ligand for the NK cell activating receptor NKp30, was targeted to create a CAR which targets multiple tumor types. B7H6 is expressed on various primary human tumors, including leukemia, lymphoma, and gastrointestinal stromal tumors (GISTs), but it is not constitutively expressed on normal tissues. B7H6-specific CAR T cells have robust cellular cytotoxicity and IFN-γ secretion when co-cultured with B7H6+ tumor cells, and they exhibit little self-reactivity to immature dendritic cells (iDCs) or pro-inflammatory monocytes. In vivo, B7H6-specific CAR T cells greatly enhanced the survival of RMA/B7H6 lymphoma bearing mice. The long-term survivor mice were protected against a B7H6-deficient tumor re-challenge. This CAR therapy also decreased tumor burden in a murine ovarian cancer model. In conclusion, B7H6-specific CARs have the potential to treat B7H6+ hematologic and solid tumors. PMID:25830550

  18. 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.

  19. 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.

  20. Dopamine receptor gene expression by enkephalin neurons in rat forebrain

    SciTech Connect

    Le Moine, C.; Normand, E.; Guitteny, A.F.; Fouque, B.; Teoule, R.; Bloch, B. )

    1990-01-01

    In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex. Hybridization with D2 and preproenkephalin A probes in adjacent sections, as well as combined hybridization with the two probes in the same sections, demonstrated that all detectable enkephalin neurons in the striatum contained the D2 receptor mRNA. Large neurons in caudate putamen, which were unlabeled with the preproenkephalin A probe and which may have been cholinergic, also expressed the D2 receptor gene. Haloperidol treatment (14 or 21 days) provoked an increase in mRNA content for D2 receptor and preproenkephalin A in the striatum. This suggests that the increase in D2 receptor number observed after haloperidol treatment is due to increased activity of the D2 gene. These results indicate that in the striatum, the enkephalin neurons are direct targets for dopamine liberated from mesostriatal neurons.

  1. 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

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

    PubMed

    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

  3. Unique antigenic gene expression at different developmental stages of Trichinella pseudospiralis.

    PubMed

    Wu, X P; Liu, X L; Wang, X L; Blaga, R; Fu, B Q; Liu, P; Bai, X; Wang, Z J; Rosenthal, B M; Shi, H N; Sandrine, L; Vallee, I; Boireau, P; Wang, F; Zhou, X N; Zhao, Y; Liu, M Y

    2013-05-20

    Parasite-induced and parasite-regulated larval capsule formation and host immunosuppression are two major characteristics that are unique in Trichinella spp. infections, but the molecule(s) and mechanism(s) that mediate these processes remain largely unknown. Trichinella pseudospiralis and Trichinella spiralis, are obviously different with respect to these two characteristics. A comparative study of these two species, in particular their antigen expression profiles at different developmental stages (the main molecules involved in the cross-talk or interaction between each parasite and its host), may help us better understand the parasite molecules and mechanisms involved. Here, we constructed cDNA libraries from T. pseudospiralis adults (Ad), newborn larvae (NBL) and muscle larvae (ML) mRNA and screened them with pig anti-T. pseudospiralis serum collected 26, 32 and 60 days post-infection (p.i.). The most abundant antigens were found to vary among life-cycle stages. Pyroglutamy peptidase 1-like and 6-phosphogluconolactonase-like genes predominated in the Ad stage and a serine protease (SS2-1-like gene) predominated in NBL similar to that observed in T. spiralis. Muscle larvae expressed proteasome activator complex subunit 3-like and 21 kDa excretory/secretory protein-like genes. This study indicated that parasites of two species may utilise different molecules and mechanisms for larvae capsule formation and host immunosuppression during their infections. Proteins of antigenic genes identified in this study may be also good candidates for diagnosis, treatment or vaccination for T. pseudospiralis infection, and also for the differential diagnosis of two species' infections. PMID:23433603

  4. Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

    PubMed

    Singh, Bijay; Maharjan, Sushila; Kim, You-Kyoung; Jiang, Tai; Islam, Mohammad Ariful; Kang, Sang-Kee; Cho, Myung-Haing; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-01

    Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational

  5. Identification of a human erythrocyte receptor for colonization factor antigen I pili expressed by H10407 enterotoxigenic Escherichia coli.

    PubMed Central

    Pieroni, P; Worobec, E A; Paranchych, W; Armstrong, G D

    1988-01-01

    We have identified a receptor for colonization factor antigen I (CFA/I) pili in human erythrocyte membranes. Erythrocyte binding assays, using whole organisms, suggested that the CFA/I receptor was a glycoprotein containing important sialic acid moieties. Subsequently, human erythrocyte membranes were extracted with lithium diiodosalicylate to obtain a soluble glycoprotein fraction from which to isolate receptors. The extracted material caused agglutination of the CFA/I+ but not the CFA/I- organisms at a protein concentration of 0.5 mg/ml. The CFA/I receptor was identified in iodinated extract by an affinity isolation procedure, using whole bacterial cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the washed, extract-coated H10407 CFA/I+ organisms revealed a band with an apparent molecular weight of 26,000 which was present in the original extract but was not observed on extract-coated H10407 CFA/I- bacteria. The addition of purified CFA/I pili reduced binding of the 26,000-molecular-weight receptor to CFA/I+ bacteria. The CFA/I-specific receptor species also bound to wheat germ agglutinin-agarose. This observation supported the suggestion that the CFA/I receptor identified in this report is a sialoglycoprotein. Images PMID:2895745

  6. The Mouse Solitary Odorant Receptor Gene Promoters as Models for the Study of Odorant Receptor Gene Choice

    PubMed Central

    Degl'Innocenti, Andrea

    2016-01-01

    Background In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice. Aim Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary) from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice. Procedures Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J), and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes) and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections. Results In the mouse genome there are eight intact solitary genes: Olfr19 (M12), Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a

  7. Genetic Analysis of the Serratia marcescens N28b O4 Antigen Gene Cluster

    PubMed Central

    Saigí, Francesc; Climent, Núria; Piqué, Núria; Sanchez, Cesar; Merino, Susana; Rubirés, Xavier; Aguilar, Alicia; Tomás, Juan M.; Regué, Miguel

    1999-01-01

    The Serratia marcescens N28b wbbL gene has been shown to complement the rfb-50 mutation of Escherichia coli K-12 derivatives, and a wbbL mutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubirés, F. Saigí, N. Piqué, N. Climent, S. Merino, S. Albertí, J. M. Tomás, and M. Regué, J. Bacteriol. 179:7581–7586, 1997). We analyzed a recombinant cosmid containing the wbbL gene by subcloning and determination of O-antigen production phenotype in E. coli DH5α by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments with S. marcescens O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when an E. coli K-12 strain with a deletion of the wb cluster was used, suggesting that the O4 wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a wecA mutant E. coli strain, suggesting that O4-antigen production is wecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of the S. marcescens N28b wbbA insertion mutant and wzm-wzt deletion mutant, we suggest that the O4 wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS4 was found downstream from the last gene in the cluster (wbbA), suggesting that an IS4-like element could have been involved in the acquisition of the O4 wb cluster. PMID:10074083

  8. Expression of complete transplantation antigens by mammalian cells transformed with truncated class I genes.

    PubMed

    Goodenow, R S; Stroynowski, I; McMillan, M; Nicolson, M; Eakle, K; Sher, B T; Davidson, N; Hood, L

    1983-02-01

    Mouse L cells transformed with the cloned class I genes of the major histocompatibility complex of the mouse express transplantation antigens with serological determinants of the donor haplotype. However, transformation with the truncated subclones of a BALB/c H-2Ld gene containing the exons encoding the external domains also leads to the production of cells which express complete cell-surface molecules. Moreover, full-length products of the foreign haplotype, as judged by serological and biochemical criteria, are generated independently of the use of carrier DNA in transformation. However, the frequency of productive transformation is substantially less than that obtained with a complete gene. The most plausible explanation for these phenomena involves homologous recombination between host chromosomal and donor class I sequences. PMID:6823314

  9. Mammalian non-classical major histocompatibility complex I and its receptors: Important contexts of gene, evolution, and immunity

    PubMed Central

    Pratheek, B. M.; Nayak, Tapas K.; Sahoo, Subhransu S.; Mohanty, Prafulla K.; Chattopadhyay, Soma; Chakraborty, Ntiya G.; Chattopadhyay, Subhasis

    2014-01-01

    The evolutionary conserved, less-polymorphic, nonclassical major histocompatibility complex (MHC) class I molecules: Qa-1 and its human homologue human leukocyte antigen-E (HLA-E) along with HLA-F, G and H cross-talk with the T-cell receptors and also interact with natural killer T-cells and other lymphocytes. Moreover, these nonclassical MHC molecules are known to interact with CD94/NKG2 heterodimeric receptors to induce immune responses and immune regulations. This dual role of Qa-1/HLA-E in terms of innate and adaptive immunity makes them more interesting. This review highlights the new updates of the mammalian nonclassical MHC-I molecules in terms of their gene organization, evolutionary perspective and their role in immunity. PMID:25400340

  10. Sir2 Paralogues Cooperate to Regulate Virulence Genes and Antigenic Variation in Plasmodium falciparum

    PubMed Central

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

    2009-01-01

    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. PMID:19402747

  11. 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

  12. Prostate stem cell antigen interacts with nicotinic acetylcholine receptors and is affected in Alzheimer's disease.

    PubMed

    Jensen, Majbrit M; Arvaniti, Maria; Mikkelsen, Jens D; Michalski, Dominik; Pinborg, Lars H; Härtig, Wolfgang; Thomsen, Morten S

    2015-04-01

    Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes and modulating their function. Hence, changes in nAChR regulatory proteins such as Lynx proteins could underlie the dysregulation of nAChRs in AD. Using Western blotting, we detected bands corresponding to the Lynx proteins prostate stem cell antigen (PSCA) and Lypd6 in human cortex indicating that both proteins are present in the human brain. We further showed that PSCA forms stable complexes with the α4 nAChR subunit and decreases nicotine-induced extracellular-signal regulated kinase phosphorylation in PC12 cells. In addition, we analyzed protein levels of PSCA and Lypd6 in postmortem tissue of medial frontal gyrus from AD patients and found significantly increased PSCA levels (approximately 70%). In contrast, no changes in Lypd6 levels were detected. In concordance with our findings in AD patients, PSCA levels were increased in the frontal cortex of triple transgenic mice with an AD-like pathology harboring human transgenes that cause both age-dependent β-amyloidosis and tauopathy, whereas Tg2576 mice, which display β-amyloidosis only, had unchanged PSCA levels compared to wild-type animals. These findings identify PSCA as a nAChR-binding protein in the human brain that is affected in AD, suggesting that PSCA-nAChR interactions may be involved in the cognitive dysfunction observed in AD. PMID:25680266

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

    PubMed

    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; Maus, Marcela V

    2016-03-01

    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

  14. 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

  15. 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

  16. Characterization of the antigenic and functional domains of a Mycoplasma synoviae variant vlhA gene.

    PubMed

    Khiari, Awatef Béjaoui; Mardassi, Boutheina Ben Abdelmoumen

    2012-05-01

    The Mycoplasma synoviae haemagglutinin gene, vlhA, encodes two major immunodominant and surface-exposed membrane proteins, MSPB and MSPA. Both products are antigenically variable but only MSPA mediates binding to erythrocytes. Previously we have shown that M. synoviae type strain WVU 1853 could express a variant vlhA gene, referred to as MS2/28.1, with a considerably shorter and divergent MSPA region. A finding that prompted detailed characterization of its antigenic and functional properties. Here we mutagenized each of the six opal codons of the variant MS2/28.1 vlhA member into tryptophan, thus allowing its expression in Escherichia coli as well as its cleavage products, MSPB and MSPA. In addition, we expressed 5 contiguous regions of MS2/28.1 extending from the last part of MSPB to the C-terminus of MSPA. Colony immunostaining with region-specific antisera mapped antigenic variation to the N-terminal half of MS2/28.1 MSPA. No haemagglutinating activity was observed for MSPB, but consistent haemadsorption inhibition was mapped to the region extending from amino acid 325 to 344. Inhibition of both haemagglutination and haemadsorption activities were obtained with sera directed against the C-terminal region of MSPA, with the highest titers (1/320 and 1/160, respectively) being recorded for its last 60 residues. Importantly, antibodies to this region also yielded the highest metabolic inhibition titer of 1/1280. Overall, aside from mapping the functional domains of a M. synoviae highly divergent haemagglutinin gene, this study shows that the C-terminal half of its MSPA region induced the highest titers of antibodies inhibiting haemagglutination, haemadsorption, and metabolism. PMID:22176762

  17. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.

    PubMed

    Singh, Harjeet; Figliola, Matthew J; Dawson, Margaret J; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E; Huls, Helen; Cooper, Laurence J N

    2013-01-01

    Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10) T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

  18. Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

    PubMed Central

    Singh, Harjeet; Figliola, Matthew J.; Dawson, Margaret J.; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E.; Huls, Helen; Cooper, Laurence J. N.

    2013-01-01

    Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

  19. Regulation of the IL-12 receptor beta2 subunit by soluble antigen and IL-12 in vivo.

    PubMed

    Galbiati, F; Rogge, L; Guéry, J C; Smiroldo, S; Adorini, L

    1998-01-01

    Continuous administration of soluble protein antigen to BALB/c mice inhibits the development of Th1 and induces selective differentiation of Th2 cells. Here we show that interleukin (IL)-12, administered together with soluble protein through a mini-osmotic pump implanted subcutaneously, not only prevents the inhibition of Th1 cell development, but stimulates higher interferon (IFN)-gamma production than in mice receiving IL-12 alone. In parallel to co-stimulation of Th1 cell development, co-administration of IL-12 blocks the Th2 response induced by soluble protein. IL-12 administered in adjuvant with antigen or intraperitoneally 2 days after the immunization does not break the inhibition of Th1 but can still decrease the Th2 response induced by pretreatment with soluble protein antigen. In contrast to IL-12, co-administration of IL-2 or IFN-gamma does not affect the diversion to Th2 induced by soluble antigen. Thus IL-12, but not IL-2 nor IFN-gamma, converts in vivo the inhibitory signal for Th1 cell development delivered by soluble antigen into an immunogenic one, while blocking a positive signal for Th2 cell differentiation. A molecular basis for the co-stimulation of Th1 priming and the prevention of Th2 differentiation by IL-12 in vivo is provided by the observation that transcripts encoding the IL-12 receptor beta2 chain, which is required for IL-12 signaling and Th1 cell development, are selectively inhibited by soluble antigen but are enhanced by IL-12 co-administration. PMID:9485201

  20. Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone.

    PubMed

    Krug, Christian; Birkholz, Katrin; Paulus, Alexander; Schwenkert, Michael; Schmidt, Patrick; Hoffmann, Nicole; Hombach, Andreas; Fey, Georg; Abken, Hinrich; Schuler, Gerold; Schuler-Thurner, Beatrice; Dörrie, Jan; Schaft, Niels

    2015-12-01

    Chimeric antigen receptor (CAR)-modified T cells emerged as effective tools in the immunotherapy of cancer but can produce severe on-target off-tissue toxicities. This risk can conceivably be overcome, at least partially, by transient transfection. The design of CARs, however, has so far not been optimized for use in non-permanent T cell modification. Here we compared the performance of T cells modified with three different first- and second-generation CARs, each specific for MCSP (HMW-MAA) which is commonly expressed by melanoma cells. Upon RNA transfer, the expression of all receptors was limited in time. The second-generation CARs, which combined CD28-CD3ζ signaling, were expressed at higher levels and more prolonged than first-generation CARs with CD3ζ only. The CD28 domain increased the cytokine production, but had only an indirect effect on the lytic capacity, by prolonging the CAR expression. Especially for the second-generation CARs, the scFv clearly impacted the level and duration of CAR expression and the T cell performance. Thus, we identified a CAR high in both expression and anti-tumor cell reactivity. T cells transfected with this CAR increased the mean survival time of mice after challenge with melanoma cells. To facilitate clinical application, this CAR was used to redirect T cells from late-stage melanoma patients by RNA transfection. These T cells mediated effective antigen-specific tumor cell lysis and release of pro-inflammatory cytokines, even after cryoconservation of the transfected T cells. Taken together, the analysis identified a CAR with superior anti-melanoma performance after RNA transfer which is a promising candidate for clinical exploration. PMID:26515978

  1. Transcription analysis, physical mapping, and molecular characterization of a nonclassical human leukocyte antigen class I gene.

    PubMed Central

    Chorney, M J; Sawada, I; Gillespie, G A; Srivastava, R; Pan, J; Weissman, S M

    1990-01-01

    The human major histocompatibility complex contains approximately 20 class I genes, pseudogenes, and gene fragments. These include the genes for the three major transplantation antigens, HLA-A, HLA-B, and HLA-C, as well as a number of other genes or pseudogenes of unknown biological significance. Most of the latter have C + G-rich sequences in their 5' ends that are unmethylated in the B-lymphoblastoid cell line 3.1.0. We investigated one of these genes, HLA-H, in more detail. The gene is, overall, strongly homologous in sequence to HLA-A but differs in several potentially significant ways, including changes in conserved promoter sequences, a single-base deletion producing a translation termination codon in exon 4, and a region of sequence divergence downstream of the transcribed portion of the gene. Nevertheless, mouse L cells transfected with the gene accumulated small amounts of apparently full-length polyadenylated RNA. A portion of this RNA begins at the transcription site predicted by analogy to certain class I cDNA clones, while another portion appears to begin shortly upstream. L cells transfected with a hybrid gene containing the first three exons of HLA-H and the last five exons of HLA-B27 accumulated full-length HLA transcripts at the same level as cells transfected with an HLA-B27 gene; both levels are at least 15- to 20-fold higher than that directed by HLA-H alone. In addition, we isolated a cDNA clone for HLA-H that contains a portion of intron 3 attached to a normally spliced sequence comprising exons 4 through 8. These results suggest that low levels of translatable mRNA for the truncated class I heavy chain encoded by HLA-H are produced under physiologic circumstances and that sequences 3' of intron 3 decrease the levels of stable transcripts. Images PMID:2294403

  2. Erasing the Epigenetic Memory and Beginning to Switch—The Onset of Antigenic Switching of var Genes in Plasmodium falciparum

    PubMed Central

    Fastman, Yair; Noble, Robert; Recker, Mario; Dzikowski, Ron

    2012-01-01

    Antigenic variation in Plasmodium falciparum is regulated by transcriptional switches among members of the var gene family, each expressed in a mutually exclusive manner and encoding a different variant of the surface antigens collectively named PfEMP1. Antigenic switching starts when the first merozoites egress from the liver and begin their asexual proliferation within red blood cells. By erasing the epigenetic memory we created parasites with no var background, similar to merozoites that egress from the liver where no var gene is expressed. Creating a null-var background enabled us to investigate the onset of antigenic switches at the early phase of infection. At the onset of switching, var transcription pattern is heterogeneous with numerous genes transcribed at low levels including upsA vars, a subtype that was implicated in severe malaria, which are rarely activated in growing cultures. Analysis of subsequent in vitro switches shows that the probability of a gene to turn on or off is not associated with its chromosomal position or promoter type per se but on intrinsic properties of each gene. We concluded that var switching is determined by gene specific associated switch rates rather than general promoter type or locus associated switch rates. In addition, we show that fine tuned reduction in var transcription increases their switch rate, indicating that transcriptional perturbation can alter antigenic switching. PMID:22461905

  3. 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

  4. 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.

  5. Induction of the nuclear I{kappa}B protein I{kappa}B-{zeta} upon stimulation of B cell antigen receptor

    SciTech Connect

    Hijioka, Kuniaki; Matsuo, Susumu; Eto-Kimura, Akiko; Takeshige, Koichiro; Muta, Tatsushi . E-mail: tmuta@biology.tohoku.ac.jp

    2007-05-04

    The nuclear I{kappa}B protein I{kappa}B-{zeta} is barely detectable in resting cells and is induced in macrophages and fibroblasts following stimulation of innate immunity via Toll-like receptors. The induced I{kappa}B-{zeta} associates with nuclear factor (NF)-{kappa}B in the nucleus and plays crucial roles in its transcriptional regulation. Here, we examined the induction of I{kappa}B-{zeta} in B lymphocytes, one of the major players in adaptive immunity. Upon crosslinking of the surface immunoglobulin complex, I{kappa}B-{zeta} mRNA was robustly induced in murine B-lymphoma cell line A20 cells. While the crosslinking activated NF-{kappa}B and induced its target gene, I{kappa}B-{alpha}, co-crosslinking of Fc{gamma} receptor IIB to the surface immunoglobulin complex inhibited NF-{kappa}B activation and the induction of I{kappa}B-{zeta} and I{kappa}B-{alpha}, suggesting critical roles for NF-{kappa}B in the induction. These results indicate that I{kappa}B-{zeta} is also induced by stimulation of B cell antigen receptor, suggesting that I{kappa}B-{zeta} is involved in the regulation of adaptive immune responses.

  6. A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

    PubMed Central

    Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

    2015-01-01

    Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

  7. Subsets of Human Dendritic Cell Precursors Express Different Toll-like Receptors and Respond to Different Microbial Antigens

    PubMed Central

    Kadowaki, Norimitsu; Ho, Stephen; Antonenko, Svetlana; de Waal Malefyt, Rene; Kastelein, Robert A.; Bazan, Fernando; Liu, Yong-Jun

    2001-01-01

    Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c+ immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-α and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-α. CD11c+ imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-α, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-α and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens. PMID:11561001

  8. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.

    PubMed

    Kadowaki, N; Ho, S; Antonenko, S; Malefyt, R W; Kastelein, R A; Bazan, F; Liu, Y J

    2001-09-17

    Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c(+) immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-alpha. CD11c(+) imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-alpha, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-alpha and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens. PMID:11561001

  9. The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells.

    PubMed

    Rappl, Gunter; Riet, Tobias; Awerkiew, Sabine; Schmidt, Annette; Hombach, Andreas A; Pfister, Herbert; Abken, Hinrich

    2012-01-01

    Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+) CD57(+) CD7(-) phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+) T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter. PMID:22292024

  10. Inhibition of antigen receptor-dependent Ca(2+) signals and NF-AT activation by P2X7 receptors in human B lymphocytes.

    PubMed

    Pippel, Anja; Beßler, Björn; Klapperstück, Manuela; Markwardt, Fritz

    2015-04-01

    One of the first intracellular signals after antigen binding by the antigen receptor of B lymphocytes is the increased intracellular Ca(2+) concentration ([Ca(2+)]i), which is followed by several intracellular signaling events like the nuclear translocation of the transcription factor NF-AT controlling the fate of B lymphocytes after their activation. Extracellular ATP, which is released from cells under several pathological conditions, is considered a danger-associated signal serving as an immunomodulator. We investigated the interaction of antigen receptor (BCR) and P2X7 receptor (P2X7R) activation on [Ca(2+)]i signaling and on nuclear translocation of the transcription factor NF-AT in human B lymphocytes. Although the P2X7R is an ATP-gated Ca(2+)-permeable ion channel, P2X7R activation inhibits the BCR-mediated [Ca(2+)]i responses. This effect is mimicked by cell membrane depolarization induced by an increase in the extracellular K(+) concentration or by application of the Na(+) ionophore gramicidin, but is abolished by stabilization of the membrane potential using the K(+) ionophore valinomycin, by extracellular Mg(2+), which is known to inhibit P2X7R-dependent effects, or by replacing Na(+) by the less P2X7R-permeable Tris(+) ion. Furthermore, P2X7R activation by ATP inhibits the BCR-dependent translocation of the transcription factor NF-ATc1 to the nucleus. We therefore conclude that extracellular ATP via the P2X7R mediates inhibitory effects on B cell activation. This may be of relevance for understanding of the activation of the BCR under pathological conditions and for the development of therapeutic strategies targeting human B lymphocytes or P2X7 receptors. PMID:25678443

  11. 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.

  12. Generation of Antigenic Diversity in Plasmodium falciparum by Structured Rearrangement of Var Genes During Mitosis

    PubMed Central

    Kekre, Mihir; Otto, Thomas D.; Faizullabhoy, Adnan; Rayner, Julian C.; Kwiatkowski, Dominic

    2014-01-01

    The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7–72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle. PMID:25521112

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

    PubMed Central

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

    2009-01-01

    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 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 epitope derived from a novel antigen termed TCRγ 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-cell Lymphocytes. PMID:18446790

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

    PubMed

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

    1993-07-15

    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. PMID:8341702

  15. 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. PMID:25962108

  16. Engineering NK cells modified with an EGFRvIII-specific chimeric antigen receptor to overexpress CXCR4 improves immunotherapy of CXCL12/SDF-1α-secreting glioblastoma

    PubMed Central

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

    2015-01-01

    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. Based on the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an EGFRvIII-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII+ glioblastoma cells in vitro and to established subcutaneous U87-MGEGFRvIII 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 to 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 to 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. PMID:25962108

  17. CD4+ T Cells and Toll-Like Receptors Recognize Salmonella Antigens Expressed in Bacterial Surface Organelles

    PubMed Central

    Bergman, Molly A.; Cummings, Lisa A.; Barrett, Sara L. Rassoulian; Smith, Kelly D.; Lara, J. Cano; Aderem, Alan; Cookson, Brad T.

    2005-01-01

    A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition. PMID:15731032

  18. 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

    2016-09-01

    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. PMID:27454490

  19. 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

    2016-06-01

    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. PMID:27131290

  20. Expression of serotonin receptor genes in cranial ganglia.

    PubMed

    Maeda, Naohiro; Ohmoto, Makoto; Yamamoto, Kurumi; Kurokawa, Azusa; Narukawa, Masataka; Ishimaru, Yoshiro; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

    2016-03-23

    Taste cells release neurotransmitters to gustatory neurons to transmit chemical information they received. Sweet, umami, and bitter taste cells use ATP as a neurotransmitter. However, ATP release from sour taste cells has not been observed so far. Instead, they release serotonin when they are activated by sour/acid stimuli. Thus it is still controversial whether sour taste cells use ATP, serotonin, or both. By reverse transcription-polymerase chain reaction and subsequent in situ hybridization (ISH) analyses, we revealed that of 14 serotonin receptor genes only 5-HT3A and 5-HT3B showed significant/clear signals in a subset of neurons of cranial sensory ganglia in which gustatory neurons reside. Double-fluorescent labeling analyses of ISH for serotonin receptor genes with wheat germ agglutinin (WGA) in cranial sensory ganglia of pkd1l3-WGA mice whose sour neural pathway is visualized by the distribution of WGA originating from sour taste cells in the posterior region of the tongue revealed that WGA-positive cranial sensory neurons rarely express either of serotonin receptor gene. These results suggest that serotonin receptors expressed in cranial sensory neurons do not play any role as neurotransmitter receptor from sour taste cells. PMID:26854841

  1. Expression of chimeric antigen receptors in natural killer cells with a regulatory-compliant non-viral method

    PubMed Central

    Li, Linhong; Liu, Linda N; Feller, Stephanie; Allen, Cornell; Shivakumar, Rama; Fratantoni, Joseph; Wolfraim, Lawrence A.; Fujisaki, Hiroyuki; Campana, Dario; Chopas, Nicholas; Dzekunov, Sergey; Peshwa, Madhusudan

    2009-01-01

    Natural killer1 cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N=5) or purified unstimulated NK cells from peripheral blood (N=4) resulted in good cell viability with eGFP expression in 85% ± 6% and 86% ± 4%, 24 hours after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19+ target cells resulting in ≥80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 hours after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer. PMID:19745843

  2. The IgM antigen receptor of B lymphocytes is associated with prohibitin and a prohibitin-related protein.

    PubMed Central

    Terashima, M; Kim, K M; Adachi, T; Nielsen, P J; Reth, M; Köhler, G; Lamers, M C

    1994-01-01

    The two major classes of antigen receptors on murine B lymphocytes, mIgM and mIgD, are both contained in a complex with two additional molecules, Ig-alpha and Ig-beta, which permit signal transduction. Accordingly, early biochemical events after antigen binding to either receptor are similar; biological effects, however, are different. Here, we describe three newly discovered intracellular proteins of 32, 37 and 41 kDa molecular mass, that are non-covalently associated with mIgM, but not with mIgD. These proteins coprecipitate with mIgM in Triton X-100 and Nonidet P-40, but not in digitonin lysates. In addition, mIgM is to some extent associated with 29 and 31 kDa proteins that are predominantly associated with mIgD (see accompanying paper). Amino acid sequencing of p32 and p37 identified p32 as mouse prohibitin; this was corroborated by Western blot analysis with antibodies specific for rat prohibitin. p37 is a newly discovered protein. cDNA clones for both proteins were isolated and sequenced. The deduced amino acid sequence of p32 is identical to that of rat prohibitin. p37 is highly homologous to p32. Since prohibitin was identified as an inhibitor of cell proliferation, its association with mIgM, but not mIgD, could explain the different biological events elicited after engagement of each receptor. Images PMID:8070406

  3. Role of the mannose receptor in phagocytosis of Enterococcus faecalis strain EC-12 by antigen-presenting cells

    PubMed Central

    Tsuruta, Takeshi; Inoue, Ryo; Nagino, Takayuki; Nishibayashi, Ryoichiro; Makioka, Yuko; Ushida, Kazunari

    2013-01-01

    Abstract The aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Fluorescein isothiocyanate (FITC)-labeled EC-12 was cocultured with peritoneal macrophage and the amount of EC-12 phagocytosed by peritoneal macrophages was measured using a microplate fluorometer. Peritoneal macrophages from toll-like receptor (TLR)2-, TLR7-, and MyD88-deficient knockout (KO) mice exhibited similar levels of EC-12 phagocytosis to those from wild-type mice. Similarly, dectin-1 neutralization of peritoneal macrophages had no effect on EC-12 phagocytosis. However, blockade of the mannose receptor (MR) significantly decreased the amount of EC-12 phagocytosed by peritoneal macrophages; the same effect was observed in bone marrow-derived macrophages and dendritic cells. Our findings suggest that MR plays a major role in EC-12 phagocytosis by the APCs. This aim of this study was to clarify the phagocytic mechanisms of a heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) by antigen-presenting cells (APCs). Our findings suggest that mannose receptor (MR) plays a major role in EC-12 phagocytosis by the APCs. PMID:23801521

  4. Chemosensory receptor genes in the Oriental tobacco budworm Helicoverpa assulta.

    PubMed

    Xu, W; Papanicolaou, A; Liu, N-Y; Dong, S-L; Anderson, A

    2015-04-01

    The Oriental tobacco budworm (Helicoverpa assulta) is a specialist herbivore moth and its larvae feed on Solanaceous plants. (Z)-9-hexadecenal (Z9-16: Ald) is the major sex pheromone component in H. assulta but the specific pheromone receptor (PR) against Z9-16: Ald has not yet been identified. In the present study, we integrated transcriptomic, bioinformatic and functional characterization approaches to investigate the chemosensory receptor genes of H. assulta. We identified seven potential PRs with 44 olfactory receptors, 18 gustatory receptors and 24 ionotropic receptors, which were further studied by in silico gene expression profile, phylogenetic analysis, reverse transcription PCR and calcium imaging assays. The candidate PR, HassOR13, showed a strong response to the minor sex pheromone component, (Z)-11-hexadecenal, but not the major component, Z9-16: Ald, in calcium imaging assays. This study provides the molecular basis for comparative studies of chemosensory receptors between H. assulta and other Helicoverpa species and will advance our understanding of the evolution and function of Lepidoptera insect chemosensation. PMID:25430896

  5. 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.

  6. NK cells engineered to express a GD2-specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

    PubMed Central

    Esser, Ruth; Müller, Tina; Stefes, Dörthe; Kloess, Stephan; Seidel, Diana; Gillies, Stephen D; Aperlo-Iffland, Christel; Huston, James S; Uherek, Christoph; Schönfeld, Kurt; Tonn, Torsten; Huebener, Nicole; Lode, Holger N; Koehl, Ulrike; Wels, Winfried S

    2012-01-01

    Abstract Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD2, which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD2-specific chimeric antigen receptor (CAR) comprising an anti-GD2 ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD2 expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD2-specific antibody or anti-idiotypic antibody occupying the CAR’s cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD2-specific NK cells was also found against primary NB cells and GD2 expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells. PMID:21595822

  7. Selection for genes encoding secreted proteins and receptors.

    PubMed Central

    Klein, R D; Gu, Q; Goddard, A; Rosenthal, A

    1996-01-01

    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. Images Fig. 1 PMID:8692953

  8. 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…

  9. T-cell receptor gene homologs are present in the most primitive jawed vertebrates.

    PubMed Central

    Rast, J P; Litman, G W

    1994-01-01

    The phylogenetic origins of T-cell immunity and T-cell antigen receptor (TCR) genes have not been established. A PCR approach using short, minimally degenerate oligodeoxynucleotide primers complementing conserved variable region segments amplifies TCR-like products from the genomic DNA of Heterodontus francisci (horned shark), a representative phylogenetically primitive cartilaginous fish. One of these products has been used as a probe to screen a Heterodontus spleen cDNA library and a clone was identified that is most related at the nucleotide sequence and predicted peptide levels to higher vertebrate TCR beta-chain genes. Genomic analyses of the TCR homologs indicate that recombining variable and joining region segments as well as constant region exons are encoded by extensive gene families, organized in the multicluster form, characteristic of both the immunoglobulin heavy- and light-chain gene loci in the cartilaginous fishes. Greater numbers of homologous products were identified when a probe complementing the putative constant region of the TCR homolog was used to screen the same cDNA library. A high degree of intergenic variation is associated with the putative variable region segments of these isolates. Direct evidence is presented for TCR-like genes, which presumably are associated with T-cell function, at the earliest stages in the phylogenetic emergence of jawed vertebrates. Images PMID:7937749

  10. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Rivoltini, L; Topalian, S L; Miki, T; Rosenberg, S A

    1994-01-01

    By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma. PMID:8170938

  11. Interleukin-1 receptor antagonist gene therapy for arthritis.

    PubMed

    Krishnan, B R

    1999-08-01

    Rheumtatoid arthritis (RA) is a crippling, autoimmune disease, and is characterized by inflammation and destruction of joint tissue. Interleukin-1 (IL-1) has been identified as a key pro-inflammatory cytokine responsible for inflammation. One of the mechanisms of regulation of activity of IL-1 is IL-1 receptor antagonist (IL-1ra)-mediated: IL-1RA competes with IL-1 for binding to the IL-1 receptor. Significant progress has been made in the potential application of IL-1ra gene therapyfor the treatment of arthritis. Various vectors have been tested for the delivery of the IL-1ra gene to the intra-articular region. Recent studies in humans have provided encouraging prospects for IL-1ra-mediated arthritis gene therapy. PMID:11713759

  12. 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

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

    PubMed

    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

  14. 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

  15. 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

  16. DNAM-1-based chimeric antigen receptors enhance T cell effector function and exhibit in vivo efficacy against melanoma

    PubMed Central

    Wu, Ming-Ru; Zhang, Tong; Alcon, Andre

    2015-01-01

    Chimeric antigen receptor (CAR) T cell therapies hold great potential for treating cancers, and new CARs that can target multiple tumor types and have the potential to target non-hematological malignancies are needed. In this study, the tumor recognition ability of a natural killer cell-activating receptor, DNAM-1 was harnessed to design CARs that target multiple tumor types. DNAM-1 ligands, PVR and nectin-2, are expressed on primary human leukemia, myeloma, ovarian cancer, melanoma, neuroblastoma, and Ewing sarcoma. DNAM-1 CARs exhibit high tumor cell cytotoxicity but low IFN-γ secretion in vitro. In contrast to other CAR designs, co-stimulatory domains did not improve the expression and function of DNAM-1 CARs. A DNAM-1/CD3zeta CAR reduced tumor burden in a murine melanoma model in vivo. In conclusion, DNAM-1-based CARs may have the potential to treat PVR and nectin-2 expressing hematological and solid tumors. PMID:25549845

  17. Identification of Significant Association and Gene-Gene Interaction of GABA Receptor Subunit Genes in Autism

    PubMed Central

    Ma, D. Q.; Whitehead, P. L.; Menold, M. M.; Martin, E. R.; Ashley-Koch, A. E.; Mei, H.; Ritchie, M. D.; DeLong, G. R.; Abramson, R. K.; Wright, H. H.; Cuccaro, M. L.; Hussman, J. P.; Gilbert, J. R.; Pericak-Vance, M. A.

    2005-01-01

    Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied to look for the genes associated with autism and their possible interactions. Single-nucleotide polymorphisms (SNPs) were screened in the following genes: GABRG1, GABRA2, GABRA4, and GABRB1 on chromosome 4p12; GABRB2, GABRA6, GABRA1, GABRG2, and GABRP on 5q34-q35.1; GABRR1 and GABRR2 on 6q15; and GABRA5, GABRB3, and GABRG3 on 15q12. Intronic and/or silent mutation SNPs within each gene were analyzed in 470 white families with autism. Initially, SNPs were used in a family-based study for allelic association analysis—with the pedigree disequilibrium test and the family-based association test—and for genotypic and haplotypic association analysis—with the genotype-pedigree disequilibrium test (geno-PDT), the association in the presence of linkage (APL) test, and the haplotype family-based association test. Next, with the use of five refined independent marker sets, extended multifactor-dimensionality reduction (EMDR) analysis was employed to identify the models with locus joint effects, and interaction was further verified by conditional logistic regression. Significant allelic association was found for markers RS1912960 (in GABRA4; P = .01) and HCV9866022 (in GABRR2; P = .04). The geno-PDT found significant genotypic association for HCV8262334 (in GABRA2), RS1912960 and RS2280073 (in GABRA4), and RS2617503 and RS12187676 (in GABRB2). Consistent with the allelic and genotypic association results, EMDR confirmed the main effect at RS1912960 (in GABRA4). EMDR also identified a

  18. Sequencing of the Escherichia coli O22 O Antigen Gene Cluster and Detection of E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and the wzy Gene in the Respective O-Antigen Gene Clusters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The O antigen gene cluster of Escherichia coli O22, an extraintestinal pathogenic E. coli (ExPEC) serogroup was sequenced, and eight open reading frames encoding O antigen sugar biosynthesis, transfer, and processing were identified. Multiplex PCR assays were developed targeting the wzx and wzy gen...

  19. 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.

  20. CRDB: database of chemosensory receptor gene families in vertebrate.

    PubMed

    Dong, Dong; Jin, Ke; 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

  1. Gene Expression Control by Glucocorticoid Receptors during Innate Immune Responses

    PubMed Central

    Xavier, Andre Machado; Anunciato, Aparecida Kataryna Olimpio; Rosenstock, Tatiana Rosado; Glezer, Isaias

    2016-01-01

    Glucocorticoids (GCs) are potent anti-inflammatory compounds that have been extensively used in clinical practice for several decades. GC’s effects on inflammation are generally mediated through GC receptors (GRs). Signal transduction through these nuclear receptors leads to dramatic changes in gene expression programs in different cell types, typically due to GR binding to DNA or to transcription modulators. During the last decade, the view of GCs as exclusive anti-inflammatory molecules has been challenged. GR negative interference in pro-inflammatory gene expression was a landmark in terms of molecular mechanisms that suppress immune activity. In fact, GR can induce varied inhibitory molecules, including a negative regulator of Toll-like receptors pathway, or subject key transcription factors, such as NF-κB and AP-1, to a repressor mechanism. In contrast, the expression of some acute-phase proteins and other players of innate immunity generally requires GR signaling. Consequently, GRs must operate context-dependent inhibitory, permissive, or stimulatory effects on host defense signaling triggered by pathogens or tissue damage. This review aims to disclose how contradictory or comparable effects on inflammatory gene expression can depend on pharmacological approach (including selective GC receptor modulators; SEGRMs), cell culture, animal treatment, or transgenic strategies used as models. Although the current view of GR-signaling integrated many advances in the field, some answers to important questions remain elusive. PMID:27148162

  2. Gene Expression Control by Glucocorticoid Receptors during Innate Immune Responses.

    PubMed

    Xavier, Andre Machado; Anunciato, Aparecida Kataryna Olimpio; Rosenstock, Tatiana Rosado; Glezer, Isaias

    2016-01-01

    Glucocorticoids (GCs) are potent anti-inflammatory compounds that have been extensively used in clinical practice for several decades. GC's effects on inflammation are generally mediated through GC receptors (GRs). Signal transduction through these nuclear receptors leads to dramatic changes in gene expression programs in different cell types, typically due to GR binding to DNA or to transcription modulators. During the last decade, the view of GCs as exclusive anti-inflammatory molecules has been challenged. GR negative interference in pro-inflammatory gene expression was a landmark in terms of molecular mechanisms that suppress immune activity. In fact, GR can induce varied inhibitory molecules, including a negative regulator of Toll-like receptors pathway, or subject key transcription factors, such as NF-κB and AP-1, to a repressor mechanism. In contrast, the expression of some acute-phase proteins and other players of innate immunity generally requires GR signaling. Consequently, GRs must operate context-dependent inhibitory, permissive, or stimulatory effects on host defense signaling triggered by pathogens or tissue damage. This review aims to disclose how contradictory or comparable effects on inflammatory gene expression can depend on pharmacological approach (including selective GC receptor modulators; SEGRMs), cell culture, animal treatment, or transgenic strategies used as models. Although the current view of GR-signaling integrated many advances in the field, some answers to important questions remain elusive. PMID:27148162

  3. Maxi-circles, glycosomes, gene transposition, expression sites, transsplicing, transferrin receptors and base J.

    PubMed

    Borst, Piet

    2016-01-01

    This is a personal story of the author of his research on trypanosomatids, covering a period of 1970-2015. Some of the highlights include the discovery of new aspects of kDNA, the mini-circle heterogeneity and the maxi-circle; the glycosome; the discovery of gene transposition as a major mechanism for antigenic variation; trans-splicing as an essential step in the synthesis of all trypanosome mRNAs; Pulsed Field Gradient gels to size-fractionate chromosome-sized DNA molecules of protozoa; the sequence of trypanosome telomeres and their growth and contraction; the first ABC-transporter of trypanosomatids, LtpgpA; the variable transferrin receptor of T. brucei and its role in Fe uptake; and base J, its structure, biosynthesis and function. PMID:27021571

  4. Hsp90-peptide complexes stimulate antigen presentation through the class II pathway after binding scavenger receptor SREC-I

    PubMed Central

    Murshid, Ayesha; Gong, Jianlin; Calderwood, Stuart K

    2016-01-01

    Molecular chaperones such as heat shock protein 90 (Hsp90) have been shown to form complexes with tumor antigens and can be used to prepare anticancer vaccines largely due to this property. Earlier studies had suggested that, mice immunized with a molecular chaperone based vaccine derived from tumors became immune to further vaccination and that both CD8+ and CD4+ T cells were activated by the chaperone vaccine in a manner dependent on scavenger receptor SREC-I. Here we have investigated mechanisms whereby SREC-I might facilitate uptake of Hsp90 conjugated peptides by APC into the MHC class II pathway for presentation to CD4+ T cells. Our studies showed that antigenic peptides associated with Hsp90 were taken up into the Class II pathway by a mechanism dependent on SREC-I binding and internalization and presented to CD4+ T cells. In addition our studies showed that SREC-I could associate with MHC class II molecules on the cell surface and in intracellular endosomes, suggesting a mechanism involving facilitated uptake of peptides into the MHC class II pathway. These studies in addition to our earlier findings showed SREC-I to play a primary role in chaperone-associated antigen uptake both through cross priming of MHC class I molecules and entry into the class II pathway. PMID:25155057

  5. 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

  6. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors.

    PubMed

    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

    2015-06-01

    Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor 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 show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials. PMID:25939063

  7. 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.

  8. Killer cell immunoglobulin like receptor gene association with tuberculosis.

    PubMed

    Pydi, Satya Sudheer; Sunder, Sharada Ramaseri; Venkatasubramanian, Sambasivan; Kovvali, Srinivas; Jonnalagada, Subbanna; Valluri, Vijaya Lakshmi

    2013-01-01

    NK cells are vital components of innate immune system and are the first cells which come into picture mediating resistance against intracellular pathogens. NK cell cytotoxicity is modulated by a wide variety of cell surface receptors that recognize and respond towards infected cells. Activation of NK cells are controlled by both inhibitory and activating receptors, encoded by KIR genes and bind to HLA ligands. Not much is known about KIR genes and their influence on the pathogenesis with M. tuberculosis infection. Our study aimed at detecting the presence of 14 KIR genes, their distribution and their association with tuberculosis. Total 77 different genotype combinations were observed which belonged to B-haplotype. Fifteen genotypes were similar to those reported in other world populations while remaining 62 were unique to this study group. Inhibitory genes KIR3DL1, KIR2DL3 and activating genes KIR2DS1, KIR2DS5 conferred susceptibility towards TB either individually or in haplotype combinations. The complimentary MHC ligands need to be tested for the functional relevance of the associated genes. PMID:23073291

  9. Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen.

    PubMed

    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

  10. 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

  11. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo

    PubMed Central

    Sommermeyer, Daniel; Hudecek, Michael; Kosasih, Paula L.; Gogishvili, Tea; Maloney, David G.; Turtle, Cameron J.; Riddell, Stanley R.

    2016-01-01

    Adoptive T-cell therapy with gene-modified T-cells expressing a tumor-reactive T-cell receptor (TCR) or chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and has shown success in the treatment of B-cell malignancies and solid tumors. In all reported trials, patients have received T-cell products comprised of random compositions of CD4+ and CD8+ naïve and memory T-cells, meaning that each patient received a different therapeutic agent. This variation might have influenced the efficacy of T-cell therapy, and complicates comparison of outcomes between different patients and across trials. We analyzed CD19 CAR-expressing effector T-cells derived from different subsets (CD4+/CD8+ naïve, central memory, effector memory). T-cells derived from each of the subsets were efficiently transduced and expanded, but showed clear differences in effector function and proliferation in vitro and in vivo. Combining the most potent CD4+ and CD8+ CAR-expressing subsets resulted in synergistic antitumor effects in vivo. We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T-cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies. PMID:26369987

  12. Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor–expressing T cells

    PubMed Central

    Lynn, Rachel C.; Poussin, Mathilde; Kalota, Anna; Feng, Yang; Low, Philip S.; Dimitrov, Dimiter S.

    2015-01-01

    T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ+ AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ+ THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34+ HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA. PMID:25887778

  13. The non-signaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity

    PubMed Central

    Hudecek, Michael; Sommermeyer, Daniel; Kosasih, Paula L.; Silva-Benedict, Anne; Liu, Lingfeng; Rader, Christoph; Jensen, Michael C.; Riddell, Stanley R.

    2015-01-01

    The use of synthetic chimeric antigen receptors (CAR) to redirect T cells to recognize tumor provides a powerful new approach to cancer immunotherapy; however the attributes of CARs that ensure optimal in vivo tumor recognition remain to be defined. Here, we analyze the influence of length and composition of IgG-derived extracellular spacer domains on the function of CARs. Our studies demonstrate that CD19-CARs with a long spacer from IgG4 hinge-CH2-CH3 are functional in vitro but lack antitumor activity in vivo due to interaction between the Fc domain within the spacer and the Fc receptor-bearing myeloid cells, leading to activation-induced T-cell death. We demonstrate that in vivo persistence and antitumor effects of CAR-T-cells with a long spacer can be restored by modifying distinct regions in the CH2 domain that are essential for Fc receptor binding. Our studies demonstrate that modifications that abrogate binding to Fc receptors are crucial for CARs in which a long spacer is obligatory for tumor recognition as shown here for a ROR1-specific CAR. These results demonstrate that the length and composition of the extracellular spacer domain that lacks intrinsic signaling function can be decisive in the design of CARs for optimal in vivo activity. PMID:25212991

  14. Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

    PubMed

    Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

    2016-06-01

    Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue. PMID:27163741

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

    PubMed Central

    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.

    2015-01-01

    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). PMID:25696001

  16. Elevated soluble CD8 antigen and soluble interleukin-2 receptors in the sera of patients with juvenile rheumatoid arthritis.

    PubMed

    Lipnick, R N; Sfikakis, P P; Klipple, G L; Tsokos, G C

    1993-07-01

    Activated T lymphocytes release various molecules including soluble CD8 (sCD8) antigen and soluble interleukin-2 receptor (sIL-2R). Elevated serum sCD8 antigen levels have been found in patients with viral infections, certain hematologic malignancies, and rheumatoid arthritis. On the other hand, elevated serum levels of sIL-2R have been found in various diseases including juvenile rheumatoid arthritis (JRA). We measured sCD8 antigen and sIL-2R levels using enzyme-linked immunosorbent assays in the sera of 49 afebrile patients with JRA (systemic 15, polyarticular 16, and pauciarticular 18) and 16 normal children. Disease activity was classified as mild, moderate, and severe. Sera from patients with severe JRA expressed statistically significant higher levels of both sCD8 and sIL-2R, whereas patients with mild disease had the lowest levels. There were no differences in the serum sCD8 and sIL-2R levels between the groups of patients with pauciarticular-, systemic-, and polyarticular-onset disease. Patients who were treated with prednisone had statistically nonsignificant higher serum levels of sCD8 and sIL-2R. A statistically significant positive correlation was found between sCD8 and sIL-2R levels, sCD8 levels and erythrocyte sedimentation rate (ESR), and sIL-2R levels and ESR. Our findings further suggest the presence of activated lymphocytes in patients with JRA and show that sCD8 antigen serum levels correlate with both serum levels of sIL-2R and ESR and thus may represent alternative indicators of disease activity. PMID:8513595

  17. [Sequence of Escherichia coli O11 O-antigen gene cluster and identification of molecular markers specific to O11].

    PubMed

    Wang, Wei; Peng, Xia; Wang, Quan; Cheng, Jian-Song; Wang, Lei

    2006-06-01

    Escherichia coli O11 belongs to Shiga toxin-producing Escherichia coli (STEC), which can cause food-borne disease, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) in humans. Because of its character of specificity, the O-antigen gene cluster provides the best material for the selection of molecular markers which can be used for rapid genotyping of bacterial strain. In this study, the E.coli O11 O-antigen gene cluster was amplified by Long-range PCR and was sequenced using Shotgun-sequencing approach. Twelve open reading frames were assigned functions on the basis of homology in the E. coli O11 O-antigen gene cluster, including UDP-N-acetyl glucosamine-4-epimerase gene (gne), genes responsible for the biosynthesis of GDP-L-fucose (gmd, fcl, gmm, manC, manB), glycosyl transferase genes, O-unit flippase gene (wzx) and O-antigen polymerase gene (wzy). By polymerase chain reaction against representative stains for all the 166 E. coli and 43 Shigella O serotypes, two genes and four pairs of primers were identified to be specific to E. coli O11. Further PCR was done to detect E. coli O11 from the environmental specimens, and the sensitivities for detecting E.coli O11 from the pork and dejecta specimens were 0.25 cfu/g and 2.5 x 10(3) cfu/g, respectively. Moreover, eight probes were designed and proved to be unique to E. coli O11, which provides the basis for a sensitive test of the rapid detection of E. coli O11 by DNA microarray method. PMID:16933598

  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. PMID:27296644

  19. 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.

  20. Characterization and allelic variation of the transporters associated with antigen processing (TAP) genes in the domestic dog (Canis lupus familiaris).

    PubMed

    Gojanovich, Gregory S; Ross, Peter; Holmer, Savannah G; Holmes, Jennifer C; Hess, Paul R

    2013-12-01

    The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8(+) T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs. PMID:23892057

  1. Characterization and allelic variation of the transporters associated with antigen processing (TAP) genes in the domestic dog (Canis lupus familiaris)

    PubMed Central

    Gojanovich, Gregory S.; Ross, Peter; Holmer, Savannah R.; Holmes, Jennifer C.; Hess, Paul R.

    2013-01-01

    The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8+ T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs. PMID:23892057

  2. [BCL1 POLYMORPHISM OF GLUCOCORTICOID RECEPTOR GENE AND RESPIRATORY DISEASES].

    PubMed

    Prystupa, L N; Garbuzova, V Yu; Kmyta, V V

    2015-01-01

    The article analyses the results of investigating the connection between BCL1-polymorphism of glucocorticoid receptor gene and respiratory diseases. Its role in increasing sensitivity to glucocorticoids is proved here. The authors investigated the association of Bcl1 polymorphism with predisposition to bronchial asthma, chronic obstructive pulmonary disease, with the nicotine addiction degree and with progressing disorders of pulmonary function in cystic fibrosis. PMID:26118026

  3. Mechanisms of oestrogen receptor (ER) gene regulation in breast cancer.

    PubMed

    Carroll, J S

    2016-07-01

    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

  4. 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. PMID:25840823

  5. Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background WC1 co-receptors belong to the scavenger receptor cysteine-rich superfamily and are encoded by a multi-gene family. Expression of particular WC1 genes defines functional subpopulations of WC1+ '' T cells. Our previous study identified partial sequences for 13 different WC1 genes by annota...

  6. Prostate stem cell antigen is an endogenous lynx1-like prototoxin that antagonizes alpha7 containing nicotinic receptors and prevents programmed cell death of parasympathetic neurons

    PubMed Central

    Hruska, Martin; Keefe, Julie; Wert, David; Tekinay, Ayse Begum; Hulce, Jonathan J.; Ibanez-Tallon, Ines; Nishi, Rae

    2010-01-01

    Vertebrate α–bungarotoxin-like molecules of the Ly-6 super family have been implicated as balancers of activity and survival in the adult nervous system. To determine whether a member of this family could be involved in the development of the avian ciliary ganglion, we identified 6 Gallus genes by their homology in structure to mouse lynx1 and lynx2. One of these genes, an ortholog of prostate stem cell antigen (PSCA), is barely detectable at embryonic day 8, prior to neuronal cell loss in the ciliary ganglion, but increases over 100-fold as the number of neurons begins to decline between E9 and E14. PSCA is highly expressed in chicken and mouse telencephalon and peripheral ganglia and correlates with expression of α7-containing nicotinic acetylcholine receptors (α7-nAChRs). Misexpressing PSCA prior to cell death in the ciliary ganglion blocks α7-nAChR activation by nicotine and rescues the choroid subpopulation from dying. Thus, PSCA, a molecule previously identified as a marker of prostate cancer, is a member of the Ly-6 neurotoxin-like family in the nervous system, and is likely to play a role as a modulator of α7 signaling induced cell death during development. PMID:19940180

  7. 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

  8. An immobilization antigen gene of the fish-parasitic protozoan Ichthyophthirius multifiliis strain ARS-6

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ichthyophthirius multifiliis (Ich) is a severe fish parasite that causes ‘white spot’ disease in many freshwater fish and leads to high mortality. The antigens on the parasite surface are involved in the antibody-mediated immobilization and hence designated as immobilization antigens (i-antigens). ...

  9. Design, engineering, and production of human recombinant t cell receptor ligands derived from human leukocyte antigen DR2.

    PubMed

    Chang, J W; Mechling, D E; Bächinger, H P; Burrows, G G

    2001-06-29

    Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (alpha1 and beta1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., Bächinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng. 12, 771-778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366-6371). Single-chain human recombinant T cell receptor ligands (RTLs) of approximately 200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85-99)) showed increased stability to thermal unfolding relative to the

  10. Characterization of a cancer/testis (CT) antigen gene family capable of eliciting humoral response in cancer patients

    PubMed Central

    Parmigiani, Raphael B.; Bettoni, Fabiana; Vibranovski, Maria D.; Lopes, Marilene H.; Martins, Waleska K.; Cunha, Isabela W.; Soares, Fernando A.; Simpson, Andrew J. G.; de Souza, Sandro J.; Camargo, Anamaria A.

    2006-01-01

    Cancer/testis (CT) antigens are immunogenic proteins expressed in normal gametogenic tissues and in different types of tumors. CT antigens are promising candidates for cancer immunotherapy, and the identification of novel CT antigens is a prerequisite for the development of cancer vaccines. We have identified a CT antigen, named CTSP-1, with partial similarity to the breast differentiation antigen NY-BR-1. CTSP-1 presents several splicing and polyadenylation variants and has a very restricted expression pattern among normal tissues. CTSP-1 is exclusively expressed in normal testis and is aberrantly expressed in 47.6% (10 of 21) of tumor cell lines and in 44.4% (75 of 169) of tumors from different histological types. The highest percentages of positive expression were observed in melanomas (59.0%) followed by prostate (58.0%) and lung (57.0%) tumors. CTSP-1 is part of a highly conserved gene family, and members of this family also have a restricted expression pattern and similar protein structure. Antibodies against members of this gene family were detected in 10% (14 of 141) of plasma samples from patients with a wide spectrum of tumors. The highest percentages of antibody response were observed in patients with prostate (20.8%), thyroid (20.0%), and breast (16.6%) tumors. Because of its very restricted expression pattern in normal tissues and immunogenicity in different types of tumors, CTSP-1 should be considered a promising candidate for cancer immunotherapy. PMID:17114284

  11. The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

    PubMed Central

    Munkley, Jennifer; Oltean, Sebastian; Vodák, Daniel; Wilson, Brian T.; Livermore, Karen E.; Zhou, Yan; Star, Eleanor; Floros, Vasileios I.; Johannessen, Bjarne; Knight, Bridget; McCullagh, Paul; McGrath, John; Crundwell, Malcolm; Skotheim, Rolf I.; Robson, Craig N.; Leung, Hing Y.; Harries, Lorna W.; Rajan, Prabhakar; Mills, Ian G.; Elliott, David J.

    2015-01-01

    Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, being significantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression. PMID:26452038

  12. Toll-Like Receptor Gene Expression during Trichinella spiralis Infection

    PubMed Central

    Kim, Sin; Park, Mi Kyung; Yu, Hak Sun

    2015-01-01

    In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (Treg) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and Treg cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP-/- MEF cells, and quite substantially decreased in TRIF-/- MEF cells. In contrast, IL-10 and TGF-β expression levels were not elevated in MyD88/TIRAP-/- MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and Treg cell mediated immune responses, although additional data are needed to convincingly prove this observation. PMID:26323841

  13. Toll-Like Receptor Gene Expression during Trichinella spiralis Infection.

    PubMed

    Kim, Sin; Park, Mi Kyung; Yu, Hak Sun

    2015-08-01

    In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (Treg) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and Treg cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP(-/-) MEF cells, and quite substantially decreased in TRIF(-/-) MEF cells. In contrast, IL-10 and TGF-β expression levels were not elevated in MyD88/TIRAP(-/-) MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and Treg cell mediated immune responses, although additional data are needed to convincingly prove this observation. PMID:26323841

  14. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    PubMed Central

    Teodorov, E.; Ferrari, M.F.R.; Fior-Chadi, D.R.; Camarini, R.; Felício, L.F.

    2012-01-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  15. T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.

    PubMed

    VanSeggelen, Heather; Hammill, Joanne A; Dvorkin-Gheva, Anna; Tantalo, Daniela G M; Kwiecien, Jacek M; Denisova, Galina F; Rabinovich, Brian; Wan, Yonghong; Bramson, Jonathan L

    2015-10-01

    Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution. PMID:26122933

  16. 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.

  17. Gene frequencies of human platelet antigens 1-5 in indigenous Australians in Western Australia.

    PubMed

    Bennett, J A; Palmer, L J; Musk, A W; Erber, W N

    2002-06-01

    The frequencies of human platelet antigen (HPA) systems vary between different racial groups; however, HPA frequency data for some racial groups are still incomplete. We report the distribution of HPA 1-5 systems in Australian Aborigines from a remote community in the north-west of Australia and compare our findings with HPA observed in a Western Australian blood donor population. Using a polymerase chain reaction (PCR) with sequence-specific primers, 185 indigenous Australians and 1000 Western Australian blood donors were genotyped for each of the HPA 1-5 systems. Comparison of gene frequencies of alleles from HPA-1, -2, -3 and -5 systems showed significant differences between Aboriginal people and Western Australian blood donors (P < 0.001). In particular, the frequency of HPA-3b (0.068) in the Australian Aboriginals, from this study, was one of the lowest reported, whilst the frequency of HPA-5b (0.246) was one of the highest for this allele. Gene frequencies were similar to those reported for central Australian Aborigines but with no other ethnic group. In conclusion, this study confirms significant differences in HPA distributions between indigenous Australians, Australian blood donors and other racial groups. These results indicate a higher potential risk of alloimmunization to HPA-1, -2 and -3 in Australian Aborigines receiving transfusion therapy from a Caucasian blood donor population, thereby having practical implications for transfusion and pregnancy risks in people of Aboriginal origin. PMID:12071877

  18. Killer immunoglobulin-like receptor and human leukocyte antigen-C genotypes in rheumatoid arthritis primary responders and non-responders to anti-TNF-α therapy.

    PubMed

    McGeough, Cathy M; Berrar, Daniel; Wright, Gary; Mathews, Clare; Gilmore, Paula; Cunningham, Rodat T; Bjourson, Anthony J

    2012-06-01

    The identification of patients who will respond to anti-tumor necrosis factor alpha (anti-TNF-α) therapy will improve the efficacy, safety, and economic impact of these agents. We investigated whether killer cell immunoglobulin-like receptor (KIR) genes are related to response to anti-TNF-α therapy in patients with rheumatoid arthritis (RA). Sixty-four RA patients and 100 healthy controls were genotyped for 16 KIR genes and human leukocyte antigen-C (HLA-C) group 1/2 using polymerase chain reaction sequence-specific oligonucleotide probes (PCR-SSOP). Each patient received anti-TNF-α therapy (adalimumab, etanercept, or infliximab), and clinical responses were evaluated after 3 months using the disease activity score in 28 joints (DAS28). We investigated the correlations between the carriership of KIR genes, HLA-C group 1/2 genes, and clinical data with response to therapy. Patients responding to therapy showed a significantly higher frequency of KIR2DS2/KIR2DL2 (67.7% R vs. 33.3% NR; P = 0.012). A positive clinical outcome was associated with an activating KIR-HLA genotype; KIR2DS2 (+) HLA-C group 1/2 homozygous. Inversely, non-response was associated with the relatively inhibitory KIR2DS2 (-) HLA-C group 1/2 heterozygous genotype. The KIR and HLA-C genotype of an RA patient may provide predictive information for response to anti-TNF-α therapy. PMID:21373785

  19. Molecular, Antigenic, and Functional Characteristics of Ferric Enterobactin Receptor CfrA in Campylobacter jejuni ▿

    PubMed Central

    Zeng, Ximin; Xu, Fuzhou; Lin, Jun

    2009-01-01

    The ferric enterobactin receptor CfrA not only is responsible for high-affinity iron acquisition in Campylobacter jejuni but also is essential for C. jejuni colonization in animal intestines. In this study, we determined the feasibility of targeting the iron-regulated outer membrane protein CfrA for immune protection against Campylobacter colonization. Alignment of complete CfrA sequences from 15 Campylobacter isolates showed that the levels of amino acid identity for CfrA range from 89% to 98%. Immunoblotting analysis using CfrA-specific antibodies demonstrated that CfrA was dramatically induced under iron-restricted conditions and was widespread and produced in 32 Campylobacter primary strains from various sources and from geographically diverse areas. The immunoblotting survey results were highly correlated with the results of an enterobactin growth promotion assay and a PCR analysis using cfrA-specific primers. Inactivation of the cfrA gene also impaired norepinephrine-mediated growth promotion, suggesting that CfrA is required for C. jejuni to sense intestinal stress hormones during colonization. Complementation of the cfrA mutant with a wild-type cfrA allele in trans fully restored the production and function of CfrA. A growth assay using purified anti-CfrA immunoglobulin G demonstrated that specific CfrA antibodies could block the function of CfrA, which diminished ferric enterobactin-mediated growth promotion under iron-restricted conditions. The inhibitory effect of CfrA antibodies was dose dependent. Immunoblotting analysis also indicated that CfrA was expressed and immunogenic in chickens experimentally infected with C. jejuni. Amino acid substitution mutagenesis demonstrated that R327, a basic amino acid that is highly conserved in CfrA, plays a critical role in ferric enterobactin acquisition in C. jejuni. Together, these findings strongly suggest that CfrA is a promising vaccine candidate for preventing and controlling Campylobacter infection in

  20. A selective defect in IgM antigen receptor synthesis and transport causes loss of cell surface IgM expression on tolerant B lymphocytes.

    PubMed Central

    Bell, S E; Goodnow, C C

    1994-01-01

    To explore the biochemical basis for maintaining immunological tolerance by functional inactivation of self-reactive B lymphocytes, transgenic mice carrying rearranged anti-lysozyme immunoglobulin transgenes and a lysozyme transgene were used as a source of large numbers of tolerant self-reactive B cells. Antigen receptors of the IgD isotype were expressed at normal levels on tolerant B cells, contained the heterodimeric MB1/B29 signalling component of the receptor complex and were structurally indistinguishable from IgD on nontolerant B cells. In contrast, cell surface expression of IgM receptor complexes on tolerant B cells was greatly reduced, despite normal expression of mRNA encoding the receptor components. Three-fold fewer immunoreactive mu heavy chains were detectable after a short period of biosynthetic labelling and the immunoreactive mu chains produced were paired with kappa light chains and assembled normally into intact receptor complexes containing the MB1/B29 heterodimer. Nascent IgM receptor complexes nevertheless failed to be processed into an endoglycosidase H-resistant form in the tolerant B cells and thus appeared to be selectively blocked in their transport from the endoplasmic reticulum to the medial Golgi. These findings demonstrate that intracellular trafficking of antigen receptor complexes is regulated by exposure to receptor stimuli at the cell surface causing a long-lasting decrease in surface receptor expression on tolerant B cells. Images PMID:8112296

  1. High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene.

    PubMed

    Hillig, R C; Coulie, P G; Stroobant, V; Saenger, W; Ziegler, A; Hülsmeyer, M

    2001-07-27

    The heterotrimeric complex of the human major histocompatibity complex (MHC) molecule HLA-A*0201, beta2-microglobulin and the decameric peptide GVYDGREHTV derived from the melanoma antigen (MAGE-A4 protein has been determined by X-ray crystallography at 1.4 A resolution. MAGE-A4 belongs to a family of genes that are specifically expressed in a variety of tumours. MAGE-A4-derived peptides are presented by MHC molecules at the cell surface to cytotoxic T-lymphocytes. As the HLA-A*0201:MAGE-A4 complex occurs only on tumour cells, it is considered to be an appropriate target for immunotherapy. The structure presented here reveals potential epitopes specific to the complex and indicates which peptide residues could be recognised by T-cell receptors. In addition, as the structure could be refined anisotropically, it was possible to describe the movements of the bound peptide in more detail. PMID:11502003

  2. Cannabinoid Receptor 1 Gene Association With Nicotine Dependence

    PubMed Central

    Chen, Xiangning; Williamson, Vernell S.; An, Seon-Sook; Hettema, John M.; Aggen, Steven H.; Neale, Michael C.; Kendler, Kenneth S.

    2009-01-01

    Context The endogenous cannabinoid system has been implicated in drug addiction in animal models. The cannabinoid receptor 1 (CNR1) gene is 1 of the 2 receptors expressed in the brain. It has been reported to be associated with alcoholism and multiple drug abuse and dependence. Objective To test the hypothesis that the CNR1 gene is associated with nicotine dependence. Design Genotype-phenotype association study. Ten single-nucleotide polymorphisms were genotyped in the CNR1 gene in 2 independent samples. For the first sample (n=688), a 3-group case-control design was used to test allele association with smoking initiation and nicotine dependence. For the second sample (n = 961), association was assessed with scores from the Fagerström Test for Nicotine Dependence (FTND). Settings Population samples selected from the Mid-Atlantic Twin Registry. Participants White patients aged 18 to 65 years who met the criteria of inclusion. Main Outcome Measures Fagerström Tolerance Questionnaire and FTND scores. Results Significant single-marker and haplotype associations were found in both samples, and the associations were female specific. Haplotype 1-1-2 of markers rs2023239-rs12720071-rs806368 was associated with nicotine dependence and FTND score in the 2 samples (P<.001 and P = .009, respectively). Conclusion Variants and haplotypes in the CNR1 gene may alter the risk for nicotine dependence, and the associations are likely sex specific. PMID:18606954

  3. Gene cloning, expression, and localization of antigen 5 in the life cycle of Echinococcus granulosus.

    PubMed

    Li, Yuzhe; Xu, Hongxu; Chen, Jiajia; Gan, Wenjia; Wu, Weihua; Wu, Weiping; Hu, Xuchu

    2012-06-01

    Antigen 5 (Ag5) has been identified as a dominant component of cyst fluid of Echinococcus granulosus and is considered as a member of serine proteases family, which in other helminth, plays an important role in the egg hatch and larva invasion. However, whether Ag5 is expressed and secreted in all life stages is unknown. In this study, according to the sequence in GenBank, we cloned and sequenced the open reading frame (ORF) of Ag5 gene from the protoscolices of E. granulosus isolated from the sheep in Qinhai Province of China, and found several substitutions and a base insert and deletion in a short region near the stop code, leading to a frameshift mutation which is conserved with the homologue of other cestode. The ORF is 1,455 bp in length, encoding 484 amino acids with a secretory signal peptide. Bioinformatics analysis predicted several phosphorylation and myristoylation sites and a N-glycosylation site and a species-specific linear B epitope in the protein. The ORF was cloned into the plasmid pET28a(+) vector and expressed in Escherichia coli . The recombinant protein was purified by affinity chromatography. Anti-rEgAg5 antiserum was prepared in rats and used to analyze the localization of Ag5 in protoscolex and adult worm by immunofluorescence technique. Results demonstrated that the Ag5 is strongly expressed in the tegument of protoscolex and the embryonic membrane of egg and surface of oncosphere; meanwhile, it is also weakly expressed in tegument of the adult. This study showed that Ag5 is expressed in all stages of life cycle, secreted from the surface of the worm and may be anchored in membrane by its myristoylation sites; these characteristics make it a candidate antigen for diagnosis and vaccine for both intermediate and definitive hosts. PMID:22200957

  4. Expression of the CMRF-35 antigen, a new member of the immunoglobulin gene superfamily, is differentially regulated on leucocytes.

    PubMed Central

    Daish, A; Starling, G C; McKenzie, J L; Nimmo, J C; Jackson, D G; Hart, D N

    1993-01-01

    A new monoclonal antibody, CMRF-35, has been generated that recognized a 224 amino acid cell surface protein which is a novel member of the immunoglobulin gene superfamily. The antibody, raised against large granular lymphocytes (LGL), stains LGL, monocytes, macrophages and granulocytes but not platelets or erythrocytes. In addition, a subset of peripheral blood T lymphocytes (26.6 +/- 13.4% CD5+ cells) and B lymphocytes (13.7 +/- 6.8% CD20+ cells) stained with CMRF-35 but tonsil T and B cells were essentially negative. Expression of the CMRF-35 antigen (Ag) on different leucocyte populations was markedly influenced by stimulation of the cells with mitogens and cytokines. Activation of peripheral blood T cells with phytohaemagglutinin (PHA), or phorbol myristate acetate (PMA) and calcium ionophore (CaI) led to a decrease in the proportion of CMRF-35+ T lymphocytes. In contrast, PHA activation of tonsil T lymphocytes resulted in an increase in CMRF-35 Ag expression (47.1 +/- 1.5% CD5 cells at 6 days). An increase in CMRF-35 Ag was also seen on phorbol ester and CaI-activated tonsil B cells. No change in CMRF-35 expression on natural killer (NK) cells occurred following activation with interleukin-2 (IL-2) but the CMRF-35 Ag was down-regulated following Fc receptor stimulation. A moderate increase in CMRF-35 expression occurred during monocyte-macrophage differentiation and the expression of the Ag on monocytes was differentially regulated by interferon-gamma (IFN-gamma). This regulation of the CMRF-35 Ag on the leucocyte surface suggests that the molecule has an important function common to diverse leucocyte types. PMID:8509141

  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. PMID:16029432

  6. T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene.

    PubMed Central

    Held, W A; Mullins, J J; Kuhn, N J; Gallagher, J F; Gu, G D; Gross, K W

    1989-01-01

    A hybrid mouse major urinary protein (MUP)/SV40 T antigen gene was microinjected into fertilized mouse embryos and the resulting transgenic mice analyzed for the regulated expression of the transgene. Available evidence indicates that the MUP gene used for the hybrid gene construct is expressed in both male and female liver and possibly mammary gland. Three different transgenic lines exhibited a consistent pattern of tissue specific expression of the transgene. As a consequence of transgene expression and T antigen synthesis in the liver, both male and female transgenic animals developed liver hyperplasia and tumors. Transgene expression and liver hyperplasia commenced at approximately 2-4 weeks of age, the same time that MUP gene expression is first detected in the liver. The expression of the transgene resulted in an immediate strong suppression of liver MUP mRNA levels but had relatively little effect on other liver specific mRNAs. From 4 to 8 weeks, the liver increased several fold in size, relative to non-transgenic littermates. Definitive tumor nodules were not apparent until 8-10 weeks. The transgene was also consistently found to be expressed in the skin sebaceous glands and the preputial gland, a modified sebaceous gland. The expression of the transgene in the skin sebaceous glands is consistent with the presence of MUP mRNA in the skin and a putative role for MUPs in the transport and excretion of small molecules. Occasional expression of the transgene in other tissues (kidney and mammary connective tissues) was also noted.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2714250

  7. 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.

  8. Localization and physical mapping of the prostate-specific membrane antigen (PSM) gene to human chromosome 11

    SciTech Connect

    Rinker-Schaeffer, C.W.; Hawkins, A.L.; Griffin, C.A.; Isaacs, J.T.

    1995-11-01

    The prostate-specific membrane antigen (PSM) was identified by the monoclonal antibody 7E11-C5.3, which was raised against the human prostatic carcinoma cell line LNCaP. The PSM antigen is expressed by normal, neoplastic, and metastatic prostatic tissues. The 2.65-kb cDNA encoding the 100-kDa PSM glycoprotein was cloned from LNCaP cells. Studies have shown that the expression of PSM is tissue-specific. In the present study monochromosomal somatic cell hybrids were used to localize the PSM gene to human chromosome 11. Using this information, initial mapping studies identified two potential PSM gene loci at 11p11.1-p13 and 11q14. Further high-stringency analysis using cosmid probes identified the 11q14 region as the location of the PSM gene. 10 refs., 2 figs.

  9. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.

    PubMed Central

    Yang, Y; Li, Q; Ertl, H C; Wilson, J M

    1995-01-01

    Recombinant adenoviruses are an attractive vehicle for gene therapy to the lung in the treatment of cystic fibrosis (CF). First-generation viruses deleted of E1a and E1b transduce genes into airway epithelial cells in vivo; however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. In this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive transfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. Our studies indicate that major histocompatibility complex class I-restricted CD8+ cytotoxic T lymphocytes are activated in response to newly synthesized antigens, leading to destruction of virus infected cells and loss of transgene expression. Major histocompatibility complex class II-associated presentation of exogenous viral antigens activates CD4+ T-helper (TH) cells of the TH1 subset and, to a lesser extent, of the TH2 subset. CD4+ cell-mediated responses are insufficient in the absence of cytotoxic T cells to completely eliminate transgene containing cells; however, they contribute to the formation of neutralizing antibodies in the airway which block subsequent adenovirus-mediated gene transfer. Definition of immunological barriers to gene therapy of cystic fibrosis should facilitate the design of rational strategies to overcome them. PMID:7884845

  10. 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

  11. In silico prediction of tumor antigens derived from functional missense mutations of the cancer gene census

    PubMed Central

    Khalili, Jahan S.; Hanson, Russell W.; Szallasi, Zoltan

    2012-01-01

    Antigen-specific immune responses against peptides derived from missense gene mutations have been identified in multiple cancers. The application of personalized peptide vaccines based on the tumor mutation repertoire of each cancer patient is a near-term clinical reality. These peptides can be identified for pre-validation by leveraging the results of massive gene sequencing efforts in cancer. In this study, we utilized NetMHC 3.2 to predict nanomolar peptide binding affinity to 57 human HLA-A and B alleles. All peptides were derived from 5,685 missense mutations in 312 genes annotated as functionally relevant in the Cancer Genome Project. Of the 26,672,189 potential 8–11 mer peptide-HLA pairs evaluated, 0.4% (127,800) display binding affinities < 50 nM, predicting high affinity interactions. These peptides can be segregated into two groups based on the binding affinity to HLA proteins relative to germline-encoded sequences: peptides for which both the mutant and wild-type forms are high affinity binders, and peptides for which only the mutant form is a high affinity binder. Current evidence directs the attention to mutations that increase HLA binding affinity, as compared with cognate wild-type peptide sequences, as these potentially are more relevant for vaccine development from a clinical perspective. Our analysis generated a database including all predicted HLA binding peptides and the corresponding change in binding affinity as a result of point mutations. Our study constitutes a broad foundation for the development of personalized peptide vaccines that hone-in on functionally relevant targets in multiple cancers in individuals with diverse HLA haplotypes. PMID:23243591

  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. PMID:27424137

  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. 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

  15. 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. PMID:27124285

  16. The effect of short-wavelength ultraviolet light on antigens, lectin receptors and the ultrastructure of Crithidia fasciculata.

    PubMed

    Santos, M A; Barros, A M; Andrade, P P; Padovan, I P

    1989-01-01

    Crithidia fasciculata is an important trypanosomatid parasite commonly affecting insects and is used extensively as a model for the study of the biochemistry, ultrastructure and organization of the kDNA network of trypanosomatids. The present study describes the evolution of UV-induced morphological changes detectable by transmission electron microscopy in Crithidia fasciculata. Although only rare and minor changes in kinetoplast DNA were demonstrable 7 h after UV irradiation, alterations of this organelle were present in almost all flagellates observed 24 h and 48 h after irradiation. Other cell structures were apparently undamaged. Ultrastructural changes in kDNA did not correspond to changes in antigenicity of protein bands in western blotting against serum from Chagas' disease patients or in the presence of 3 different lectin receptors on the surface of the parasite. PMID:2553178

  17. CD19-Targeted chimeric antigen receptor-modified T-cell immunotherapy for B-cell malignancies.

    PubMed

    Turtle, C J; Riddell, S R; Maloney, D G

    2016-09-01

    Chimeric antigen receptors (CARs) comprise a tumor-targeting moiety, often in the form of a single chain variable fragment derived from a monoclonal antibody, fused to one or more intracellular T-cell signaling sequences. Lymphodepletion chemotherapy followed by infusion of T cells that are genetically modified to express a CD19-specific CAR is a promising therapy for patients with refractory CD19(+) B-cell malignancies, producing rates of complete remission that are remarkably high in acute lymphoblastic leukemia and encouraging in non-Hodgkin lymphoma and chronic lymphocytic leukemia. Responses are often durable, although additional studies are needed to define the role of CAR-T cell immunotherapy in the context of other treatments. CAR-modified T-cell immunotherapy can be complicated by cytokine release syndrome and neurologic toxicity, which in most cases are manageable and reversible. Here we review recent clinical trial data and discuss issues for the field. PMID:27170467

  18. Novel meningococcal 4CMenB vaccine antigens - prevalence and polymorphisms of the encoding genes in Neisseria gonorrhoeae.

    PubMed

    Hadad, Ronza; Jacobsson, Susanne; Pizza, Mariagrazia; Rappuoli, Rino; Fredlund, Hans; Olcén, Per; Unemo, Magnus

    2012-09-01

    The first cross-protective Neisseria meningitidis vaccine (focus on serogroup B), the protein-based 4 component meningococcus serogroup B (4CMenB), includes the New Zealand outer membrane vesicle and three main genome-derived neisserial antigens (GNAs). These GNAs are fHbp (fused to GNA2091), NHBA (fused to GNA1030) and NadA. In this study, the prevalence and polymorphisms of the nucleotide and amino acid sequences of the 4CMenB antigens in a temporally and geographically diverse collection of N. gonorrhoeae isolates (n = 111) were investigated. All the examined GNA genes, except the nadA gene, were present in all gonococcal isolates. However, 25 isolates contained premature stop codons in the fHbp gene and/or the nhba gene, resulting in truncated proteins. Compared with the 4CMenB antigen sequences in reference strain MC58, the gonococcal strains displayed 67.0-95.4% and 60.9-94.9% identity in nucleotide sequence and amino acid sequence, respectively, in the equivalent GNA antigens. The absence of NadA, lack of universal expression of fHbp and NHBA and the uncertainty regarding the surface exposure of fHbp as well as the function of NHBA in N. gonorrhoeae will likely limit the use of the identical 4CMenB antigens in a gonococcal vaccine. However, possible cross-immunity of 4CMenB with gonococci and expression and function of the equivalent gonococcal GNAs, as well as of more appropriate GNAs for a gonococcal vaccine, need to be further examined. PMID:22882265

  19. Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes

    PubMed Central

    Preston, Gavin C; Sinclair, Linda V; Kaskar, Aneesa; Hukelmann, Jens L; Navarro, Maria N; Ferrero, Isabel; MacDonald, H Robson; Cowling, Victoria H; Cantrell, Doreen A

    2015-01-01

    Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses. PMID:26136212

  20. 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

  1. 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.

  2. Armed Oncolytic Virus Enhances Immune Functions of Chimeric Antigen Receptor-Modified T Cells in Solid Tumors

    PubMed Central

    Nishio, Nobuhiro; Diaconu, Iulia; Liu, Hao; Cerullo, Vincenzo; Caruana, Ignazio; Hoyos, Valentina; Bouchier-Hayes, Lisa; Savoldo, Barbara; Dotti, Gianpietro

    2014-01-01

    The clinical efficacy of chimeric antigen receptor (CAR)-redirected T cells remains marginal in solid tumors compared to leukemias. Failures have been attributed to insufficient T-cell migration and to the highly immunosuppressive milieu of solid tumors. To overcome these obstacles, we have combined CAR-T cells with an oncolytic virus (OV) armed with the chemokine RANTES and the cytokine IL-15, reasoning that the modified OV will have both a direct lytic effect on infected malignant cells and facilitate migration and survival of CAR-T cells. Using neuroblastoma (NB) as a tumor model we found that the adenovirus Ad5Δ24 exerted a potent, dose-dependent, cytotoxic effect on tumor cells, while CAR-T cells specific for the tumor antigen GD2 (GD2.CAR-T cells) were not damaged. When used in combination, Ad5Δ24 directly accelerated the caspase pathways in tumor cells exposed to CAR-T cells, while the intratumoral release of both RANTES and IL-15 attracted CAR-T cells and promoted their local survival, respectively, increasing the overall survival of tumor bearing mice. These preclinical data support the use of this innovative biological platform of immunotherapy for solid tumors. PMID:25060519

  3. Armed oncolytic virus enhances immune functions of chimeric antigen receptor-modified T cells in solid tumors.

    PubMed

    Nishio, Nobuhiro; Diaconu, Iulia; Liu, Hao; Cerullo, Vincenzo; Caruana, Ignazio; Hoyos, Valentina; Bouchier-Hayes, Lisa; Savoldo, Barbara; Dotti, Gianpietro

    2014-09-15

    The clinical efficacy of chimeric antigen receptor (CAR)-redirected T cells remains marginal in solid tumors compared with leukemias. Failures have been attributed to insufficient T-cell migration and to the highly immunosuppressive milieu of solid tumors. To overcome these obstacles, we have combined CAR-T cells with an oncolytic virus armed with the chemokine RANTES and the cytokine IL15, reasoning that the modified oncolytic virus will both have a direct lytic effect on infected malignant cells and facilitate migration and survival of CAR-T cells. Using neuroblastoma as a tumor model, we found that the adenovirus Ad5Δ24 exerted a potent, dose-dependent, cytotoxic effect on tumor cells, whereas CAR-T cells specific for the tumor antigen GD2 (GD2.CAR-T cells) were not damaged. When used in combination, Ad5Δ24 directly accelerated the caspase pathways in tumor cells exposed to CAR-T cells, whereas the intratumoral release of both RANTES and IL15 attracted CAR-T cells and promoted their local survival, respectively, increasing the overall survival of tumor-bearing mice. These preclinical data support the use of this innovative biologic platform of immunotherapy for solid tumors. Cancer Res; 74(18); 5195-205. ©2014 AACR. PMID:25060519

  4. 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. PMID:26056165

  5. Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells.

    PubMed

    Pinz, K; Liu, H; Golightly, M; Jares, A; Lan, F; Zieve, G W; Hagag, N; Schuster, M; Firor, A E; Jiang, X; Ma, Y

    2016-03-01

    Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs. PMID:26526988

  6. From humble beginnings to success in the clinic: Chimeric antigen receptor-modified T-cells and implications for immunotherapy.

    PubMed

    Firor, Amelia E; Jares, Alexander; Ma, Yupo

    2015-08-01

    In the past 50 years, disease burden has steadily shifted from infectious disease to cancer. Standard chemotherapy has long been the mainstay of cancer medical management, and despite vast efforts towards more targeted and personalized drug therapy, many cancers remain refractory to treatment, with high rates of relapse and poor prognosis. Recent dramatic immunotherapy clinical trials have demonstrated that engineering T-cells with chimeric antigen receptors (CARs) to target CD19 can lead to complete remission in relapsed or refractory B-cell malignancies, generating a great deal of enthusiasm in the field. Here we provide a comprehensive overview of the history of adoptive T-cell therapy, including CARs, in solid tumors as well as hematologic malignancies. CAR therapy has the potential to fundamentally transform cancer treatment with specific and even personalized targeting of tissue- and tumor-specific antigens. However, before CARs become standard first-line treatment modalities, critical issues regarding efficacy, combinatorial regimens, and mechanisms of treatment failure and toxicity will need to be addressed. PMID:25956686

  7. Expression of immunoglobulin receptors with distinctive features indicating antigen selection by marginal zone B cells from human spleen.

    PubMed

    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)IgM(high)sIgD(low)CD23(low/-)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

  8. A High-avidity WT1-reactive T-Cell Receptor Mediates Recognition of Peptide and Processed Antigen but not Naturally Occurring WT1-positive Tumor Cells.

    PubMed

    Jaigirdar, Adnan; Rosenberg, Steven A; Parkhurst, Maria

    2016-04-01

    Wilms tumor gene 1 (WT1) is an attractive target antigen for cancer immunotherapy because it is overexpressed in many hematologic malignancies and solid tumors but has limited, low-level expression in normal adult tissues. Multiple HLA class I and class II restricted epitopes have been identified in WT1, and multiple investigators are pursuing the treatment of cancer patients with WT1-based vaccines and adoptively transferred WT1-reactive T cells. Here we isolated an HLA-A*0201-restricted WT1-reactive T-cell receptor (TCR) by stimulating peripheral blood lymphocytes of healthy donors with the peptide WT1:126-134 in vitro. This TCR mediated peptide recognition down to a concentration of ∼0.1 ng/mL when pulsed onto T2 cells as well as recognition of HLA-A*0201 target cells transfected with full-length WT1 cDNA. However, it did not mediate consistent recognition of many HLA-A*0201 tumor cell lines or freshly isolated leukemia cells that endogeneously expressed WT1. We dissected this pattern of recognition further and observed that WT1:126-134 was more efficiently processed by immunoproteasomes compared with standard proteasomes. However, pretreatment of WT1 tumor cell lines with interferon gamma did not appreciably enhance recognition by our TCR. In addition, we highly overexpressed WT1 in several leukemia cell lines by electroporation with full-length WT1 cDNA. Some of these lines were still not recognized by our TCR suggesting possible antigen processing defects in some leukemias. These results suggest WT1:126-134 may not be a suitable target for T-cell based tumor immunotherapies. PMID:26938944

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

    PubMed

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

    2002-11-01

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

  10. Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

    PubMed Central

    Favoreel, Herman W.; Hoebeke, Inge; Delrue, Iris; Dewerchin, Hannah; Verdonck, Frank; Verhasselt, Bruno; Cox, Eric; Nauwynck, Hans J.

    2011-01-01

    Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages. PMID:21359217

  11. Cloning and sequence analysis of the Schistosoma mansoni membrane glycoprotein antigen gene GP22.

    PubMed

    el-Sherbeini, M; Ramadan, N; Bostian, K A; Knopf, P M

    1991-11-01

    A family of Schistosoma mansoni proteins (18-22 kDa, pI 5.3-5.8) are biosynthesized in juvenile worms and immunoprecipitated by antibodies uniquely present in protective Fischer rat antiserum. A cDNA clone, lambda gt11-40, expressing epitopes common to this protein family was used to obtain a genomic DNA clone, by hybridization with a lambda gt11-40 oligonucleotide probe. In the 1.37 kb of genomic DNA sequenced, an open reading frame of 182 amino acids was identified on the strand corresponding to lambda gt11-40 coding sequences, and those of identical independently isolated cDNA clones defining a 25-kDa surface membrane glycoprotein. The new S. mansoni gene is termed GP22. There are two candidate promoters, confirmed by primer extension studies with worm RNA. Promoter 1 (P1) is preceded by a G + C-rich region and potential CAAT sequences, and is to the 5'-side of P2. Transcription from P1 is initiated at 2 different sites, apparently producing mRNAs with different translation start sites (ATG). Decoding these mRNAs yields protein products of 182 (P1), 175 (P1), 140 (P2) and 136 (P2) amino acids. The polypeptides share the following features: a hydrophobic segment near the carboxy terminus sufficient to span a lipid bilayer, with a consensus sequence for thio-esterification by a fatty acid; an external domain containing 2 potential N-linked glycosylation sites; and a candidate leucine-zipper motif, suggesting the protein may exist as a dimer on the worm surface. While sharing these common features in their carboxy terminal regions, the three proteins differ in the length and properties of their amino termini. The 140-amino acid protein has a short hydrophobic amino terminus, while the 175- and 182-amino acid proteins have more extensive hydrophobic sequences, each preceded by a hydrophilic amino terminal sequence. The heterogeneity observed in 2-dimensional gels of the antigen may be explained in part by the size and charge differences among the proteins deduced

  12. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    SciTech Connect

    Levy, J.R.; Olefsky, J.M.

    1988-05-05

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4/sup 0/C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37/sup 0/C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

  13. Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM.

    PubMed Central

    Wienands, J; Hombach, J; Radbruch, A; Riesterer, C; Reth, M

    1990-01-01

    Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain. Images Fig. 3. Fig. 5. Fig. 6. Fig. 7. PMID:2303036

  14. Cloning and sequencing of the gene for alpha antigen from Mycobacterium avium and mapping of B-cell epitopes.

    PubMed Central

    Ohara, N; Matsuo, K; Yamaguchi, R; Yamazaki, A; Tasaka, H; Yamada, T

    1993-01-01

    The complete nucleotide sequence of alpha antigen secreted from Mycobacterium avium (A-alpha) was determined. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-alpha. Comparisons between A-alpha and alpha antigens of Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasii showed highly homologous regions which suggested a conserved functional domain and two less-homologous regions. Serological analysis of recombinant A-alpha, expressed by a series of deletion constructs, indicated the possibility that A-alpha carries at least six B-cell epitopes. The three antigenic determinants were common to Mycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there are three species-specific epitopes. Images PMID:7681039

  15. Chimeric Antigen Receptors With Mutated IgG4 Fc Spacer Avoid Fc Receptor Binding and Improve T Cell Persistence and Antitumor Efficacy

    PubMed Central

    Jonnalagadda, Mahesh; Mardiros, Armen; Urak, Ryan; Wang, Xiuli; Hoffman, Lauren J; Bernanke, Alyssa; Chang, Wen-Chung; Bretzlaff, William; Starr, Renate; Priceman, Saul; Ostberg, Julie R; Forman, Stephen J; Brown, Christine E

    2015-01-01

    The success of adoptive therapy using chimeric antigen receptor (CAR)–expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here, we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (FcγRs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble FcγRs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid FcγR binding would improve CAR+ T cell engraftment and antitumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)) or incorporated a CH2 deletion (CD19Rch2Δ). These mutations reduced binding to soluble FcγRs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2Δ T cells exhibited improved persistence and more potent CD19-specific antilymphoma efficacy in NSG mice. Together, these studies suggest that optimal CAR function may require the elimination of cellular FcγR interactions to improve T cell persistence and antitumor responses. PMID:25366031

  16. 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.

  17. 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

  18. Epigenetic regulation of the formyl peptide receptor 2 gene.

    PubMed

    Simiele, Felice; Recchiuti, Antonio; Patruno, Sara; Plebani, Roberto; Pierdomenico, Anna Maria; Codagnone, Marilina; Romano, Mario

    2016-10-01

    Lipoxin (LX) A4, a main stop signal of inflammation, exerts potent bioactions by activating a specific G protein-coupled receptor, termed formyl peptide receptor 2 and recently renamed ALX/FPR2. Knowledge of the regulatory mechanisms that drive ALX/FPR2 gene expression is key for the development of innovative anti-inflammatory pharmacology. Here, we examined chromatin patterns of the ALX/FPR2 gene. We report that in MDA-MB231 breast cancer cells, the ALX/FPR2 gene undergoes epigenetic silencing characterized by low acetylation at lysine 27 and trimethylation at lysine 4, associated with high methylation at lysine 27 of histone 3. This pattern, which is consistent with transcriptionally inaccessible chromatin leading to low ALX/FPR2 mRNA and protein expression, is reversed in polymorphonuclear leukocytes that express high ALX/FPR2 levels. Activation of p300 histone acetyltransferase and inhibition of DNA methyltransferase restored chromatin accessibility and significantly increased ALX/FPR2 mRNA transcription and protein levels in MDA-MB231 cells, as well as in pulmonary artery endothelial cells. In both cells types, changes in the histone acetylation/methylation status enhanced ALX/FPR2 signaling in response to LXA4. Collectively, these results uncover unappreciated epigenetic regulation of ALX/FPR2 expression that can be exploited for innovative approaches to inflammatory disorders. PMID:27424221

  19. 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.

  20. 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

  1. 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

    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

  2. Molecular Characterization of the Escherichia coli Serogroup O117, O126, and O146 O Antigen Gene Clusters and Development of PCR Assays Targeting Serogroup O117-, O126-, and O146-Specific DNA Sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The O antigen gene clusters of Escherichia coli serogroups O117, O126, and O146 were sequenced, and eleven, ten, and eleven open reading frames (ORFs) were identified, respectively. Genes required for O antigen sugar biosynthesis, sugar transfer and sugar processing in these O-antigen gene clusters...

  3. Study of Toll-like receptor gene loci in sarcoidosis

    PubMed Central

    Schürmann, M; Kwiatkowski, R; Albrecht, M; Fischer, A; Hampe, J; Müller-Quernheim, J; Schwinger, E; Schreiber, S

    2008-01-01

    Sarcoidosis is a multi-factorial systemic disease of granulomatous inflammation. Current concepts of the aetiology include interactions of unknown environmental triggers with an inherited susceptibility. Toll-like receptors (TLRs) are main components of innate immunity and therefore TLR genes are candidate susceptibility genes in sarcoidosis. Ten members of the human TLR gene family have been identified and mapped to seven chromosomal segments. The aim of this study was to investigate all known TLR gene loci for genetic linkage with sarcoidosis and to follow positive signals with different methods. We analysed linkage of TLR gene loci to sarcoidosis by use of closely flanking microsatellite markers in 83 families with 180 affected siblings. We found significant linkage between sarcoidosis and markers of the TLR4 gene locus on chromosome 9q (non-parametric linkage score 2·63, P = 0·0043). No linkage was found for the remaining TLR gene loci. We subsequently genotyped 1203 sarcoidosis patients from 997 families, 1084 relatives and 537 control subjects for four single nucleotide polymorphisms of TLR4, including Asp299Gly and Thr399Ile. This genotype data set was studied by case–control comparisons and transmission disequilibrium tests, but showed no significant results. In summary, TLR4 − w ith significant genetic linkage results − appears to be the most promising member of the TLR gene family for further investigation in sarcoidosis. However, our results do not confirm the TLR4 polymorphisms Asp299Gly and Thr399Ile as susceptibility markers. Our results rather point to another as yet unidentified variant within or close to TLR4 that might confer susceptibility to sarcoidosis. PMID:18422738

  4. Diverse growth hormone receptor gene mutations in Laron syndrome

    SciTech Connect

    Berg, M.A.; Francke, U. ); Gracia, R.; Rosenbloom, A.; Toledo, S.P.A. ); Chernausek, S. ); Guevara-Aguirre, J. ); Hopp, M. ); Rosenbloom, A.; Argente, J. ); Toledo, S.P.A. )

    1993-05-01

    To better understand the molecular genetic basis and genetic epidemiology of Laron syndrome (growth-hormone insensitivity syndrome), the authors analysed the growth-hormone receptor (GHR) genes of seven unrelated affected individuals from the United States, South America, Europe, and Africa. They amplified all nine GHR gene exons and splice junctions from these individuals by PCR and screened the products for mutations by using denaturing gradient gel electrophoresis (DGGE). They identified a single GHR gene fragment with abnormal DGGE results for each affected individual, sequenced this fragment, and, in each case, identified a mutation likely to cause Laron syndrome, including two nonsense mutations (R43X and R217X), two splice-junction mutations, (189-1 G to T and 71+1 G to A), and two frameshift mutations (46 del TT and 230 del TA or AT). Only one of these mutations, R43X, has been previously reported. Using haplotype analysis, they determined that this mutation, which involves a CpG dinucleotide hot spot, likely arose as a separate event in this case, relative to the two prior reports of R43X. Aside from R43X, the mutations identified are unique to patients from particular geographic regions. Ten GHR gene mutations have now been described in this disorder. The authors conclude that Laron syndrome is caused by diverse GHR gene mutations, including deletions, RNA processing defects, translational stop codons, and missense codons. All the identified mutations involve the extracellular domain of the receptor, and most are unique to particular families or geographic areas. 35 refs., 3 figs., 1 tab.

  5. Determination of serotyping antigens, clonal analysis and genetic characterization of the 4CMenB vaccine antigen genes in invasive Neisseria meningitidis from Western Canada, 2009 to 2013.

    PubMed

    Law, Dennis K S; Zhou, Jianwei; Deng, Saul; Hoang, Linda; Tyrrell, Gregory; Horsman, Greg; Wylie, John; Tsang, Raymond S W

    2014-11-01

    This study examined invasive Neisseria meningitidis recovered from invasive meningococcal disease (IMD) cases in Western Canada between 2009 and 2013. A total of 161 isolates from individual IMD cases were analysed for serogroup, serotype, serosubtype, PorA genotype, multi-locus sequence type and nucleotide sequence of their 4CMenB vaccine antigen genes. Sixty-nine isolates were serogroup B (MenB), 47 were serogroup Y (MenY), 22 were serogroup C (MenC), 19 were serogroup W (MenW), three were serogroup E and one was non-encapsulated. MenC, MenY and MenW were mainly clonal, represented primarily by clonal complex (cc) 11, cc23 or cc167, and cc22, respectively. In contrast, MenB were composed of eight different ccs together with 11 isolates not assigned to any known cc. Antigenic analysis and PorA genotyping confirmed the heterogeneity of MenB isolates, while such results supported the clonal nature of most MenC, MenY and MenW isolates. Thirty-four (21.1%) isolates had at least one gene that encoded one matching vaccine protein component of the 4CMenB vaccine (i.e. PorA P1.4; fHbp variant 1.1; NHBA peptide 2; and NadA-1, -2, or -3). An additional 18 isolates had genes that encoded variant 1 or subfamily B factor H binding proteins of this same vaccine. PMID:25165123

  6. Peripherin: an islet antigen that is cross-reactive with nonobese diabetic mouse class II gene products.

    PubMed Central

    Boitard, C; Villa, M C; Becourt, C; Gia, H P; Huc, C; Sempe, P; Portier, M M; Bach, J F

    1992-01-01

    The nonobese diabetic (NOD) mouse, in which major histocompatibility complex genes may be involved in the susceptibility to diabetes, has been developed as a model of autoimmune diabetes. The NOD mouse expresses I-A-encoded class II major histocompatibility complex antigens, which differ from those of other mouse haplotypes by the presence of a serine at position 57 of the A beta chain. Identifying islet autoantigens may help elucidate the role of class II antigens in the activation of autoreactive T cells and, thus, in the development of diabetes. We have detected autoantibodies directed against a 58-kDa islet cell antigen in NOD mice but not in other strains, including lupus-prone mice. Apart from insulin-secreting cells, the 58-kDa antigen was only found to be expressed by neuroblastoma cells and was identified as peripherin, an intermediate filament protein previously characterized in well-defined neuronal populations. This autoantigen cross-reacted with I-Anod class II antigens, suggesting that it may contribute to defective self-tolerance of islet beta cells in the NOD mouse. Images PMID:1729686

  7. Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells

    PubMed Central

    2014-01-01

    Background The development of immunotherapy has led to significant progress in the treatment of metastatic cancer, including the development of genetic engineering technologies that redirect lymphocytes to recognize and target a wide variety of tumor antigens. Chimeric antigen receptors (CARs) are hybrid proteins combining antibody recognition domains linked to T cell signaling elements. Clinical trials of CAR-transduced peripheral blood lymphocytes (PBL) have induced remission of both solid organ and hematologic malignancies. Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target antigen that is overexpressed in multiple cancer histologies including melanoma, triple-negative breast cancer, glioblastoma, mesothelioma and sarcoma. Methods CSPG4 expression in cancer cell lines was assayed using flow cytometry (FACS) and reverse-transcription PCR (RT-PCR). Immunohistochemistry was utilized to assay resected melanomas and normal human tissues (n = 30) for CSPG4 expression and a reverse-phase protein array comprising 94 normal tissue samples was also interrogated for CSPG4 expression. CARs were successfully constructed from multiple murine antibodies (225.28S, TP41.2, 149.53) using second generation (CD28.CD3ζ) signaling domains. CAR sequences were cloned into a gamma-retroviral vector with subsequent successful production of retroviral supernatant and PBL transduction. CAR efficacy was assayed by cytokine release and cytolysis following coculture with target cell lines. Additionally, glioblastoma stem cells were generated from resected human tumors, and CSPG4 expression was determined by RT-PCR and FACS. Results Immunohistochemistry demonstrated prominent CSPG4 expression in melanoma tumors, but failed to demonstrate expression in any of the 30 normal human tissues studied. Two of 94 normal tissue protein lysates were positive by protein array. CAR constructs demonstrated cytokine secretion and cytolytic function after co-culture with tumor cell lines

  8. Evolution of the chicken Toll-like receptor gene family: A story of gene gain and gene loss

    PubMed Central

    Temperley, Nicholas D; Berlin, Sofia; Paton, Ian R; Griffin, Darren K; Burt, David W

    2008-01-01

    Background Toll-like receptors (TLRs) perform a vital role in disease resistance through their recognition of pathogen associated molecular patterns (PAMPs). Recent advances in genomics allow comparison of TLR genes within and between many species. This study takes advantage of the recently sequenced chicken genome to determine the complete chicken TLR repertoire and place it in context of vertebrate genomic evolution. Results The chicken TLR repertoire consists of ten genes. Phylogenetic analyses show that six of these genes have orthologs in mammals and fish, while one is only shared by fish and three appear to be unique to birds. Furthermore the phylogeny shows that TLR1-like genes arose independently in fish, birds and mammals from an ancestral gene also shared by TLR6 and TLR10. All other TLRs were already present prior to the divergence of major vertebrate lineages 550 Mya (million years ago) and have since been lost in certain lineages. Phylogenetic analysis shows the absence of TLRs 8 and 9 in chicken to be the result of gene loss. The notable exception to the tendency of gene loss in TLR evolution is found in chicken TLRs 1 and 2, each of which underwent gene duplication about 147 and 65 Mya, respectively. Conclusion Comparative phylogenetic analysis of vertebrate TLR genes provides insight into their patterns and processes of gene evolution, with examples of both gene gain and gene loss. In addition, these comparisons clarify the nomenclature of TLR genes in vertebrates. PMID:18241342

  9. 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

  10. 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. PMID:25986534

  11. 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.

  12. Evolution of galanin receptor genes: insights from the deuterostome genomes.

    PubMed

    Liu, Z; Xu, Y; Wu, L; Zhang, S

    2010-08-01

    Galanin exerts its biological activities through three different G protein-coupled receptors, Galr1, Galr2 and Galr3. To obtain insights into the evolution of Galrs, we searched the genomes of the deuterostomes by extensive BLAST survey and phylogenetic analyses. The Galr2 and Galr3 share similar genomic structures, and most of them are composed of 2 exons and 1 intron. However, most of Galr1 are composed of 3 extrons and 2 introns. We did not detect the typical Galr genes in the genomic databases of invertebrate deutserotomes, but three Galr1/Alstr homologs and two Galr1/Gpr151 homologs in amphioxus, two Galr1/Gpr151 homologs in sea squirt and one Galr1/Gpr151 homologs in sea urchin were identified. It is highly possible that the Galr genes in vertebrates may evolve from the homologous genes of Galr1/Alstr/Gpr151 in invertebrate deuterostomes. We also proposed that Galr3 genes were the products of Galr2 duplication during evolution, while Galr2 genes may evolve from Galr1. PMID:20476798

  13. A homologue of the recombination-dependent growth gene, rdgC, is involved in gonococcal pilin antigenic variation.

    PubMed Central

    Mehr, I J; Long, C D; Serkin, C D; Seifert, H S

    2000-01-01

    Neisseria gonorrhoeae pilin undergoes high-frequency changes in primary amino acid sequence that aid in the avoidance of the host immune response and alter pilus expression. The pilin amino acid changes reflect nucleotide changes in the expressed gene, pilE, which result from nonreciprocal recombination reactions with numerous silent loci, pilS. A series of mini-transposon insertions affecting pilin antigenic variation were localized to three genes in one region of the Gc chromosome. Mutational analysis with complementation showed that a Gc gene with sequence similarity to the Escherichia coli rdgC gene is involved in pilus-dependent colony phase variation and in pilin antigenic variation. Furthermore, we show that the Gc rdgC homologue is transcriptionally linked in an operon with a gene encoding a predicted GTPase. The inability to disrupt expression of this gene suggests it is an essential gene (engA, essential neisserial GTPase). While some of the transposon mutations in rdgC and insertions in the 5'-untranslated portion of engA showed a growth defect, all transposon insertions investigated conferred an aberrant cellular morphology. Complementation analysis showed that the growth deficiencies are due to the interruption of RdgC expression and not that of EngA. The requirement of RdgC for efficient pilin variation suggests a role for this protein in specialized DNA recombination reactions. PMID:10655208

  14. INDUCTION OF ACTIVATION ANTIGENS ON HUMAN NATURAL KILLER CELLS MODIFIED THROUGH THE FC-Y RECEPTOR

    EPA Science Inventory

    NK cells, defined here as lymphocytes bearing the CD16 Ag found on the NK cell Fc-y receptor (FcR), are known to enter a proliferative and activated state in response to stimulation with IL-2 as assessed by clonal expansion, short-term DNA synthesis, and de novo expression of lym...

  15. Immunogenicity of recombinant BCGs expressing predicted antigenic epitopes of bovine viral diarrhea virus E2 gene.

    PubMed

    Liu, Dongxu; Lu, Huijun; Shi, Kun; Su, Fengyan; Li, Jianming; Du, Rui

    2014-10-01

    To develop a vaccine to prevent diseases caused by Mycobacterium tuberculosis and bovine viral diarrhea virus (BVDV) simultaneously, recombinant Bacillus Calmette-Guerin (rBCG) vaccines expressing different regions of the BVDV E2 gene were constructed. Using DNASTAR 6.0 software, potential antigenic epitopes were predicted, and six regions were chosen to generate recombinant plasmids with the pMV361 vector (pMV361-E2-1, pMV361-E2-2, pMV361-E2-3, pMV361-E2-4, pMV361-E2-5 and pMV361-E2-6, respectively). The recombinant plasmids were transformed into BCG, and protein expression was thermally induced at 45 °C. Mice were immunized with 5 × 10(6) CFU/200 µL of each rBCG strain. Compared with other groups, BVDV E2 specific antibody titers were higher in mice immunized with rBCG-E2-6. Ratios and numbers of CD4+, CD8+ and IL-12 expressing spleen lymphocytes of the rBCG-E2-6 group also were higher than those of other groups. Thus, the rBCG-E2-6 vaccine showed the highest immunogenicity of all groups based on the humoral and cellular responses to vaccination. PMID:25135492

  16. 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

  17. Detection of Cytotoxic T-Lymphocyte Associated Antigen-4 Gene Polymorphism in Type 1 Diabetes Mellitus.

    PubMed

    Arafa, Roshdan M; Desouky, Somaya M; Emam, Sherin M; Abed, Neveen Tawfik; Mohamed, Sahar Y

    2015-01-01

    Type 1 diabetes is one of the most common chronic childhood illnesses. Interplay between genetic susceptibility and environmental factors is thought to provide the fundamental element for the disease. It has been shown that more than 40 genetic loci are associated with T1DM. Important one among these is the CTLA-4. This work aimed to detect Cytotoxic T Lymphocyte-associated antigen 4 (CTLA-4) gene polymorphism in patients with type 1 diabetes mellitus T1DM using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to clarify its role in the susceptibility to T1DM. The study was carried out on forty unrelated Egyptian children with TIDM. Twenty unrelated healthy children were enrolled as a control group. Blood samples were collected from patients and control groups and subjected to CTLA-4 gene polymorphism analysis using polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). CTLA-4 G allele and GG homozygous genotype were significantly increased in T1DM patients than in control group (P < 0.001, P = 0.002 respectively). There was significant association between the three CTLA-4 genotypes (AA, AG, GG) and diabetic complications (p = 0.002), AG and GG polymorphisms were associated with complications of diabetes with ratio 84.6% and 100% respectively. While no association was found with sex, weight, height, risk factors of diabetes or insulin treatment. It was concluded that there is a strong association between AG polymorphism and T1DM (P = 0.002). PMID:26415372

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

    PubMed Central

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

    2016-01-01

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

  19. Crosstalk of carcinoembryonic antigen and transforming growth factor-β via their receptors: comparing human and canine cancer.

    PubMed

    Jensen-Jarolim, Erika; Fazekas, Judit; Singer, Josef; Hofstetter, Gerlinde; Oida, Kumiko; Matsuda, Hiroshi; Tanaka, Akane

    2015-05-01

    There is accumulating evidence that the transforming growth factor beta (TGF-β) and nuclear factor kappa-B (NFκB) pathways are tightly connected and play a key role in malignant transformation in cancer. Immune infiltration by regulatory T- and B-lymphocytes (Tregs, Bregs) has recently gained increased attention for being an important source of TGF-β. There is a plethora of studies examining the pro-tumorigenic functions of carcinoembryonic antigen (CEA), but its receptor CEAR is far less studied. So far, there is a single connecting report that TGF-β also may signal through CEAR. The crosstalk between cancer tissues is further complicated by the expression of CEAR and TGF-β receptors in stromal cells, and implications of TGF-β in epithelial-mesenchymal transition. Furthermore, tumor-infiltrating Tregs and Bregs may directly instruct cancer cells by secreting TGF-β binding to their CEAR. Therefore, both TGF-β and CEA may act synergistically in breast cancer and cause disease progression, and NFκB could be a common crossing point between their signaling. CEAR, TGF-β1-3, TGF-β-R types I-III and NFκB class I and II molecules have an outstanding human-canine sequence identity, and only a canine CEA homolog has not yet been identified. For these reasons, the dog may be a valid translational model patient for investigating the crosstalk of the interconnected CEA and TGF-β networks. PMID:25832000

  20. Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein.

    PubMed

    Qin, Haiying; Cho, Monica; Haso, Waleed; Zhang, Ling; Tasian, Sarah K; Oo, Htoo Zarni; Negri, Gian Luca; Lin, Yongshun; Zou, Jizhong; Mallon, Barbara S; Maude, Shannon; Teachey, David T; Barrett, David M; Orentas, Rimas J; Daugaard, Mads; Sorensen, Poul H B; Grupp, Stephan A; Fry, Terry J

    2015-07-30

    Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL. PMID:26041741

  1. 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. PMID:26298772

  2. A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing

    PubMed Central

    Prapa, Malvina; Caldrer, Sara; Spano, Carlotta; Bestagno, Marco; Golinelli, Giulia; Grisendi, Giulia; Petrachi, Tiziana; Conte, Pierfranco; Horwitz, Edwin M.; Campana, Dario

    2015-01-01

    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. PMID:26298772

  3. B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events.

    PubMed Central

    Liossis, S N; Kovacs, B; Dennis, G; Kammer, G M; Tsokos, G C

    1996-01-01

    To understand the molecular mechanisms that are responsible for the B cell overactivity that is observed in patients with SLE, we have conducted experiments in which the surface immunoglobulin (sIg)-mediated early cell signaling events were studied. The anti-sIgM-mediated free intracytoplasmic calcium ([Ca2+]i) responses were significantly higher in SLE B cells compared with responses of normal individuals and to those of patients with other systemic autoimmune rheumatic diseases. The anti-IgD mAb induced [Ca2+]i responses were also higher in lupus B cells than in controls. The magnitude of anti-sIgM-mediated Ca2+ release from intracellular stores was also increased in B cells from SLE patients compared with normal controls. The amount of inositol phosphate metabolites produced upon crosslinking of sIgM was slightly higher in patients with lupus than in normal controls, although the difference was not statistically significant. In contrast, the degree of anti-sIgM-induced protein tyrosine phosphorylation was obviously increased in lupus patients. Our study demonstrates clearly for the first time that SLE B cells exhibit aberrant early signal transduction events, including augmented calcium responses after crosslinking of the B cell receptor and increased antigen-receptor-mediated phosphorylation of protein tyrosine residues. Because the above abnormalities did not correlate with disease activity or treatment status, we propose that they may have pathogenic significance. PMID:8958217

  4. 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

  5. T−B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3ζ subunit of the T-cell antigen receptor complex

    PubMed Central

    Lauritsen, Jens Peter H.; Cooney, Myriah; Parrott, Roberta E.; Sajaroff, Elisa O.; Win, Chan M.; Keller, Michael D.; Carpenter, Jeffery H.; Carabana, Juan; Krangel, Michael S.; Sarzotti, Marcella; Zhong, Xiao-Ping; Wiest, David L.; Buckley, Rebecca H.

    2007-01-01

    CD3ζ is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T−B+NK+ severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3ζ gene. The few T cells present contained no detectable CD3ζ protein, expressed low levels of cell surface CD3ε, and were nonfunctional. CD4+CD8−CD3εlow, CD4−CD8+CD3εlow, and CD4−CD8−CD3εlow cells were detected in the periphery, and the patient also exhibited an unusual population of CD56−CD16+ NK cells with diminished cytolytic activity. Additional studies demonstrated that retrovirally transduced patient mutant CD3ζ cDNA failed to rescue assembly of nascent complete TCR complexes or surface TCR expression in CD3ζ-deficient MA5.8 murine T-cell hybridoma cells. Nascent transduced mutant CD3ζ protein was also not detected in metabolically labeled MA5.8 cells, suggesting that it was unstable and rapidly degraded. Taken together, these findings provide the first demonstration that complete CD3ζ deficiency in humans can cause SCID by preventing normal TCR assembly and surface expression. PMID:17170122

  6. Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2

    PubMed Central

    Morgan, Richard A; Yang, James C; Kitano, Mio; Dudley, Mark E; Laurencot, Carolyn M; Rosenberg, Steven A

    2010-01-01

    In an attempt to treat cancer patients with ERBB2 overexpressing tumors, we developed a chimeric antigen receptor (CAR) based on the widely used humanized monoclonal antibody (mAb) Trastuzumab (Herceptin). An optimized CAR vector containing CD28, 4-1BB, and CD3ζ signaling moieties was assembled in a γ-retroviral vector and used to transduce autologous peripheral blood lymphocytes (PBLs) from a patient with colon cancer metastatic to the lungs and liver, refractory to multiple standard treatments. The gene transfer efficiency into autologous T cells was 79% CAR+ in CD3+ cells and these cells demonstrated high-specific reactivity in in vitro coculture assays. Following completion of nonmyeloablative conditioning, the patient received 1010 cells intravenously. Within 15 minutes after cell infusion the patient experienced respiratory distress, and displayed a dramatic pulmonary infiltrate on chest X-ray. She was intubated and despite intensive medical intervention the patient died 5 days after treatment. Serum samples after cell infusion showed marked increases in interferon-γ (IFN-γ), granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10, consistent with a cytokine storm. We speculate that the large number of administered cells localized to the lung immediately following infusion and were triggered to release cytokine by the recognition of low levels of ERBB2 on lung epithelial cells. PMID:20179677

  7. First evidence for functional vomeronasal 2 receptor genes in primates.

    PubMed

    Hohenbrink, Philipp; Mundy, Nicholas I; Zimmermann, Elke; Radespiel, Ute

    2013-02-23

    Two classes of vomeronasal receptor genes, V1R and V2R, occur in vertebrates. Whereas, V1R loci are found in a wide variety of mammals, including primates, intact V2R genes have thus far only been described in rodents and marsupials. In primates, the V2R repertoire has been considered degenerate. Here, we identify for the first time two intact V2R loci in a strepsirrhine primate, the grey mouse lemur (Microcebus murinus), and demonstrate their expression in the vomeronasal organ. Putatively functional orthologues are present in two other strepsirrhines, whereas, both loci are pseudogenes in a range of anthropoid species. The functional significance of the loci is unknown, but positive selection on one of them is consistent with an adaptive role in pheromone detection. Finally, conservation of V2R loci in strepsirrhines is notable, given their high diversity and role in MUP and MHC detection in rodents. PMID:23269843

  8. Relationship between serum carcinoembryonic antigen level and epidermal growth factor receptor mutations with the influence on the prognosis of non-small-cell lung cancer patients

    PubMed Central

    Cai, Zuxun

    2016-01-01

    Objective To investigate the relationship between serum carcinoembryonic antigen (CEA) level and epidermal growth factor receptor (EGFR) gene mutations in non-small-cell lung cancer (NSCLC) patients and to analyze the influence of CEA level on postoperative survival time in lung cancer patients. Methods A total of 296 patients who were treated in Thoracic Surgery Department of Henan Provincial Chest Hospital from September 2011 to September 2013 were recruited. The level of tumor markers, such as CEA, was determined before the surgery, and EGFR gene mutations were detected after surgery. Thereby, the relationship between tumor makers, including CEA, and EGFR mutation and its influence on prognosis could be investigated. Results Among 296 patients, the positive rate of EGFR gene mutation was 37.84% (112/296); the mutation occurred more frequently in nonsmokers, adenocarcinoma patients, women, and patients aged <60 years (P<0.05). Both tumor markers and chemosensitivity indicators were related to the profile of EGFR mutations. Elevated squamous cell carcinoma and Cyfra21-1 as well as positively expressed ERCC1 were more common in patients with wild-type EGFR (P<0.05), whereas increased CEA level was observed more frequently in patients with EGFR gene mutation (P=0.012). The positive rate of EGFR gene mutations was higher as the serum CEA level increased, that is, the positive rate in patients with serum CEA level <5, 5–20, and >20 μg/L was 39.81%, 45.32%, and 65.47%, respectively (P=0.004). Logistic regression analysis showed that CEA level was an independent factor in predicting EGFR gene mutations, and serum CEA level was also an independent factor in affecting the prognosis of NSCLC patients, as the overall 2-year survival rate was 73.86% in elevated CEA group and 86.43% in normal group (P<0.01). Conclusion The prognosis of NSCLC patients receiving resection can be predicted according to serum CEA level, which is associated with EGFR mutations in NSCLC patients

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

    PubMed Central

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

    2015-01-01

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

  10. Chimeric antigen receptors: a costimulatory boost promotes immunological memory to B-cell malignancies.

    PubMed

    Peggs, Karl S

    2011-11-01

    Cellular immunotherapies offer perhaps the best proof of concept of the ability of immune cells to eradicate malignancy. However, the majority of these data derive from either the use of expanded tumor-infiltrating lymphocytes to treat metastatic melanoma or the use of donor lymphocytes in relapsed hematological malignancies following allogeneic hematopoietic stem cell transplantation. Genetic engineering of T cells to redirect specificity against tumor-associated antigens potentially overcomes one of the major hurdles to more widespread application. Despite early evidence of possible activity, limited in vivo expansion and persistence of adoptively transferred cells has been associated with disappointing clinical efficacy. A report from Porter and colleagues now demonstrates more fully the potential of such genetic modification in a patient with chronic lymphocytic leukemia, illustrating that when delivered in the right setting with appropriate costimulatory signaling, these cells can expand and persist, and in doing so effect quite remarkable anti-tumor activity. PMID:22050014

  11. 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. PMID:26968708

  12. Genome-wide association replicates the association of Duffy antigen receptor for chemokines (DARC) polymorphisms with serum monocyte chemoattractant protein-1 (MCP-1) levels in Hispanic children.

    PubMed

    Voruganti, V Saroja; Laston, Sandra; Haack, Karin; Mehta, Nitesh R; Smith, C Wayne; Cole, Shelley A; Butte, Nancy F; Comuzzie, Anthony G

    2012-12-01

    Obesity is associated with a chronic low inflammatory state characterized by elevated levels of chemokines. Monocyte chemoattractant protein-1 (MCP-1) is a member of the cysteine-cysteine (CC) chemokine family and is increased in obesity. The purpose of this study was to identify loci regulating serum MCP-1 in obese Hispanic children from the Viva La Familia Study. A genome-wide association (GWA) analysis was performed in 815 children, ages 4-19 years, using genotypes assayed with the Illumina HumanOmni1-Quad v1.0 BeadChips. All analyses were performed in SOLAR using a linear regression-based test under an additive model of allelic effect, while accounting for the relatedness of family members via a kinship variance component. The strongest association for MCP-1 levels was found with a non-synonymous single nucleotide polymorphism (SNP), rs12075, resulting in an amino acid substitution (Asp42Gly) in the Duffy antigen receptor for chemokines (DARC) gene product (minor allele frequency=43.6%, p=1.3 × 10(-21)) on chromosome 1. Four other DARC SNPs were also significantly associated with MCP-1 levels (p<10(-16)-10(-6)). The Asp42Gly variant was associated with higher levels of MCP-1 and accounted for approximately 10% of its variability. In addition, MCP-1 levels were significantly associated with SNPs in chemokine receptor 3 (CCR3) and caspase recruitment domain family, member 9 (CARD9). In summary, the association of the DARC Asp42Gly variant with MCP-1 levels replicates previous GWA results substantiating a potential role for DARC in the regulation of pro-inflammatory cytokines. PMID:23017229

  13. Enhancer RNAs participate in androgen receptor-driven looping that selectively enhances gene activation.

    PubMed

    Hsieh, Chen-Lin; Fei, Teng; Chen, Yiwen; Li, Tiantian; Gao, Yanfei; Wang, Xiaodong; Sun, Tong; Sweeney, Christopher J; Lee, Gwo-Shu Mary; Chen, Shaoyong; Balk, Steven P; Liu, Xiaole Shirley; Brown, Myles; Kantoff, Philip W

    2014-05-20

    The androgen receptor (AR) is a key factor that regulates the behavior and fate of prostate cancer cells. The AR-regulated network is activated when AR binds enhancer elements and modulates specific enhancer-promoter looping. Kallikrein-related peptidase 3 (KLK3), which codes for prostate-specific antigen (PSA), is a well-known AR-regulated gene and its upstream enhancers produce bidirectional enhancer RNAs (eRNAs), termed KLK3e. Here, we demonstrate that KLK3e facilitates the spatial interaction of the KLK3 enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. KLK3e carries the core enhancer element derived from the androgen response element III (ARE III), which is required for the interaction of AR and Mediator 1 (Med1). Furthermore, we show that KLK3e processes RNA-dependent enhancer activity depending on the integrity of core enhancer elements. The transcription of KLK3e was detectable and its expression is significantly correlated with KLK3 (R(2) = 0.6213, P < 5 × 10(-11)) and KLK2 (R(2) = 0.5893, P < 5 × 10(-10)) in human prostate tissues. Interestingly, RNAi silencing of KLK3e resulted in a modest negative effect on prostate cancer cell proliferation. Accordingly, we report that an androgen-induced eRNA scaffolds the AR-associated protein complex that modulates chromosomal architecture and selectively enhances AR-dependent gene expression. PMID:24778216

  14. Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses.

    PubMed

    Parker, Lauren; Wharton, Stephen A; Martin, Stephen R; Cross, Karen; Lin, Yipu; Liu, Yan; Feizi, Ten; Daniels, Rodney S; McCauley, John W

    2016-06-01

    Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity. PMID:26974849

  15. 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

    2016-07-01

    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. PMID:27365313

  16. Identification of Actinobacillus pleuropneumoniae Genes Preferentially Expressed During Infection Using In Vivo-Induced Antigen Technology (IVIAT).

    PubMed

    Zhang, Fei; Zhang, Yangyi; Wen, Xintian; Huang, Xiaobo; Wen, Yiping; Wu, Rui; Yan, Qigui; Huang, Yong; Ma, Xiaoping; Zhao, Qin; Cao, Sanjie

    2015-10-01

    Porcine pleuropneumonia is an infectious disease caused by Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo, is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens expressed in vivo during A. pleuropneumoniae infection, using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A. pleuropneumoniae genes. Based on sequence analysis, proteins encoded by these genes were involved in metabolism, replication, transcription regulation, and signal transduction. Moreover, three function-unknown proteins were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro. IVI (in vivoinduced) genes that were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae. PMID:26059519

  17. 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

  18. Vitamin D receptor gene polymorphisms in breast cancer.

    PubMed

    Buyru, Nur; Tezol, Ayda; Yosunkaya-Fenerci, Elif; Dalay, Nejat

    2003-12-31

    Breast cancer is the leading cause of cancer death among women around the world and its incidence is annually increasing. The vitamin D receptor (VDR) gene is a member of the nuclear receptor superfamily, which is expressed in breast tissue and known to modulate the rate of cell proliferation. Association between the VDR gene polymorphisms and cancer development has been suggested by several studies. However, the relationship between VDR polymorphisms and breast cancer is controversial and has not been confirmed by all studies. The purpose of this study was to investigate the genotype frequencies and association of the VDR Bsm I and Taq I polymorphisms with breast cancer in Turkish patients. In this study, 78 patients with breast cancer and 27 healthy individuals were enrolled. The prevalence of the VDR Taq I and Bsm I alleles and the genotype frequencies in patients with breast cancer was similar to that in the normal population. Our data indicate that no significant differences exist between the patients and control subjects. PMID:14749534

  19. The dopamine D3 receptor gene and posttraumatic stress disorder.

    PubMed

    Wolf, Erika J; Mitchell, Karen S; Logue, Mark W; Baldwin, Clinton T; Reardon, Annemarie F; Aiello, Alison; Galea, Sandro; Koenen, Karestan C; Uddin, Monica; Wildman, Derek; Miller, Mark W

    2014-08-01

    The dopamine D3 receptor (DRD3) gene has been implicated in schizophrenia, autism, and substance use-disorders and is related to emotion reactivity, executive functioning, and stress-responding, processes impaired in posttraumatic stress disorder (PTSD). The aim of this candidate gene study was to evaluate DRD3 polymorphisms for association with PTSD. The discovery sample was trauma-exposed White, non-Hispanic U.S. veterans and their trauma-exposed intimate partners (N = 491); 60.3% met criteria for lifetime PTSD. The replication sample was 601 trauma-exposed African American participants living in Detroit, Michigan; 23.6% met criteria for lifetime PTSD. Genotyping was based on high-density bead chips. In the discovery sample, 4 single nucleotide polymorphisms (SNPs), rs2134655, rs201252087, rs4646996, and rs9868039, showed evidence of association with PTSD and withstood correction for multiple testing. The minor alleles were associated with reduced risk for PTSD (OR range = 0.59 to 0.69). In the replication sample, rs2251177, located 149 base pairs away from the most significant SNP in the discovery sample, was nominally associated with PTSD in men (OR = 0.32). Although the precise role of the D3 receptor in PTSD is not yet known, its role in executive functioning and emotional reactivity, and the sensitivity of the dopamine system to environmental stressors could potentially explain this association. PMID:25158632

  20. The Dopamine D3 Receptor Gene and Posttraumatic Stress Disorder

    PubMed Central

    Wolf, Erika J.; Mitchell, Karen S.; Logue, Mark W.; Baldwin, Clinton T.; Reardon, Annemarie F.; Aiello, Alison; Galea, Sandro; Koenen, Karestan C.; Uddin, Monica; Wildman, Derek; Miller, Mark W.

    2014-01-01

    The dopamine D3 receptor (DRD3) gene has been implicated in schizophrenia, autism, and substance use-disorders and is related to emotion reactivity, executive functioning, and stress-responding, processes impaired in posttraumatic stress disorder (PTSD). This aim of this candidate gene study was to evaluate DRD3 polymorphisms for association with PTSD. The discovery sample was trauma-exposed white, non-Hispanic veterans and their trauma-exposed intimate partners (N = 491); 60% met criteria for lifetime PTSD. The replication sample was 601 trauma-exposed African American participants; 24% met criteria for lifetime PTSD. Genotyping was based on high-density bead chips. In the discovery sample, four single nucleotide polymorphisms (SNPs), rs2134655, rs201252087, rs4646996, and rs9868039, showed evidence of association with PTSD and withstood correction for multiple testing. The minor alleles were associated with reduced risk for PTSD (odds ratio range: 0.59 – 0.69). In the replication sample, rs2251177, located 149 base pairs away from the most significant SNP in the discovery sample, was nominally associated with PTSD in men (odds ratio: 0.32). Although the precise role of the D3 receptor in PTSD is not yet known, its role in executive functioning and emotional reactivity, and the sensitivity of the dopamine system to environmental stressors, could potentially explain this association. PMID:25158632

  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. Oxytocin receptor and vasopressin receptor 1a genes are respectively associated with emotional and cognitive empathy.

    PubMed

    Uzefovsky, F; Shalev, I; Israel, S; Edelman, S; Raz, Y; Mankuta, D; Knafo-Noam, A; Ebstein, R P

    2015-01-01

    Empathy is the ability to recognize and share in the emotions of others. It can be considered a multifaceted concept with cognitive and emotional aspects. Little is known regarding the underlying neurochemistry of empathy and in the current study we used a neurogenetic approach to explore possible brain neurotransmitter pathways contributing to cognitive and emotional empathy. Both the oxytocin receptor (OXTR) and the arginine vasopressin receptor 1a (AVPR1a) genes contribute to social cognition in both animals and humans and hence are prominent candidates for contributing to empathy. The following research examined the associations between polymorphisms in these two genes and individual differences in emotional and cognitive empathy in a sample of 367 young adults. Intriguingly, we found that emotional empathy was associated solely with OXTR, whereas cognitive empathy was associated solely with AVPR1a. Moreover, no interaction was observed between the two genes and measures of empathy. The current findings contribute to our understanding of the distinct neurogenetic pathways involved in cognitive and emotional empathy and underscore the pervasive role of both oxytocin and vasopressin in modulating human emotions. PMID:25476609

  3. Evolutionary dynamics of olfactory and other chemosensory receptor genes in vertebrates

    PubMed Central

    Niimura, Yoshihito

    2007-01-01

    The numbers of functional olfactory receptor (OR) genes in humans and mice are about 400 and 1,000 respectively. In both humans and mice, these genes exist as genomic clusters and are scattered over almost all chromosomes. The difference in the number of genes between the two species is apparently caused by massive inactivation of OR genes in the human lineage and a substantial increase of OR genes in the mouse lineage after the human–mouse divergence. Compared with mammals, fishes have a much smaller number of OR genes. However, the OR gene family in fishes is much more divergent than that in mammals. Fishes have many different groups of genes that are absent in mammals, suggesting that the mammalian OR gene family is characterized by the loss of many group genes that existed in the ancestor of vertebrates and the subsequent expansion of specific groups of genes. Therefore, this gene family apparently changed dynamically depending on the evolutionary lineage and evolved under the birth-and-death model of evolution. Study of the evolutionary changes of two gene families for vomeronasal receptors and two gene families for taste receptors, which are structurally similar, but remotely related to OR genes, showed that some of the gene families evolved in the same fashion as the OR gene family. It appears that the number and types of genes in chemosensory receptor gene families have evolved in response to environmental needs, but they are also affected by fortuitous factors. PMID:16607462

  4. 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. PMID:24586993

  5. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation.

    PubMed

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-09-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  6. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation

    PubMed Central

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-01-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  7. Chimeric antigen receptor T cells targeting HERV-K inhibit breast cancer and its metastasis through downregulation of Ras

    PubMed Central

    Zhou, Fuling; Krishnamurthy, Janani; Wei, Yongchang; Li, Ming; Hunt, Kelly; Johanning, Gary L; Cooper, Laurence JN; Wang-Johanning, Feng

    2015-01-01

    We have previously reported that human endogenous retrovirus-K (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) for cancer vaccines, and that its antibodies (mAbs) possess antitumor activity against cancer. In this study, a chimeric antigen receptor (CAR) specific for HERV-K env protein (K-CAR) was generated using anti-HERV-K mAb. K-CAR T cells from peripheral blood mononuclear cells (PBMCs) of 9 breast cancer (BC) patients and 12 normal donors were able to inhibit growth of, and to exhibit significant cytotoxicity toward, BC cells but not MCF-10A normal breast cells. The antitumor effects in cancer cells were significantly reduced when control T cells were used, or the expression of HERV-K was knocked down by an shRNA. Secretion of multiple cytokines, including IFNγ, TNF-α, and IL-2, was significantly enhanced in culture media of BC cells treated with K-CARs. Significantly reduced tumor growth and tumor weight was observed in xenograft models bearing MDA-MB-231 or MDA-MB-435.eB1 BC cells. Importantly, the K-CAR prevented tumor metastasis to other organs. Furthermore, downregulation of HERV-K expression in tumors of mice treated with K-CAR correlated with upregulation of p53 and downregulation of MDM2 and p-ERK. Importantly, the expression of HERV-K env protein in metastatic tumor tissues treated with K-CAR T cells correlated with the expression of Ras. Our results indicate that HERV-K env protein is an oncoprotein and may play an important role in tumorigenesis related to p53 and Ras signaling pathways. Anti-HERV-K treatment, including K-CAR treatment, shows potential for immunotherapy of BC. PMID:26451325

  8. Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

    PubMed Central

    Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

    2016-01-01

    Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

  9. Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

    PubMed Central

    DebRoy, Chitrita; Fratamico, Pina M.; Roberts, Elisabeth; Davis, Michael A.; Liu, Yanhong

    2005-01-01

    The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 106 and 108 CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55. PMID:16085897

  10. 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

  11. Mechanistic Assessment of PD-1H Coinhibitory Receptor-Induced T-Cell Tolerance to Allogeneic Antigens1

    PubMed Central

    Flies, Dallas B.; Higuchi, Tomoe; Chen, Lieping

    2015-01-01

    PD-1H is a recently identified cell surface co-inhibitory molecule of the B7/CD28 immune modulatory gene family. We showed previously that single injection of a PD-1H agonistic monoclonal antibody (mAb) protected mice from graft versus host disease (GVHD). We report here two distinct mechanisms operate in PD-1H-induced T cell tolerance. First, signaling via PD-1H co-inhibitory receptor potently arrests allo-reactive donor T cells from activation and expansion in the initiation phase. Second, donor regulatory T cells are subsequently expanded to maintain long-term tolerance and GVHD suppression. Our study reveals the crucial function of PD-1H as a co-inhibitory receptor on allo-reactive T cells and its function in the regulation of T cell tolerance. Therefore, PD-1H may be a target for the modulation of allo-reactive T cells in GVHD and transplantation. PMID:25917101

  12. Receptor protein kinase gene encoded at the self-incompatibility locus

    DOEpatents

    Nasrallah, June B.; Nasrallah, Mikhail E.; Stein, Joshua

    1996-01-01

    Described herein is a S receptor kinase gene (SRK), derived from the S locus in Brassica oleracea, having a extracellular domain highly similar to the secreted product of the S-locus glycoprotein gene.

  13. Molecular characterization of thyroid hormone receptor beta from Schistosoma japonicum and assessment of its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice

    PubMed Central

    2012-01-01

    Background Thyroid hormones (TH) modulate growth, development and differentiation and metabolic processes by interacting with thyroid hormone receptors (THRs). The purpose of this study was to identify a novel thyroid hormone receptor beta encoding gene of Schistosoma japonicum (SjTHRβ) and to investigate its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice. Methods The full-length cDNA sequence of SjTHRβ, its gene organization, and its transcript levels were characterized, and the phylogenetic relationship between THR, RAR and RXR from other organisms were analysis, the ability of this protein binding to a conserved DNA core motif, and its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice were evaluated. Results The SjTHRβ cDNA was cloned, verified by 5’ and 3’ Rapid Amplification of cDNA Ends and shown to be polyadenylated at the 3’end, suggesting the transcript is full-length. SjTHRβ is homologous to THRs from other species and has a predicted conservative DNA binding domain and ligand binding domain that normally characterizes these receptors. A comparative quantitative PCR analysis showed that SjTHRβ was the highest expressed in 21d worms and the lowest in 7 d and 13 d schistosomula. The cDNA corresponding to DNA binding domain (SjTHRβ-DBD) and ligand binding domain (SjTHRβ-LBD) were cloned and subsequently expressed in E coli. The expressed proteins were used to immunize mice and generate specific serum against recombinant SjTHRβ (rSjTHRβ). Western blotting revealed that anti-rSjTHRβ-LBD serum recognized two protein bands in extracts from 21 d worm with molecular sizes of approximately 95 kDa and 72 kDa. Electrophoretic mobility shift assay (EMSA) analysis showed that rSjTHRβ-DBD could bind to a conserved DNA core motif. Immunization of BALB/c mice with rSjTHRβ-LBD could induce partial protective efficacy(27.52% worm reduction and 29.50% liver eggs reduction)against schistosome

  14. Transcriptional Characterization of Porcine Leptin and Leptin Receptor Genes.

    PubMed

    Pérez-Montarelo, Dafne; Fernández, Almudena; Barragán, Carmen; Noguera, Jose L; Folch, Josep M; Rodríguez, M Carmen; Ovilo, Cristina; Silió, Luis; Fernández, Ana I

    2013-01-01

    The leptin (LEP) and its receptor (LEPR) regulate food intake and energy balance through hypothalamic signaling. However, the LEP-LEPR axis seems to be more complex and its expression regulation has not been well described. In pigs, LEP and LEPR genes have been widely studied due to their relevance. Previous studies reported significant effects of SNPs located in both genes on growth and fatness traits. The aim of this study was to determine the expression profiles of LEP and LEPR across hypothalamic, adipose, hepatic and muscle tissues in Iberian x Landrace backcrossed pigs and to analyze the effects of gene variants on transcript abundance. To our knowledge, non porcine LEPR isoforms have been described rather than LEPRb. A short porcine LEPR isoform (LEPRa), that encodes a protein lacking the intracellular residues responsible of signal transduction, has been identified for the first time. The LEPRb isoform was only quantifiable in hypothalamus while LEPRa appeared widely expressed across tissues, but at higher levels in liver, suggesting that both isoforms would develop different roles. The unique LEP transcript showed expression in backfat and muscle. The effects of gene variants on transcript expression revealed interesting results. The LEPRc.1987C>T polymorphism showed opposite effects on LEPRb and LEPRa hypothalamic expression. In addition, one out of the 16 polymorphisms identified in the LEPR promoter region revealed high differential expression in hepatic LEPRa. These results suggest a LEPR isoform-specific regulation at tissue level. Conversely, non-differential expression of LEP conditional on the analyzed polymorphisms could be detected, indicating that its regulation is likely affected by other mechanisms rather than gene sequence variants. The present study has allowed a transcriptional characterization of LEP and LEPR isoforms on a range of tissues. Their expression patterns seem to indicate that both molecules develop peripheral roles apart from

  15. A New O-Antigen Gene Cluster Has a Key Role in the Virulence of the Escherichia coli Meningitis Clone O45:K1:H7▿

    PubMed Central

    Plainvert, Céline; Bidet, Philippe; Peigne, Chantal; Barbe, Valérie; Médigue, Claudine; Denamur, Erick; Bingen, Edouard; Bonacorsi, Stéphane

    2007-01-01

    A new highly pathogenic clone of Escherichia coli meningitis strains harboring the unusual serogroup O45 has recently emerged in France. To gain insight into the pathogenicity of this new clone, we investigated the possible role of antigen O45 in the virulence of strain S88 (O45:K1:H7), representative of this emerging clone. We first showed that the S88 O-antigen gene cluster sequence differs from that of O45 in the reference strain E. coli 96-3285, suggesting that the two O45 polysaccharides, while probably sharing a community of epitopes, represent two different antigens. The unique functional organization of the two O-antigen gene clusters and the low DNA sequence homology of the orthologous genes suggest that the two loci originated from a common ancestor and have since undergone multiple recombination events. Phylogenetic analysis based on the flanking gene gnd sequences indicates that the S88 antigen O45 (O45S88) gene cluster may have been acquired, at least in part, from another member of the Enterobacteriaceae. Mutagenesis of the O45S88 antigen gene cluster was used for functional analysis of the loci and revealed the crucial role of the O polysaccharide in S88 virulence in a neonatal rat meningitis model. We also developed a PCR method to specifically identify the O45S88 antigen gene cluster. Together, our findings suggest that horizontal acquisition of a new O-antigen gene cluster, at least partly from another species, may have been a key event in the emergence and virulence of the E. coli O45:K1:H7 clone in France. PMID:17905975

  16. A Novel Family of Human Leukocyte Antigen Class II Receptors May Have Its Origin in Archaic Human Species*

    PubMed Central

    Temme, Sebastian; Zacharias, Martin; Neumann, Jürgen; Wohlfromm, Sebastian; König, Angelika; Temme, Nadine; Springer, Sebastian; Trowsdale, John; Koch, Norbert

    2014-01-01

    HLA class II α and β chains form receptors for antigen presentation to CD4+ T cells. Numerous pairings of class II α and β subunits from the wide range of haplotypes and isotypes may form, but most of these combinations, in particular those produced by isotype mixing, yielded mismatched dimers. It is unclear how selection of functional receptors is achieved. At the atomic level, it is not known which interactions of class II residues regulate selection of matched αβ heterodimers and the evolutionary origin of matched isotype mixed dimer formation. In this study we investigated assembly of isotype-mixed HLA class II α and β heterodimers. Assembly and carbohydrate maturation of various HLA-class II isotype-mixed α and β subunits was dependent on the groove binding section of the invariant chain (Ii). By mutation of polymorphic DPβ sequences, we identified two motifs, Lys-69 and GGPM-(84–87), that are engaged in Ii-dependent assembly of DPβ with DRα. We identified five members of a family of DPβ chains containing Lys-69 and GGPM 84–87, which assemble with DRα. The Lys/GGPM motif is present in the DPβ sequence of the Neanderthal genome, and this ancient sequence is related to the human allele DPB1*0401. By site-directed mutagenesis, we inspected Neanderthal amino acid residues that differ from the DPB1*0401 allele and aimed to determine whether matched heterodimers are formed by assembly of DPβ mutants with DRα. Because the *0401 allele is rare in the sub-Saharan population but frequent in the European population, it may have arisen in modern humans by admixture with Neanderthals in Europe. PMID:24214983

  17. Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.

    PubMed

    Kuramitsu, S; Ohno, M; Ohka, F; Shiina, S; Yamamichi, A; Kato, A; Tanahashi, K; Motomura, K; Kondo, G; Kurimoto, M; Senga, T; Wakabayashi, T; Natsume, A

    2015-10-01

    The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses. PMID:26450624

  18. The Dopamine D2 Receptor Gene, Perceived Parental Support, and Adolescent Loneliness: Longitudinal Evidence for Gene-Environment Interactions

    ERIC Educational Resources Information Center

    van Roekel, Eeske; Goossens, Luc; Scholte, Ron H. J.; Engels, Rutger C. M. E.; Verhagen, Maaike

    2011-01-01

    Background: Loneliness is a common problem in adolescence. Earlier research focused on genes within the serotonin and oxytocin systems, but no studies have examined the role of dopamine-related genes in loneliness. In the present study, we focused on the dopamine D2 receptor gene (DRD2). Methods: Associations among the DRD2, sex, parental support,…

  19. Haptenic oligosaccharides in antigenic variants of mycobacterial C-mycosides antagonize lipid receptor activity for mycobacteriophage D4 by masking a methylated rhamnose.

    PubMed Central

    Dhariwal, K R; Liav, A; Vatter, A E; Dhariwal, G; Goren, M B

    1986-01-01

    The simple apolar C-mycosides, i.e., structurally well-defined hydrophobic glycopeptidolipids of several Mycobacterium species (see diagram below), were earlier shown to behave as receptors for adsorption of mycobacteriophage D4. This phage is usually virulent for Mycobacterium smegmatis. More complex, polar C-mycosides with additional carbohydrate substituents attached solely to the deoxytalose have recently been described. They are the highly specific serotyping antigens discovered by W. B. Schaefer--lipids which characterize members of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex. Both kinds are depicted in the structure below: (Formula: see text) where X equals H (for simple, apolar C-mycosides) and X equals small oligosaccharides (for antigenic forms; more complex, polar C-mycosides). The present investigations showed that the purified polar antigenic lipids exhibit considerably less adsorptive activity for D4 than do the apolar C-mycosides. Thus, the haptenic oligosaccharides are believed to shield the site in the molecule that the phage recognizes, and the blocking is reinforced by the specific antibodies that the antigens elicit. Although the MAIS serovars usually also produce the phage-reactive apolar C-mycosides, they are not permissive hosts for D4, nor do whole cells adsorb the phage. We suggest that in these species the apolar forms are probably "covered" at the cell surface by the antigenic lipids. Therefore, these antigenic mycosides may play a putative role in virulence of the MAIS members by protecting these mycobacteria from their own potential pathogen. The results of chemical transformations at specific sites of the mycoside core coupled with studies of simple synthetic lipid glycosides indicated that the principal phage receptor activity resides in the terminal methylated rhamnose (see diagram). It is this sugar which is evidently masked by the (seemingly remote) haptenic oligosaccharides

  20. DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

    PubMed

    Liu, Yang; Blanchfield, Lori; Ma, Victor Pui-Yan; Andargachew, Rakieb; Galior, Kornelia; Liu, Zheng; Evavold, Brian; Salaita, Khalid

    2016-05-17

    T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response. PMID:27140637

  1. 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

  2. Structural and Functional Studies of Ig[alpha beta] and Its Assembly with the B Cell Antigen Receptor

    SciTech Connect

    Radaev, Sergei; Zou, Zhongcheng; Tolar, Pavel; Nguyen, Khanh; Nguyen, AnhThao; Krueger, Peter D.; Stutzman, Nicole; Pierce, Susan; Sun, Peter D.

    2010-11-09

    The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Ig{beta} form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Ig{alpha} displays a similar fold as Ig{beta}. An Ig{alpha}{beta} heterodimer model was generated based on the unique disulfide-bonded Ig{beta} dimer. Solution binding studies showed that the extracellular domains of Ig{alpha}{beta} preferentially recognize the constant region of BCR with chain specificity, suggesting a role for Ig{alpha}{beta} to enhance BCR chain signaling. Cluster mutations on Ig{alpha}, Ig{beta}, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the C4 domain of mIgM. These studies provide the first structural model for understanding BCR function.

  3. 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.

  4. 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

  5. Increased numbers of T lymphocytes with gamma delta-positive antigen receptors in a subgroup of individuals with pulmonary sarcoidosis.

    PubMed Central

    Balbi, B; Moller, D R; Kirby, M; Holroyd, K J; Crystal, R G

    1990-01-01

    Individuals with sarcoidosis were evaluated for preferential usage of T cells with the gamma delta-positive (+) type of T cell antigen receptor. Compared with normal subjects (n = 19), the group with sarcoidosis had increased numbers of CD3+ alpha beta-negative (-) T cells in the blood (normal, 58 +/- 12 cells/microliters; sarcoid, 192 +/- 45 cells/microliters, P less than 0.05) and in the epithelial lining fluid of the lung (normal, 78 14 cells/microliters; sarcoid, 240 +/- 60 cells/microliters, P less than 0.04) and a concomitant elevated number of blood and lung CD3+ gamma delta+ T cells, owing to a striking increase in the number of CD3+ gamma delta+ T cells in a subgroup (7 of 20) of sarcoid individuals. The elevated numbers of sarcoid blood gamma delta+ T lymphocytes were mostly Ti gamma A+ and delta TCS1-, a pattern also seen in normal individuals, consistent with the majority of gamma delta+ T cells expressing one gamma-chain variable region, V gamma 9. The observation of an increase in the total gamma delta+ T cell numbers in a sarcoid subgroup suggests that various specific stimuli may trigger the expansion of different T cell subpopulations within different groups of individuals with sarcoidosis. Images PMID:2110187

  6. The receptor for interleukin-17E is induced by Th2 cytokines in antigen-presenting cells.

    PubMed

    Gratchev, A; Kzhyshkowska, J; Duperrier, K; Utikal, J; Velten, F W; Goerdt, S

    2004-09-01

    Interleukin-17E (IL-17E) (IL-25) is a recently identified cytokine capable to induce Th2-associated cytokine production (IL-5 and IL-13) and T helper 2 (Th2)-type pathologies in animal models. The IL-17E-responsive cell population in vivo was described to be a further uncharacterized non-T-, non-B-splenic accessory cell. Despite the identification of IL-17BR as the receptor for IL-17E, the cell population expressing IL-17BR has hitherto not been identified. Here, we show that human monocyte-derived Th2-skewed antigen-presenting cells (APC2) express membrane-bound and soluble forms of IL-17BR on the mRNA and protein level upon stimulation with IL-4, IL-10, IL-13 or transforming growth factor-betain vitro. These results indicate that IL-17BR-expressing APC2s may mediate the development of the IL-17E-mediated immunological reaction patterns observed in vivo. PMID:15320879

  7. 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

  8. P2X7 receptor activation impairs exogenous MHC class I oligopeptides presentation in antigen presenting cells.

    PubMed

    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

  9. Inhibition of Intracellular Transport of B Cell Antigen Receptor Complexes by Kaposi's Sarcoma–Associated Herpesvirus K1

    PubMed Central

    Lee, Bok-Soo; Alvarez, Xavier; Ishido, Satoshi; Lackner, Andrew A.; Jung, Jae U.

    2000-01-01

    The B cell antigen receptor (BCR) is a large complex that consists of a disulfide-linked tetramer of two transmembrane heavy (μ) chains and two light (λ or κ) chains in association with a heterodimer of Igα and Igβ. Kaposi's sarcoma–associated herpesvirus (KSHV) encodes a transforming protein called K1, which has structural and functional similarity to Igα and Igβ. We demonstrate that K1 downregulates the expression of BCR complexes on the surface. The NH2-terminal region of K1 specifically interacts with the μ chains of BCR complexes, and this interaction retains BCR complexes in the endoplasmic reticulum, preventing their intracellular transport to the cell surface. Thus, KSHV K1 resembles Igα and Igβ in its ability to induce signaling and to interact with μ chains of the BCR. However, unlike Igα and Igβ, which interact with μ chains to direct BCR complexes to the cell surface, K1 interacts with μ chains to block the intracellular transport of BCR complexes to the cell surface. These results demonstrate a unique feature of the K1 transforming protein, which may confer virus-infected cells with a long-term survival advantage. PMID:10880522

  10. Quantitative Time-Resolved Fluorescence Imaging of Androgen Receptor and Prostate-Specific Antigen in Prostate Tissue Sections.

    PubMed

    Krzyzanowska, Agnieszka; Lippolis, Giuseppe; Helczynski, Leszek; Anand, Aseem; Peltola, Mari; Pettersson, Kim; Lilja, Hans; Bjartell, Anders

    2016-05-01

    Androgen receptor (AR) and prostate-specific antigen (PSA) are expressed in the prostate and are involved in prostate cancer (PCa). The aim of this study was to develop reliable protocols for reproducible quantification of AR and PSA in benign and malignant prostate tissue using time-resolved fluorescence (TRF) imaging techniques. AR and PSA were detected with TRF in tissue microarrays from 91 PCa patients. p63/ alpha-methylacyl-CoA racemase (AMACR) staining on consecutive sections was used to categorize tissue areas as benign or cancerous. Automated image analysis was used to quantify staining intensity. AR intensity was significantly higher in AMACR+ and lower in AMACR- cancer areas as compared with benign epithelium. The PSA intensity was significantly lower in cancer areas, particularly in AMACR- glands. The AR/PSA ratio varied significantly in the AMACR+ tumor cells as compared with benign glands. There was a trend of more rapid disease progression in patients with higher AR/PSA ratios in the AMACR- areas. This study demonstrates the feasibility of developing reproducible protocols for TRF imaging and automated image analysis to study the expression of AR and PSA in benign and malignant prostate. It also highlighted the differences in AR and PSA protein expression within AMACR- and AMACR+ cancer regions. PMID:27026295

  11. Variants in the vitamin D receptor gene and asthma

    PubMed Central

    Wjst, Matthias

    2005-01-01

    Background Early lifetime exposure to dietary or supplementary vitamin D has been predicted to be a risk factor for later allergy. Twin studies suggest that response to vitamin D exposure might be influenced by genetic factors. As these effects are primarily mediated through the vitamin D receptor (VDR), single base variants in this gene may be risk factors for asthma or allergy. Results 951 individuals from 224 pedigrees with at least 2 asthmatic children were analyzed for 13 SNPs in the VDR. There was no preferential transmission to children with asthma. In their unaffected sibs, however, one allele in the 5' region was 0.5-fold undertransmitted (p = 0.049), while two other alleles in the 3' terminal region were 2-fold over-transmitted (p = 0.013 and 0.018). An association was also seen with bronchial hyperreactivity against methacholine and with specific immunoglobulin E serum levels. Conclusion The transmission disequilibrium in unaffected sibs of otherwise multiple-affected families seem to be a powerful statistical test. A preferential transmission of vitamin D receptor variants to children with asthma could not be confirmed but raises the possibility of a protective effect for unaffected children. PMID:15651992

  12. Progesterone receptor gene variants and risk of endometrial cancer

    PubMed Central

    O'Mara, Tracy A.; Fahey, Paul; Ferguson, Kaltin; Marquart, Louise; Lambrechts, Diether; Despierre, Evelyn; Vergote, Ignace; Amant, Frederic; Hall, Per; Liu, Jianjun; Czene, Kamila; Rebbeck, Timothy R.; Ahmed, Shahana; Dunning, Alison M.; Gregory, Catherine S.; Shah, Mitul; Webb, Penelope M.; Spurdle, Amanda B.

    2011-01-01

    Prolonged excessive estrogen exposure unopposed by progesterone is widely accepted to be a risk factor for endometrial cancer development. The physiological function of progesterone is dependent upon the presence of its receptor [progesterone receptor (PGR)] and several studies have reported single nucleotide polymorphisms (SNPs) in the PGR gene to be associated with endometrial cancer risk. We sought to confirm the associations with endometrial cancer risk previously reported for four different PGR polymorphisms. A maximum of 2888 endometrial cancer cases and 4483 female control subjects from up to three studies were genotyped for four PGR polymorphisms (rs1042838, rs10895068, rs11224561 and rs471767). Logistic regression with adjustment for age, study, ethnicity and body mass index was performed to calculate odds ratios (ORs) and associated 95% confidence intervals (CIs) and P-values. Of the four SNPs investigated, only rs11224561 in the 3′ region of the PGR gene was found to be significantly associated with endometrial cancer risk. The A allele of the rs11224561 SNP was associated with increased risk of endometrial cancer (OR per allele 1.31; 95% CI 1.12–1.53, P = 0.001, adjusted for age and study), an effect of the same magnitude and direction as reported previously. We have validated the endometrial cancer risk association with a tagSNP in the 3′ untranslated region of PGR previously reported in an Asian population. Replication studies will be required to refine the risk estimate and to establish if this, or a correlated SNP, is the underlying causative variant. PMID:21148628

  13. An Expression Refinement Process Ensures Singular Odorant Receptor Gene Choice.

    PubMed

    Abdus-Saboor, Ishmail; Al Nufal, Mohammed J; Agha, Maha V; Ruinart de Brimont, Marion; Fleischmann, Alexander; Shykind, Benjamin M

    2016-04-25

    Odorant receptor (OR) gene choice in mammals is a paradigmatic example of monogenic and monoallelic transcriptional selection, in which each olfactory sensory neuron (OSN) chooses to express one OR allele from over 1,000 encoded in the genome [1-3]. This process, critical for generation of the circuit from nose to brain [4-6], is thought to occur in two steps: a slow initial phase that randomly activates a single OR allele, followed by a rapid feedback that halts subsequent expression [7-14]. Inherent in this model is a finite failure rate wherein multiple OR alleles may be activated prior to feedback suppression [15, 16]. Confronted with more than one receptor, the neuron would need to activate a refinement mechanism to eliminate multigenic OR expression and resolve unique neuronal identity [16], critical to the generation of the circuit from nose to olfactory bulb. Here we used a genetic approach in mice to reveal a new facet of OR regulation that corrects adventitious activation of multiple OR alleles, restoring monogenic OR expression and unique neuronal identity. Using the tetM71tg model system, in which the M71 OR is expressed in >95% of mature OSNs and potently suppresses the expression of the endogenous OR repertoire [10], we provide clear evidence of a post-selection refinement (PSR) process that winnows down the number of ORs. We further demonstrate that PSR efficiency is linked to OR expression level, suggesting an underlying competitive process and shedding light on OR gene switching and the fundamental mechanism of singular OR choice. PMID:27040780

  14. Melanoma risk is associated with vitamin D receptor gene polymorphisms.

    PubMed

    Zeljic, Katarina; Kandolf-Sekulovic, Lidija; Supic, Gordana; Pejovic, Janko; Novakovic, Marijan; Mijuskovic, Zeljko; Magic, Zvonko

    2014-06-01

    Previous studies have reported that vitamin D receptor (VDR) gene polymorphisms are associated with the occurrence of various cancers, including melanoma. The aim of the current study was to investigate the association of VDR gene polymorphisms with melanoma risk, clinicopathological characteristics, and vitamin D levels. The study group included 117 patients (84 patients with superficial spreading melanoma and 33 patients with nodular melanoma). The control group included 122 sex-matched and age-matched healthy-blood donors of the same ethnicity. VDR gene polymorphisms FokI, EcoRV, TaqI, and ApaI were genotyped by real-time PCR. In 60 patients, the total 25-hydroxyvitamin D levels were evaluated in serum samples by direct chemiluminescence. Associations among parameters were considered to be significant if the P value was less than 0.05. Significant differences in the frequencies of VDR genotypes were observed between cases and the control group for FokI and TaqI polymorphisms (P<0.0001; P=0.005, respectively). Heterozygous Ff as well as mutant FF genotypes of the FokI polymorphism were associated with increased melanoma risk compared with the wild-type form [odds ratio (OR)=3.035, P=0.003; OR=9.276, P<0.0001, respectively]. A significantly increased melanoma risk was observed for the heterozygous Tt (OR=2.302, P=0.011) and the mutated variant tt (OR=3.697, P=0.003) of the TaqI polymorphism in comparison with the wild-type genotype. None of the polymorphisms studied was associated with clinicopathological characteristics and vitamin D serum level. Our results suggest that FokI and TaqI polymorphisms in the VDR gene may be considered as potential biomarkers for melanoma susceptibility. Low vitamin D levels in melanoma patients indicate the need for vitamin D supplementation. PMID:24638155

  15. Association between the vitamin D receptor gene polymorphism and osteoporosis

    PubMed Central

    Wu, Ju; Shang, De-Peng; Yang, Sheng; Fu, Da-Peng; Ling, Hao-Yi; Hou, Shuang-Shuang; Lu, Jian-Min

    2016-01-01

    The influence of the vitamin D receptor (VDR) gene for the risk of osteoporosis remains to be elucidated. The aim of the present study was to understand the distribution of various single-nucleotide polymorphisms (SNPs) within the VDR gene and its association with the risk of osteoporosis. In total, 378 subjects without a genetic relationship were recruited to the study between January 2013 and July 2015. The subjects were divided into three groups, which were the normal (n=234), osteoporosis (n=65) and osteoporosis with osteoporotic fracture (n=79) groups. Three pertinent SNPs of the VDR gene rs17879735 (ApaI, Allele A/a, SNP C>A) were examined with polymerase chain reaction-restriction fragment length polymorphism. The bone mineral density (BMD) of the lumbar spine (L2-L4), femoral neck, Ward's and Tro was measured using dual-energy X-ray absorptiometry. The distributions of genotype frequencies aa, AA and Aa were 48.68, 42.86 and 8.46%, separately. Following analysis of each site, BMD, body mass index (BMI) and age, BMD for each site was negatively correlated with age (P<0.01) and positively correlated with BMI (P<0.01). Correction analysis revealed that there were significant differences in the Ward's triangle BMD among each genotype (P<0.05), in which the aa genotype exhibited the lower BMD (P<0.05). No significant difference was identified among the different genotypes in the occurrence of osteoporosis with osteoporotic fracture (P>0.05). In conclusion, these indicated that the VDR gene ApaI polymorphisms had an important role in the osteoporosis risk. PMID:27446548

  16. 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

  17. Thymic expression of a T-cell receptor targeting a tumor-associated antigen coexpressed in the thymus induces T-ALL

    PubMed Central

    Cui, Yongzhi; Onozawa, Masahiro; Garber, Haven R.; Samsel, Leigh; Wang, Ziyao; McCoy, J. Philip; Burkett, Sandra; Wu, Xiaolin; Aplan, Peter D.

    2015-01-01

    T-cell receptors (TCRs) and chimeric antigen receptors recognizing tumor-associated antigens (TAAs) can now be engineered to be expressed on a wide array of immune effectors. Engineered receptors targeting TAAs have most commonly been expressed on mature T cells, however, some have postulated that receptor expression on immune progenitors could yield T cells with enhanced potency. We generated mice (survivin-TCR-transgenic [Sur-TCR-Tg]) expressing a TCR recognizing the immunodominant epitope (Sur20-28) of murine survivin during early stages of thymopoiesis. Spontaneous T-cell acute lymphoblastic leukemia (T-ALL) occurred in 100% of Sur-TCR-Tg mice derived from 3 separate founders. The leukemias expressed the Sur-TCR and signaled in response to the Sur20-28 peptide. In preleukemic mice, we observed increased cycling of double-negative thymocytes expressing the Sur-TCR and increased nuclear translocation of nuclear factor of activated T cells, consistent with TCR signaling induced by survivin expression in the murine thymus. β2M−/− Sur-TCR-Tg mice, which cannot effectively present survivin peptides on class I major histocompatibility complex, had significantly diminished rates of leukemia. We conclude that TCR signaling during the early stages of thymopoiesis mediates an oncogenic signal, and therefore expression of signaling receptors on developing thymocytes with specificity for TAAs expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis. PMID:25814528

  18. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

    PubMed

    Beringer, Dennis X; Kleijwegt, Fleur S; Wiede, Florian; van der Slik, Arno R; Loh, Khai Lee; Petersen, Jan; Dudek, Nadine L; Duinkerken, Gaby; Laban, Sandra; Joosten, Antoinette; Vivian, Julian P; Chen, Zhenjun; Uldrich, Adam P; Godfrey, Dale I; McCluskey, James; Price, David A; Radford, Kristen J; Purcell, Anthony W; Nikolic, Tatjana; Reid, Hugh H; Tiganis, Tony; Roep, Bart O; Rossjohn, Jamie

    2015-11-01

    Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition. PMID:26437244

  19. Fibronectin and asialoglyprotein receptor mediate hepatitis B surface antigen binding to the cell surface.

    PubMed

    Yang, Jing; Wang, Feng; Tian, Linlin; Su, Jing; Zhu, Xiangqian; Lin, Li; Ding, Xiaoran; Wang, Xuejun; Wang, Shengqi

    2010-06-01

    Both fibronectin and the asialoglycoprotein receptor (ASGPR) have been identified by some investigators as partners for hepatitis B virus (HBV) envelope proteins. Because fibronectin is a natural ligand for ASGPR, we speculated that HBV might attach to ASGPR expressed on the hepatocyte surface via fibronectin. To test this hypothesis, we first confirmed by co-immunoprecipitation that ASGPR, fibronectin and HBsAg bind to each other in HepG2.2.15 cells, and possible binding domains were identified by GST pull-down. In addition, by measuring binding of HBsAg to cells, we found that ASGPR and fibronectin enhanced the binding capability of HBsAg to HepG2 cells, and even to 293T and CHO cells, which normally do not bind HBV. In conclusion, our findings suggest that both fibronectin and ASGPR mediate HBsAg binding to the cell surface, which provides further evidence for the potential roles of these two proteins in mediating HBV binding to liver cells. PMID:20364278

  20. Trypanosoma cruzi and Its Soluble Antigens Induce NET Release by Stimulating Toll-Like Receptors

    PubMed Central

    Diniz, Larissa Figueiredo Alves; Souza, Priscila Silva Sampaio; Pinge-Filho, Phileno; Toledo, Karina Alves

    2015-01-01

    Neutrophils release fibrous traps of DNA, histones, and granule proteins known as neutrophil extracellular traps (NETs), which contribute to microbicidal killing and have been implicated in autoimmunity. The role of NET formation in the host response to nonbacterial pathogens is not well-understood. In this study, we investigated the release of NETs by human neutrophils upon their interaction with Trypanosoma cruzi (Y strain) parasites. Our results showed that human neutrophils stimulated by T. cruzi generate NETs composed of DNA, histones, and elastase. The release occurred in a dose-, time-, and reactive oxygen species-dependent manner to decrease trypomastigote and increase amastigote numbers of the parasites without affecting their viability. NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4. In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested. Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi. We suggest that contact of T. cruzi with NETs during Chagas’s disease can limit infection by affecting the infectivity/pathogenicity of the parasite. PMID:26431537

  1. Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis.

    PubMed

    Niarchos, Athanasios; Zouridakis, Marios; Douris, Vassilis; Georgostathi, Assimina; Kalamida, Dimitra; Sotiriadis, Alexandros; Poulas, Konstantinos; Iatrou, Kostas; Tzartos, Socrates J

    2013-01-01

    We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies. PMID:24376846

  2. Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein.

    PubMed Central

    Compton, S R; Stephensen, C B; Snyder, S W; Weismiller, D G; Holmes, K V

    1992-01-01

    Like most coronaviruses, the coronavirus mouse hepatitis virus (MHV) exhibits strong species specificity, causing natural infection only in mice. MHV-A59 virions use as a receptor a 110- to 120-kDa glycoprotein (MHVR) in the carcinoembryonic antigen (CEA) family of glycoproteins (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G. S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; and R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). The role of virus-receptor interactions in determining the species specificity of MHV-A59 was examined by comparing the binding of virus and antireceptor antibodies to cell lines and intestinal brush border membranes (BBM) from many species. Polyclonal antireceptor antiserum (anti-MHVR) raised by immunization of SJL/J mice with BALB/c BBM recognized MHVR specifically in immunoblots of BALB/c BBM but not in BBM from adult SJL/J mice that are resistant to infection with MHV-A59, indicating a major difference in epitopes between MHVR and its SJL/J homolog which does not bind MHV (7). Anti-MHVR bound to plasma membranes of MHV-susceptible murine cell lines but not to membranes of human, cat, dog, monkey, or hamster cell lines. Cell lines from these species were resistant to MHV-A59 infection, and only the murine cell lines tested were susceptible. Pretreatment of murine fibroblasts with anti-MHVR prevented binding of radiolabeled virions to murine cells and prevented virus infection. Solid-phase virus-binding assays and virus overlay protein blot assays showed that MHV-A59 virions bound to MHVR on intestinal BBM from MHV-susceptible mouse strains but not to proteins on intestinal BBM from humans, cats, dogs, pigs, cows, rabbits, rats, cotton rats, or chickens. In immunoblots of BBM from these species, both polyclonal and monoclonal antireceptor antibodies that block MHV-A59 infection of murine cells recognized only the murine CEA-related glycoprotein

  3. Liver X Receptor Gene Polymorphisms in Tuberculosis: Effect on Susceptibility

    PubMed Central

    Liu, Li-rong; Yue, Jun; Zhao, Yan-lin; Xiao, He-ping

    2014-01-01

    Objectives The Liver X receptors (LXRs), Liver X receptor A (LXRA) and Liver X receptor B (LXRB), regulate lipid metabolism and antimicrobial response. LXRs have a crucial role in the control of Mycobacterium tuberculosis (M.tb). Lacking LXRs mice is more susceptibility to infection M.tb, developing higher bacterial burdens and an increase in the size and number of granulomatous lesions. We aimed to assess the associations between single nucleotide polymorphisms (SNPs) in LXRs and risk of tuberculosis. Methods We sequenced the LXRs genes to detect SNPs and to examine genotypic frequencies in 600 patients and 620 healthy controls to investigate for associations with tuberculosis (TB) in the Chinese Han population. DNA re-sequencing revealed eight common variants in the LXRs genes. Results The G allele of rs1449627 and the T allele of rs1405655 demonstrated an increased risk of developing TB (p<0.001, p = 0.002), and the T allele of rs3758673, the T allele of rs2279238, and the C allele of rs1449626 in LXRA and the C allele of rs17373080, the G allele of rs2248949, and the C allele of rs1052677 in LXRB were protective against TB patients compared to healthy controls (p = 0.0002, p = 0.006, p<0.001, p = 0.004, p = 0.008, p = 0.003, respectively). All SNP genotypes were significantly associated with TB. An estimation of the frequencies of haplotypes revealed two potential risk haplotypes,GGCG in LXRB (p = 0.004,) and TTCG in LXRA (p<0.001, p = 0.004). Moreover, three protective haplotypes, TTAT and CCAT in LXRA and CATC in LXRB, were significantly “protective” (p = 0.008, p<0.001, p = 0.031) for TB. Furthermore, we determined that the LXRs SNPs were nominally associated with the clinical pattern of disease. Conclusions Our study data supported that LXRs play a fundamental role in the genetic susceptibility to TB and to different clinical patterns of disease. Thus, further investigation is required in larger populations and in

  4. Modulation of Cellular and Viral Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

    PubMed Central

    Renne, Rolf; Barry, Chris; Dittmer, Dirk; Compitello, Nicole; Brown, Patrick O.; Ganem, Don

    2001-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)—and from NF-κB-dependent reporter genes—was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection. PMID:11119614

  5. Expression of the human ABCC6 gene is induced by retinoids through the retinoid X receptor

    SciTech Connect

    Ratajewski, Marcin; Bartosz, Grzegorz; Pulaski, Lukasz . E-mail: lpulaski@cbm.pan.pl

    2006-12-01

    Mutations in the human ABCC6 gene are responsible for the disease pseudoxanthoma elasticum, although Physiological function or substrate of the gene product (an ABC transporter known also as MRP6) is not known. We found that the expression of this gene in cells of hepatic origin (where this gene is predominantly expressed in the body) is significantly upregulated by retinoids, acting as agonists of the retinoid X receptor (RXR) rather than the retinoid A receptor (RAR). The direct involvement of this nuclear receptor in the transcriptional regulation of ABCC6 gene expression was confirmed by transient transfection and chromatin immunoprecipitation assays. This constitutes the first direct proof of previously suggested involvement of nuclear hormone receptors in ABCC6 gene expression and the first identification of a transcription factor which may be relevant to regulation of ABCC6 level in tissues and in some PXE patients.

  6. Leptin receptor gene polymorphisms in severely pre-eclamptic women.

    PubMed

    Rigó, János; Szendei, György; Rosta, Klára; Fekete, Andrea; Bögi, Krisztina; Molvarec, Attila; Rónai, Zsolt; Vér, Agota

    2006-09-01

    Variants of the leptin receptor gene (LEPR) may modulate the effect of elevated serum leptin levels in pre-eclampsia. The aim of our study was to evaluate the LEPR gene polymorphisms Lys109Arg (A109G) and Gln223Arg (A223G) in severely pre-eclamptic women. In a case-control study, we analyzed blood samples from 124 severely pre-eclamptic patients and 107 healthy control women by the polymerase chain reaction-restriction fragment length polymorphism method. The P