DOCSLIB.ORG

Precursor B Cells Transformed by Epstein-Barr Virus Undergo

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Precursor B Cells Transformed by Epstein-Barr Virus Undergo Proc. Nati. Acad. Sci. USA Vol. 85, pp. 875-879, February 1988 Immunology Precursor B cells transformed by Epstein-Barr virus undergo sterile plasma-cell differentiation: J-chain expression without immunoglobulin (hnmunoglobulin gene rearrangement/transormation/X chromosome-linked agammaglobulinemia) HIROMI KUBAGAWA*t, PETER D. BURROWSt, CARLO E. GRossI*, JIRI MESTECKYt, AND MAX D. COOPERO§ Departments of *Pathology, tMicrobiology and §Pediatrics, The Cellular Immunobiology Unit of the Tumor Institute, The Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294 Communicated by Marian E. Koshland, October 12, 1987 ABSTRACT Human bone marrow cells were depleted of polymeric immunoglobulin, is increased as a function of B lymphocytes to enrich for precursor B cells that could be plasma-cell maturation (4, 5). transformed with Epstein-Barr virus. Transformed immuno- The molecular details of this scheme of mammalian B-cell globulin-negative precursors either maintained their immuno- development have been deduced largely from studies of globulin genes in the germ-line configuration or had under- transformed mouse cell lines. To analyze changes in geno- gone DJ or abortive VDJ rearrangements (V, D, and J type and phenotype that occur during B-lineage develop- represent variable, diversity, and joining gene segments). All ment in humans, we have used Epstein-Barr virus (EBV) to cell lines and their derivative clones, even those with no rescue clones of early B-lineage cells. detectable immunoglobulin gene rearrangements, generated subpopulations of cells that produced high levels ofjoining (J) MATERIALS AND METHODS chain when analyzed by immunoprecipitation after biosyn- thetic labeling and by blot hybridization of cytoplasmic RNA. EBV Transformation of Early Pre-B Cells. Bone marrow Morphologic and immunofluorescence analyses revealed that specimens were obtained with parental consent from five J-chain production was confined to clonal progeny that had boys (ages 2-11 years) with X chromosome-linked agamma- exited the cell cycle to undergo plasma-cell differentiation. globulinemia (XLA) and from four fetuses (14-16 weeks of Analysis of cell surface antigens revealed expression of several gestation) aborted for reasons of maternal health. Mononu- B-cell maturational markers, including complement receptor clear cells from fetal marrow samples were depleted of sIg+ ype 2 (CR2) and plasma cell antigen 1 (PCA-1). Epstein-Barr cells by fluorescence-activated cell sorting (FACS) or by virus can thus transform B-cell progenitors, allowing them to "panning" over anti-Ig-coated plastic dishes (6, 7). The proliferate and undergo terminal B-cell differentiation coupled frequencies of pre-B and B cells were 5.2% and 4.0% before with J-chain expression. These events appear to occur inde- sorting and 7.0% and <0.1% after sorting; corresponding pendently of the immunoglobulin gene status of the trans- values were 8.0%o and 3.8% before panning and 8.3% and formed cells. 0.3% after panning. Wells containing p. + pre-B cells were infected with EBV produced by the B95.8 marmoset cell line During differentiation along the B-cell pathway, a lymphoid (8, 9). EBV-transformed cells were subcloned by limiting stem cell undergoes a series of somatic rearrangements of dilution (9). germ-line immunoglobulin gene segments to generate func- Immunofluorescence Analysis of Cells. Fixed smears of tional immunoglobulin heavy (H)- and light (L)-chain genes. EBV-transformed cells were examined for J-chain expres- The initial rearrangement brings together diversity (D) and sion by two-color immunofluorescence (10) using polyclonal joining (J) gene segments in the H-chain locus on both antibodies (11) and monoclonal antibodies (mAb). mAb chromosomes, and this is followed by joining of a variable NF-11 [IgGl(K) isotype], made against a V,, preparation of (V) gene segment to DJ. VL to JL joining may take place an IgM(A) human myeloma (12) and subsequently shown by subsequently in the L-chain loci (1). immunoprecipitation analysis to have J-chain reactivity These initial gene rearrangements to generate H- and (data not shown), was used with rhodamine-labeled goat L-chain genes occur in bone marrow pre-B cells and result in antibodies specific for mouse immunoglobulin (Southern the emergence of the primary antigen-reactive cells of the B Biotechnology Associates, Birmingham, AL) to detect J- lineage, the surface-IgM-positive (sIgM+) B lymphocytes, chain expression. Cells were counterstained with fluoresce- which are then seeded to the peripheral lymphoid tissues (2). in-labeled goat antibodies specific for human immunoglobu- These cells are small and have a high nucleus/cytoplasm lin (10). Viable cells were similarly stained for surface ratio, and their scant cytoplasm is nearly devoid of organ- antigens and analyzed with a FACS IV (Becton Dickinson). elles. Stimulation with antigen can result in their activation Electron Microscopic Analysis. Cells fixed with 1.25% and differentiation into plasma cells with abundant cyto- glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5) were plasm containing a well-developed Golgi apparatus and washed, postfixed with 1% osmium tetraoxide, dehydrated rough endoplasmic reticulum. This morphological matura- with ethanol, and embedded in Spurr medium (13). Ultrathin tion is paralleled by a shift from production of the membrane form of immunoglobulin to the secreted form (3). Large Abbreviations: H, heavy; L, light; V, variable gene segment; D, amounts of immunoglobulin are then secreted by the termi- diversity gene segment; J, joining gene segment; C, constant gene nally differentiated plasma cells. The expression of joining segment; Am and A., H chains of membrane and secreted forms of (J) chain, a 15-kDa protein involved in the assembly of IgM, respectively; sIg, surface immunoglobulin; EBV, Epstein- Barr virus; XLA, X chromosome-linked agammaglobulinemia; mAb, monoclonal antibody(ies). The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: University of payment. This article must therefore be hereby marked "advertisement" Alabama at Birmingham, T263 Tumor Institute, University Station, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Birmingham, AL 35294. 875 Downloaded by guest on September 27, 2021 876 Immunology: Kubagawa et al. Proc. Natl. Acad. Sci. USA 85 (1988) sections stained with uranyl acetate/lead citrate were exam- limited subpopulation (8-22%) of cells within each of the ined with a Philips EM 201 electron microscope. clones expressed cytoplasmic J chains (Table 1). Biosynthetic Analysis of Immunoglobulln and J-Chain Mol- The morphology of the cells in these lines and subclones ecules. Cells (5-10 x 106) were cultured for 8 hr at 370C in 1 differed from that characteristic of normal or leukemic pre-B ml of methionine- and cysteine-free RPMI 1640 medium cells; a spectrum of cell types ranging from lymphoblastoid containing 10%o dialyzed fetal bovine serum, [35S]methionine to plasmacytoid morphology was seen in each clone. J (100 MCi), and [35S]cysteine (100 MCi; 1 1ACi = 37 kBq). For chains were expressed strongly in the cytoplasm ofcells with inhibition of N-linked glycosylation, cells were incubated plasmacytoid morphology but weakly, if at all, by cells with with tunicamycin (2.5 ,ug/ml) for 1.5 hr at 370C, washed, and lymphoblastoid morphology within the clone (Fig. 1). More- resuspended in medium containing [35S]methionine and over, in two cell lines examined by electron microscopy and [35S]cysteine for an additional 8 hr in the presence of immunofluorescence, the proportion of J+ cells correlated tunicamycin. Radiolabeled cells were solubilized with 1% closely (± 3%) with the proportion of cells having fully Nonidet P-40 in 0.15 M NaCl/50 mM Tris HCl buffer (pH developed rough endoplasmic reticulum characteristic of 7.4) containing 20 mM E-aminocaproic acid, 0.01% soybean cells with plasmacytoid features (Fig. 1). Cells within these trypsin inhibitor, 20 mM iodoacetamide, and 1 mM phenyl- null cell clones also expressed cell-surface differentiation methylsulfonyl fluoride. Solid-phase immunoisolation was antigens that are characteristically displayed by mature B performed in microtiter wells coated with a rat mAb to cells (e.g., HB2, ref. 21; CR2/EBV receptor, ref. 6; and mouse K chain and then with mouse mAbs to human J-chain HB7, ref. 22), activated B cells (BAC-1, ref. 23), or plasma or immunoglobulin-isotypic determinants (10). Antibody- cells (PCA-1, ref. 24) (Table 1). They also expressed HLA- bound molecules were dissociated by incubation with 2% DR determinants but did not express CALLA, interleukin-2 NaDodSO4/5% 2-mercaptoethanol for 30 min at 37°C, re- receptors, or T-cell antigens (CD1, CD3) in detectable solved by NaDodSO4/PAGE, and autoradiographed. amounts. DNA Blot Analysis. High molecular weight DNA from cell In contrast, J chain was not demonstrable by immunofluo- clones, placenta, and tonsillar T and B cells (12) was rescence in the spontaneously transformed pre-B leukemia digested to completion with restriction endonucleases, elec- cell lines 697 and 207, which are morphologically similar to trophoresed in 0.7% agarose, and transferred to Hybond normal pre-B cells (25), or in several established lines of nylon membranes (Amersham). The membranes were hy- T-cells (MOLT4, MOLT-3, HSB-2), myelomonocytic (HL- bridized at 65°C for 15 hr with nick-translated 32P-labeled 60, U937), or erythroid (K562) lineage. DNA probes: a 3.9-kilobase (kb) Bgl II JH probe (14, 15), a J-Chain Biosynthesis by Null Cell Clones. To examine the 1.3-kb EcoRI ,u constant-region (C,.) probe (14), a 2.0-kb Sac molecular nature of J chains produced by EBV-transformed I JK probe (16), a 3.6-kb EcoRI-HindIII CA2 (Ke-Oz-) null cells, the cells were metabolically labeled and analyzed probe (17), a 3.15-kb BamHI DH probe (a kind gift of S. by immunoprecipitation with a mAb to J chain.
Load more

Recommended publications
  • Cell Surface Markers in Acute Lymphoblastic Leukemia* F
    ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 10, No. 3 Copyright © 1980, Institute for Clinical Science, Inc. Cell Surface Markers in Acute Lymphoblastic Leukemia* f G. BENNETT HUMPHREY, M.D., REBECCA BLACKSTOCK, Ph .D., AND JANICE FILLER, M.S. University of Oklahoma, Health Sciences Center, Oklahoma City, OK 73126 ABSTRACT During the last nine years, two important methodologies have been used to characterize the cell surfaces of normal lymphocytes and malignant lym­ phoblasts. Normal mature T-cells have a receptor for sheep erythrocytes (E+) while mature B-cells bear membrane-bound immunoglobulin molecules (slg+). These two findings can be used to divide acute lymphoblastic leukemia of childhood into three major groups; B-cell leukemia (slg+ E -), which is rare (approximately 2 percent) and has the poorest prognosis, T-cell leukemia (slg~, E +) which is more common (10 percent) but also has a poor prognosis and null cell leukemia (slg~, E~) which is the most common (85 percent) and has the best prognosis. By the use of additional immunological methods, subgroups within T-cell leukemia and null cell leukemia have also been proposed. One of the most valuable of these additional methods is the detection of surface antigens. Three of the more commonly detected antigens currently being evaluated are (1) common leukemia antigen (cALL), (2) a normal B Lymphocyte antigen the la antigen (la) which is not generally expressed on most T lympho­ cytes and (3) a normal T lymphocyte antigen (T) not expressed on B lympho­ cytes. Within null cell leukemia, the most commonly identified and proba­ bly the largest subgroup is Ia+, cALL+, T”, E _, slg-.
    [Show full text]
  • Separation and Functional Studies of the Human Lymphokine-Activated Killer Cell Kevan Roberts, Michael T
    [CANCER RESEARCH 47, 4366-4371, August 15, 1987] Separation and Functional Studies of the Human Lymphokine-activated Killer Cell Kevan Roberts, Michael T. Lotze,1 and Steven A. Rosenberg Surgery Branch, National Cancer Institute, NÃŒH,Betnesda,Maryland 20892 ABSTRACT undefined. The purification of cells responsible for LAK activity was desirable to further assess their morphology, phenotype, Cell separation studies were undertaken in an attempt to purify the and target cell binding properties prior to and following IL-2 lymphokine-activated killer (LAK) precursor cell. Null cells, prepared stimulation. by the sequential depletion of monocytes, T- and B-lymphocytes from human peripheral blood mononuclear cells, were found to be potent mediators of LAK activity. Such preparations were Leu-11* but Leu-4" MATERIALS AND METHODS and displayed high levels of natural killer activity. Incubation of these cells with recombinant interleukin 2 (11-2) for periods in excess of 24 h Media. All cell lines were maintained in suspension culture using RPMI 1640 supplemented with 10% FBS, 100 ng/m\ penicillin, 100 induced LAK lysis of fresh tumor targets which were resistant to lysis Mg/ml streptomycin, and 1% glutamine. CM consisted of RPMI 1640 by unstimulated null effectors. In contrast, lymphocytes which formed high affinity rosettes with sheep RBC (I-* lymphocytes) were poor supplemented with 10% human AB serum, antibiotics, and glutamine. Conjugation buffer, at pH 7.3, was prepared by adding 683 mg of NaCl, mediators of both natural killer and LAK activity. Interleukin 2 stimu lated null cells, retained a Leu-ll*, I.t-u-4 phenotype, and expressed 142 mg of MgCl2 6H2O, and 66 mg of CaCl2-6H2O to 450 ml of PBS supplemented with 50 ml of FBS, antibiotics, and 10 HIM4-(2-hydrox- only low levels of receptors for II,-2 and transferrin.
    [Show full text]
  • Genotyping in Patients with Acute Ischemic Stroke
    Tuttolomondo et al. Journal of Neuroinflammation (2019) 16:88 https://doi.org/10.1186/s12974-019-1469-5 RESEARCH Open Access HLA and killer cell immunoglobulin-like receptor (KIRs) genotyping in patients with acute ischemic stroke Antonino Tuttolomondo1*† , Domenico Di Raimondo1†, Rosaria Pecoraro6,7, Alessandra Casuccio2, Danilo Di Bona5, Anna Aiello3, Giulia Accardi3, Valentina Arnao4, Giuseppe Clemente1, Vittoriano Della Corte8, Carlo Maida1, Irene Simonetta1, Calogero Caruso3, Rosario Squatrito6, Antonio Pinto1 and on behalf of KIRIIND (KIR Infectious and Inflammatory Diseases) Collaborative Group Abstract Introduction: In humans, a major component of natural killer (NK) and T cell target recognition depends on the surveillance of human leukocyte antigen (HLA) class I molecules by killer immunoglobulin-like receptors (KIRs). Aims: To implement the knowledge about the immunological genetic background of acute ischemic stroke susceptibility in relation to the frequency of the KIR genes and HLA alleles. Methods: Subjects with acute ischemic stroke and subjects without stroke were genotyped for the presence of KIR genes and of the three major KIR ligand groups, HLA-C1, HLA-C2, and HLA-Bw4, both HLA-B and HLA-A loci. Results: Between November 2013 and February 2016, consecutive patients with acute ischemic stroke were recruited. As healthy controls, we enrolled subjects without acute ischemic stroke. Subjects with acute ischemic stroke in comparison with controls showed a higher frequency of 2DL3, 2DL5B, 2DS2, and 2DS4 KIR genes and a lower frequency of HLA-B-Bw4I alleles. Subjects without acute ischemic stroke showed a higher frequency of interaction between KIR 2DS2 and HLAC2. We also observed a higher frequency of 2DL3 and 2 DL4 KIR genes in subjects with atherosclerotic (LAAS) subtype.
    [Show full text]
  • Table of Contents (PDF)
    126 (2) J Immunol 1981; 126:393-810; ; http://www.jimmunol.org/content/126/2.citation This information is current as of October 1, 2021. Downloaded from Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists http://www.jimmunol.org/ • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: by guest on October 1, 2021 http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE JOURNAL OF IMMUNOLOGY Volume 126/Number 2 Contents CELLULAR IMMUNOLOGY K. Kudo, A. H. Sehon, and R. J. 403 The Role of Antigen-Presenting Cells in the IgE Antibody Response. I. The Induction Schwenk of High Titer IgE Antibody Responses in IgE High-Responder and Low-Responder Mice by the Administration of Antigen-Pulsed Macrophages in the Absence of Adjuvants D. A. Hubbard, W. Y. Lee, and A. 407 Suppression of the Anti-DNP IgE Response with Tolerogenic Conjugates of DNP H. Sehon with Polyvinyl Alcohol. I. Specific Suppression of the Anti-DNP IgE Response W. Y. Lee and A. H. Sehon 41 4 Suppression of the Anti-DNP IgE Response with Tolerogenic Conjugates of DNP with Polyvinyl Alcohol.
    [Show full text]
  • Expression of Ligands for Activating Natural Killer Cell Receptors on Cell
    Tremblay-McLean et al. BMC Immunology (2019) 20:8 https://doi.org/10.1186/s12865-018-0272-x RESEARCHARTICLE Open Access Expression of ligands for activating natural killer cell receptors on cell lines commonly used to assess natural killer cell function Alexandra Tremblay-McLean1,2, Sita Coenraads1, Zahra Kiani1,2, Franck P. Dupuy1 and Nicole F. Bernard1,2,3,4* Abstract Background: Natural killer cell responses to virally-infected or transformed cells depend on the integration of signals received through inhibitory and activating natural killer cell receptors. Human Leukocyte Antigen null cells are used in vitro to stimulate natural killer cell activation through missing-self mechanisms. On the other hand, CEM.NKr.CCR5 cells are used to stimulate natural killer cells in an antibody dependent manner since they are resistant to direct killing by natural killer cells. Both K562 and 721.221 cell lines lack surface major histocompatibility compatibility complex class Ia ligands for inhibitory natural killer cell receptors. Previous work comparing natural killer cell stimulation by K562 and 721.221 found that they stimulated different frequencies of natural killer cell functional subsets. We hypothesized that natural killer cell function following K562, 721.221 or CEM.NKr.CCR5 stimulation reflected differences in the expression of ligands for activating natural killer cell receptors. Results: K562 expressed a higher intensity of ligands for Natural Killer G2D and the Natural Cytotoxicity Receptors, which are implicated in triggering natural killer cell cytotoxicity. 721.221 cells expressed a greater number of ligands for activating natural killer cell receptors. 721.221 expressed cluster of differentiation 48, 80 and 86 with a higher mean fluorescence intensity than did K562.
    [Show full text]
  • Dissection of Distinct Human Immunoregulatory T-Cell Subsets by A
    Proc. Nati. Acad. Sci. USA Vol. 78, No. 5, pp. 3160-3164, May 1981 Immunology Dissection of distinct human immunoregulatory T-cell subsets by a monoclonal antibody recognizing a cell surface antigen with wide tissue distribution (T- and B-cell lines/T- and B-cell leukemias/helper and suppressor T cells) OSCAR H. IRIGOYEN*, PHILIP V. RIZZOLO*, YOLENE THOMAS*, MARTIN E. HEMLERt, HARRY H-L SHEN* STEVEN M. FRIEDMAN*, JACK L. STROMINGERt, AND LEONARD CHESS* *Department of Medicine, Division of Rheumatology, Columbia University, College of Physicians and Surgeons, New York, New York 10032; and tSidney Farber Cancer Institute, Boston, Massachusetts 02115 Contributed by Jack L. Strominger, December 31, 1980 ABSTRACT A monoclonal antibody, PVR-1, was obtained wide tissue distribution but is preferentially expressed on pre- after hybridization of X63Ag8.653 murine myeloma cells with cursors of helper T lymphocytes. spleen cells from a mouse immunized with human lymphocytes. It recognizes a 175,000- to 185,000-dalton surface antigen present on -"80% ofnormal human peripheral T lymphocytes, 50% ofnon- MATERIALS AND METHODS T non-B cells, and <10% ofB cells as determined by complement- dependent microcytotoxicity. It is also present on various leukemia Lymphocytes, Cell Lines, and Leukemic Cells. Fresh pe- T cells, on some but not all T lymphoblastoid cell lines, and on a ripheral blood mononuclear cells were isolated from healthy small fraction of some B lymphoblastoid cell lines. Some B-cell human volunteers by Ficoll/diatrizoate density gradient cen- chronic lymphocytic leukemia cells also express the PVR-11 anti- trifugation. Highly enriched populations of T and B cells were gen.
    [Show full text]
  • Separation of Functional Subsets of Human T Cells by a Monoclonal Antibody (Hybridoma/T Cell Antigens/Helper T Cells) ELLIS L
    Proc. NatI. Acad. Sci. USA Vol. 76, No. 8, pp. 4061-4065, August 1979 Immunology Separation of functional subsets of human T cells by a monoclonal antibody (hybridoma/T cell antigens/helper T cells) ELLIS L. REINHERZ*t, PATRICK C. KUNGt, GIDEON GOLDSTEIN1, AND STUART F. SCHLOSSMAN* *Division of Tumor Immunology, Sidney Farber Cancer Institute-Harvard Medical School, Boston, Massachusetts 02115; and tDivision of Immunosciences, Ortho Pharmaceutical Corporation, Raritan, New Jersey 08869 Communicated by Philip Levine, May 18, 1979 ABSTRACT A monoclonal antibody was produced to the equivalent of the murine Lyl inducer (helper) cell popu- human peripheral blood T cells. This hybridoma antibody, lation (1). termed OKT4, was reactive by indirect immunofluorescence with only 55-60% of the peripheral blood T cell population (OKT4+) and unreactive with normal B cells, null cells, and MATERIALS AND METHODS macrophages. The OKT4- T cell population contained the previously described TH2+ subset that has been shown to con- Production of Monoclonal Antibodies. (i) Immunization tain cytotoxic/suppressor cells. With cell-sorter separation of and somatic cell hybridization. An 8-week-old female CAF1 OKT4+ and OKT4- cells, it was shown that these T cell subsets mouse (Jackson Laboratory) was immunized intraperitoneally were functionally discrete. Both- gave proliferative responses with 2 X 107 purified human peripheral T cells in phosphate- with concanavalin A, alloantigens, and phytohemagglutinin, buffered saline at 14-day intervals. Four days after the third although OKT4+ cells were much more responsive to the latter. OKT4f cells alone responded to soluble antigens whereas immunization, the spleen was removed and a single-cell sus- OKT4- cells alone were cytotoxic after alloantigenic sensiti- pension was made.
    [Show full text]
  • Characterization of the Expression Profiles of Ligands to Activating Natural Killer Cell Receptors on the HLA-Null Cell Lines K5
    Characterization of the expression profiles of ligands to activating Natural Killer cell receptors on the HLA-null cell lines K562 and 721.221 and on HIV-1 infected or non-infected CD4+ T cells Alexandra Tremblay-McLean Department of Experimental Medicine McGill University A thesis submitted to the Faculty of Graduate Studies and Research In fulfilment of the requirements for the degree of Master of Science © Alexandra Tremblay-McLean February 2017 1 Characterization of the expression profiles of ligands to activating Natural 2 Killer cell receptors on the HLA-null cell lines K562 and 721.221 and on 3 HIV-1 infected or non-infected CD4+ T cells 4 Alexandra Tremblay-McLean 5 Abstract 6 Natural Killer (NK) cells direct anti-viral responses through a process dependent on the 7 integration of signals received from inhibitory and activating NK receptors (iNKR and aNKR). 8 NK cells can be activated by autologous HIV-infected CD4 T cells (iCD4) to inhibit HIV 9 replication. While iNKR and the downregulation of their HLA ligands on iCD4s have been 10 investigated, the contribution of aNKR and their ligands on iCD4 to NK cell activation and 11 subsequent anti-viral responses remain unclear. Additionally, previous work from our lab showed 12 that the HLA-null cell lines 721.221 (721) and K562 activate different frequencies and functional 13 subsets of NK cells. These cell lines do not express iNKR ligands, but their aNKR ligand profile 14 may differ in a manner that explains the how they activate NK cell differentially. In this thesis, I 15 will describe experiments that characterized the aNKR profile of iCD4 and HLA null cells to 16 improve our understanding of the way in which these cells activate NK cells.
    [Show full text]
  • Membrane Antigen on Epstein-Barr Virus-Infected Human B Cells Recognized by a Monoclonal Antibody (Hybridoma/Immunofluorescence/Cytotoxic T Cells) S
    Proc. Natd Acad. Sci. USA Vol. 79, pp. 2649-2653, April 1982 Immunology Membrane antigen on Epstein-Barr virus-infected human B cells recognized by a monoclonal antibody (hybridoma/immunofluorescence/cytotoxic T cells) S. F. SLOVIN*, D. M. FRISMANt, C. D. TSOUKAS*, I. ROYSTONt, S. M. BAIRDt, S. B. WORMSLEYt, D. A. CARSON*, AND J. H. VAUGHAN*t of Clinical Research, Scripps Clinic and Research Foundation, and tDepartments of Pathology and Medicine, University of California at San Diego, *DepajrtnentLa Jolla, California 92037 Communicated by Ernest Beutler, December 31, 1981 ABSTRACT This paper describes a monoclonal antibody Cloning of Hybridoma B532. The monoclonal B532 was sub- (B532) that detects a membrane antigen present on 295% of the cloned by limiting dilution in a 96-well plate in the presence B cells from lines carrying the Epstein-Barr virus (EBV) genome. ofa normal murine spleen cell feeder layer. Ten subelones were Evidence suggesting that B532 is EBV-related was originally ob- retested and all showed the same binding characteristics as with tained by using a cell-binding radioassay with different cell line the original clone. Three of these were grown up to 106 cells substrates. Immunofluorescence and cell-sorter analysis con- per ml in 200-ml portions. These cells were then frozen down, firmed that the antigen was present in high density on all EBV- and the supernatants were used as the monoclonal antibody. infected lymphoblastoid B-cell lines, but not on EBV-negative The monoclonal antibody is an IgG1 protrein. For routine use, B-, T-, myeloid, or null cell lines. Isolated normal peripheral blood it was B and T lymphocytes and monocytes failed to bind B532.
    [Show full text]
  • A Human Thymus-Leukemia Antigen Defined by Hybridoma Monoclonal
    Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6552-6556, December 1979 Immunology A human thymus-leukemia antigen defined by hybridoma monoclonal antibodies (acute lymphocytic leukemia/cell surface/tumor antigens) RONALD LEVY, JEANETTE DILLEY, ROBERT I. Fox, AND ROGER WARNKE The Howard Hughes Medical Institute Laboratories, and the Departments of Medicine and Pathology, Stanford University Medical Center, Stanford, California 94305 Communicated by Henry Kaplan, July 9, 1979 ABSTRACT A series of mouse hybridomas producing mo- MATERIALS AND METHODS noclonal antibodies against human acute lymphocytic leukemia (ALL) cells was generated and screened for tumor specificity. Human Cells. The leukemia cells used for immunization and Among 1200 primary cultures, 60 produced an antibody that for screening of antibodies were derived from the peripheral could distinguish between the immunizing leukemia cells and blood of a child (Dom) with T-cell ALL. The patient had a an isologous B lymphoblastoid cell line. Of these, two produced mediastinal mass and a blood lymphoblast cell count of 460,000 an antibody that detects an antigen expressed preferentially on ALL cells and on a subpopulation of normal cells found in the per mm3. Greater than 95% of these blast cells formed heat- cortex of the thymus. Other normal human lymphoid cells from stable sheep erythrocyte rosettes (7). Leukemia cells from patient lymph nodes, spleen, bone marrow, and peripheral blood express Dom and a series of other patients were purified from peri- only low levels of this antigen. High levels of this "thymus- pheral blood or bone marrow or Ficoll-Hypaque sedimenta- leukemia" antigen were found on T-ALL cells, T-ALL-derived tion (8) and stored in 10% dimethyl sulfoxide under liquid N2.
    [Show full text]
  • Surface Features of Human Natural Killer Cells and Antibody-Dependent Cytotoxic Cells
    J. Cell Sd. 77, 27-46 (1985) 27 Printed in Great Britain © The Company of Biologists Limited 1985 SURFACE FEATURES OF HUMAN NATURAL KILLER CELLS AND ANTIBODY-DEPENDENT CYTOTOXIC CELLS CLAIRE M. PAYNE1*, ALEC LINDE1, RUTH KIBLER2, BONNIE POULOS2, LEWIS GLASSER1 AND ROGER FIEDERLEIN1 'Department of Pathology and 2 Department of Microbiology and Immunology, University of Arizona, College of Medicine, 1501 N. Campbell Avenue, Tucson, Arizona 85724, U.SA. SUMMARY The purpose of the present study was to examine the surface features of purified large granular lymphocytes (LGLs) (natural killer (NK) cells, antibody-dependent cytotoxic lymphoid (ADCL) cells, K-cells, Fcy( + ) third population (non-T, non-B) lymphoid cells, Tr cells) by scanning electron microscopy (SEM) and to compare their surface features with granulocytes, monocytes and Fcy(—) lymphoid cells that were all fixed for SEM under identical conditions. We have determined that 72-80 % of LGLs enriched by rosette formation with sensitized erythrocytes or using Percoll gradients, have a complex microvillous surface (CMS) pattern identical to that of lymphocytes. The LGL fraction appears by SEM to represent a morphologically homogeneous population of cells. Monocytes prepared for SEM under identical conditions had distinct surface folds and granulocytes displayed numerous broad-based ridge-like profiles. The majority of lymphoid cells in an unfrac- tioned population have a CMS pattern when incubated at room temperature (25 °C) before fixation, and a sparse microvillous surface (SMS) pattern when incubated at body temperature (37°C). Ficoll-Hypaque (FH) also had a direct effect on the cell surface pattern. Over half of the unfrac- tionated lymphoid cells displayed a CMS pattern after cells were washed free of FH and incubated at 37 CC before fixation.
    [Show full text]
  • Relative Frequencies and Sites of Presentation of Lymphoid Neoplasms in a Community Hospital According to the Revised European-American Classification
    Hematopathology / FREQUENCIES AND SITES OF PRESENTATION OF LYMPHOID NEOPLASMS IN A COMMUNITY HOSPITAL Relative Frequencies and Sites of Presentation of Lymphoid Neoplasms in a Community Hospital According to the Revised European-American Classification James D. Siebert, MD,1-2 DebraA. Mulvaney, MLT(ASCP) ,J<2 Kari L. Potter, HT(ASCP),1'2 Paul A. S. Fishkin, MD,L3 and Francois J. Geoffroy, MDJ3 Key Words: Lymphoma; Lymphoid neoplasms; REAL classification; Immunophenotype Downloaded from https://academic.oup.com/ajcp/article/111/3/379/1758571 by guest on 27 September 2021 Abstract In 1994, the International Lymphoma Study Group Relative frequencies for common subtypes in the proposed a revised European-American classification of revised European-American classification of lymphoid lymphoid neoplasms (REAL classification), which catego­ neoplasms (REAL classification) have been reported. rizes lymphoma variants based on morphologic features, We determined the relative frequencies and sites of immunophenotype, and clinical findings.1 The classifica­ presentation of REAL subtypes at a 700-bed community tion represents a significant departure from the Working hospital in central Illinois. A database was used to Formulation because of the use of immunophenotype, the identify and prospectively catalogue all newly omission of some Working Formulation entities, and the diagnosed lymphoid neoplasms from July 1, 1995 to recognition of newly characterized lymphoid neoplasms.2 March 1, 1998. The approach to diagnosis and Data about the new classification are being accrued. REAL subtyping incorporated morphologic features, classification subtyping is highly reproducible among immunophenotype, and clinical findings according to expert pathologists, it is most accurate when immunopheno­ criteria proposed in the REAL classification.
    [Show full text]