DOCSLIB.ORG

Defining Origins of Malignant B Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Defining Origins of Malignant B Cells Leukemia (2009) 23, 2075–2080 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE Defining origins of malignant B cells: a new circulating normal human IgM þ D þ B-cell subset lacking CD27 expression and displaying somatically mutated IGHV genes as a relevant memory population N Weston-Bell1, M Townsend1, G Di Genova1, F Forconi2 and SS Sahota1 1Cancer Sciences Division, School of Medicine, University of Southampton, Southampton, UK and 2Sezione Ematologia e Trapianti, Universita` di Siena, Siena, Italy In probing the cell of origin in malignant B cells, an imprint of In normal B cells, a memory of antigen is imprinted somatic hypermutation (SHM) in immunoglobulin (Ig) variable (V) irreversibly by SHM in Ig V genes during T-cell-dependent region genes delineates antigen encounter, and identifying the encounter through germinal centers (GCs) in secondary lym- precise pathway generating SHM in the normal B-cell counterpart 1 becomes relevant. SHM remains the definitive memory imprint in phoid organs. Isotype class switch recombination can also normal human B cells, but CD27 expression also delineates occur in the GCs by deleting specified constant region coding memory. Recently, dye extrusion adenosine triphosphate-bind- sequences.1 Although both SHM and class switch recombina- ing transporter assays identified circulating isotype-switched tion have generally been regarded as markers of memory B cells, memory B cells that lacked CD27, yet exhibited low levels of SHM. a seminal finding was the identification of CD27 as an To extend findings, we report a pre-switched CD27Àve circulating apparently robust phenotypic marker able to discriminate memory B-cell population in normal blood using comparable 2 assays, and isolated CD19 þ IgM þ D þ CD27Àve cells (499% between human naive and memory B cells. The circulating þ purity) for the analysis of IGHV5/IGHV3-IGHM transcripts. Of ‘classical’ CD27 memory pool in that study was shown to these (n ¼ 334), B78% were germ line and naive B cell derived. comprise mutated IgM þ D þ , IgM þ DÀve and IgG þ or IgA þ Strikingly, 21.9% of the transcripts were mutated. They B cells.2 However, SHM can also occur through T-independent showed 3–5 mutations (13.5% of sequences) and 45 mutations mechanisms ectopically at non-GC sites,3 and this may impact (8.4% of sequences) per transcript. Accrual of mutations in a subset of CD19 þ IgM þ D þ CD27Àve cells define a new circulating crucially on deciphering a role for instance, for infection-related pre-switched memory B-cell pool, present in substantial antigens in clonal tumor origins. numbers in the population harboring naive B cells. These In malignant B cells, there are instances when the presence of CD19 þ IgM þ D þ CD27Àve memory B cells may have a distinct SHM is confounding, as it does not correlate with the expected lineage and function, and seem relevant to understanding origins classical memory phenotype. This is illustrated by hairy cell of malignant B cells, in particular those of hairy cell leukemia leukemia (HCL) cells that exhibit mutated IGHV genes but are cells, which display mutated V genes yet lack CD27 expression. CD27Àve.4,5 In addition, in IgM-expressing Waldenstrom’s Leukemia (2009) 23, 2075–2080; doi:10.1038/leu.2009.178; Àve published online 24 September 2009 macroglobulinemia, mutated tumor cells are seen in CD27 6,7 Keywords: CD27; normal B-cell memory; somatic hypermutation; fractions, and it has been proposed that origins of this disease hairy cell leukemia may be from ‘unusual’ memory B cells.6 Interestingly, more recent observations indicate that the normal human B-cell memory pool may as yet not be mapped fully. These studies have examined the association of CD27 expression as an absolute requirement in normal human Introduction memory B cells. Pivotal to the studies is an assay based on dye extrusion by the adenosine triphosphate-binding cassette Defining the cell of origin that causes B-cell tumors is a central subfamily B1 (ABCB1) transporter pump, which is functionally question in understanding the pathogenesis of malignant B cells. 8 restricted to naive B cells. When loaded with a suitable dye, Allied to this are questions of a role for antigen in selecting such ABCB1-positive naive B cells extrude the dye and retain a low a cell, what the nature of antigen might be and whether it is þ level of dye fluorescence, whereas CD27 memory B cells are presented in the context of T-cell help or through T-independent 8 pump negative and retain much higher levels of dye. Using this pathways. We have been addressing these questions by focusing þ Àve assay, a population of IgG but CD27 B cells could be on the analysis of immunoglobulin (Ig) variable (V) region genes identified as ABCB1-negative in normal human blood lympho- in malignant B cells, as these genes encode the antigen-binding Àve þ Àve cytes. A subset of this ABCB1 IgG CD27 population was domains of the surface Ig molecule and can reveal important þ þ indeed shown to be functionally comparable with IgG CD27 aspects of clonal history of the cell of origin. The most striking 8 þ Àve memory B cells. It is noteworthy that these IgG CD27 cells aspect is whether such a cell has undergone somatic hyper- were not initially examined for V gene mutations. However, two mutation (SHM) in response to antigen, then generating þ Àve parallel studies established that the new memory IgG CD27 questions of where SHM may have occurred and in response population indeed displayed SHM, but at a lower level than did to which stimuli. 9,10 classical switched memory B cells. A relevance for these IgG þ CD27Àve cells also emerged in B-cell disease, specifically Correspondence: Dr SS Sahota, Cancer Sciences Division, School of for systemic lupus erythematosus in which they represent a Medicine, Southampton University Hospitals, Tremona Road, South- major circulating compartment.10 ampton SO16 6YD, UK. E-mail: [email protected] As yet, however, it is not known whether a pre-switched Àve Received 23 December 2008; revised 3 July 2009; accepted 13 July CD27 component also exists in the normal human circula- 2009; published online 24 September 2009 tion, with a parallel mutational imprint. This is of relevance Human IgM þ D þ memory B cells lacking CD27 N Weston-Bell et al 2076 for understanding the CD27Àve memory B-cell pool more 5 mg/ml (Dako) and streptavidin-perCP-cy5.5 at 2 mg/ml (BD) at fully as well as for defining origins of B cells in malignancy. 4 1C for 30 min. Isotype antibody control labeling was In this study, we report the IGHV mutational status performed as described above. Separation of both ABCB1- of CD19 þ IgM þ D þ CD27Àve cells in normal circulating positive and ABCB1-negative cells was established using the B lymphocytes. ABCB1 transporter inhibitor. As MTG was never entirely extruded from cells, MTG-stained cells were spiked with unstained cells to confirm the correct acquisition voltage. Materials and methods CD19 þ IgM þ D þ cells were then analyzed by MTG retention and CD27 expression as reported.8 Analysis and isolation of circulating B lymphocytes by flow cytometry procedures Peripheral blood mononuclear cells were isolated from healthy Isolation of RNA and DNA for IGHV analysis donors after obtaining informed consent using Lymphoprep The highly purified CD19 þ IgD þ CD27Àve,CD19þ IgD þ CD27 þ (Axis Shield, Bicton Industrial Park, UK), and were analyzed and and CD19 þ IgDÀveCD27 þ populations were used for RNA cell sorted by flow cytometry using a FACSAria (BD Biosciences, extraction using the RNeasy Mini Kit (Qiagen, Crawley, UK), Oxford, UK). The two donors for cell sorted fractions for and cDNA was synthesized using SuperScript II Reverse molecular analysis were aged 23 and 36 years, respectively. Transcriptase (Invitrogen), both in accordance with the For flow cytometry, 2–4 Â 107 peripheral blood mononuclear manufacturers’ instructions. Using cDNA, IGHM transcripts cells were incubated with a-CD19-perCP-cy5.5 at 1:10 (BD were specifically amplified with downstream Cm primers. Pharmingen, Oxford, UK), a-IgM-phycoerythrin (PE) at 10 mg/ml IGHV5-IGHM gene transcripts were amplified from (Dako, Ely, UK), a-IgD-FITC (a-IgD-fluorescein isothiocyanate) sorted CD19 þ IgD þ CD27Àve, CD19 þ IgD þ CD27 þ and at 16 mg/ml (Dako) and a-CD27-allophycocyanin (APC) at 1:10 CD19 þ IgDÀveCD27 þ fractions by semi-nested reverse tran- (Biolegend, San Diego, CA, USA), or with relevant isotype scriptase-PCR, using in the first round a combination of IGHV5 antibody controls in FACS buffer (1% bovine serum albumin, leader (50-atggggtcaaccgccatcctcg) or IGHV3 leader (50-ccatg 4mM EDTA (ethylenediaminetetraacetic acid) and 0.15 mM gagtttgggctgagctgg) primers, respectively, with downstream sodium azide in phosphate-buffered saline) for 30 min at 4 1C. IGHM100 30 (50-ggagaaagtgatggagtcgg), followed in the second 0 Isotype antibody control labeling was performed with mIgG1,k- round of amplification by IGHV5 framework-1 (5 -gaggtg 0 perCP-cy5.5 (BD, Pharmingen), mIgG1,k-APC (BD, Pharmin- cagctgngtcagtctg) or IGHV3 framework-1 (5 -gaggtgcagctggtg 4,7,11 gen), rF(ab)2-FITC (Dako, Pharmingen) and rF(ab)2-PE (Dako) at sagtcyg) with IGHM100 as previously described. PCR equivalent concentrations to relevant staining antibody. Quad- products were cloned and individual plasmids with IGHV- rants defining positive and negative staining were positioned IGHM inserts were sequenced as described.4,7,11 Taq error was according to isotype control staining. calculated in a 350–400 bp stretch of the IGHM constant region Cells were gated first as lymphocytes by forward and side (excluding primer sequences) using semi-nested IGHV5 or scatter, and then CD19 þ IgM þ /Àve B cells were analyzed for IGHV3 upstream primers, as described above, with IGHM370 CD27 and IgD expression.
Load more

Recommended publications
  • Tacrolimus Prevents TWEAK-Induced PLA2R Expression in Cultured Human Podocytes
    Journal of Clinical Medicine Article Tacrolimus Prevents TWEAK-Induced PLA2R Expression in Cultured Human Podocytes Leticia Cuarental 1,2, Lara Valiño-Rivas 1,2, Luis Mendonça 3, Moin Saleem 4, Sergio Mezzano 5, Ana Belen Sanz 1,2 , Alberto Ortiz 1,2,* and Maria Dolores Sanchez-Niño 1,2,* 1 IIS-Fundacion Jimenez Diaz, Universidad Autonoma de Madrid, Fundacion Renal Iñigo Alvarez de Toledo-IRSIN, 28040 Madrid, Spain; [email protected] (L.C.); [email protected] (L.V.-R.); [email protected] (A.B.S.) 2 Red de Investigación Renal (REDINREN), Fundacion Jimenez Diaz, 28040 Madrid, Spain 3 Nephrology Department, Centro Hospitalar Universitário São João, 4200-319 Porto, Portugal; [email protected] 4 Bristol Renal, University of Bristol, Bristol BS8 1TH, UK; [email protected] 5 Laboratorio de Nefrologia, Facultad de Medicina, Universidad Austral de Chile, 5090000 Valdivia, Chile; [email protected] * Correspondence: [email protected] (A.O.); [email protected] (M.D.S.-N.); Tel.: +34-91-550-48-00 (A.O. & M.D.S.-N.) Received: 29 May 2020; Accepted: 7 July 2020; Published: 10 July 2020 Abstract: Primary membranous nephropathy is usually caused by antibodies against the podocyte antigen membrane M-type phospholipase A2 receptor (PLA2R). The treatment of membranous nephropathy is not fully satisfactory. The calcineurin inhibitor tacrolimus is used to treat membranous nephropathy, but recurrence upon drug withdrawal is common. TNF superfamily members are key mediators of kidney injury. We have now identified key TNF receptor superfamily members in podocytes and explored the regulation of PLA2R expression and the impact of tacrolimus.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • CD226 T Cells Expressing the Receptors TIGIT and Divergent Phenotypes of Human Regulatory
    The Journal of Immunology Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226 Christopher A. Fuhrman,*,1 Wen-I Yeh,*,1 Howard R. Seay,* Priya Saikumar Lakshmi,* Gaurav Chopra,† Lin Zhang,* Daniel J. Perry,* Stephanie A. McClymont,† Mahesh Yadav,† Maria-Cecilia Lopez,‡ Henry V. Baker,‡ Ying Zhang,x Yizheng Li,{ Maryann Whitley,{ David von Schack,x Mark A. Atkinson,* Jeffrey A. Bluestone,‡ and Todd M. Brusko* Regulatory T cells (Tregs) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4+FOXP3+Helios+ thymic-derived Tregs and CD4+FOXP3+Helios2 T cells, followed by comparison with CD4+FOXP32Helios2 T conventional cells. These analyses revealed that the coinhibitory receptor T cell Ig and ITIM domain (TIGIT) was highly expressed on thymic-derived Tregs. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4+CD25+CD127lo/2 T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Tregs after activation and in vitro expansion, and is associated with lineage stability and suppressive capacity. Conversely, the CD226+TIGIT2 population was associated with reduced Treg purity and suppressive capacity after expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Tregs in health and disease.
    [Show full text]
  • Downloaded and Further Processed with the R Programming Language ( and Bioconductor ( Software
    bioRxiv preprint doi: https://doi.org/10.1101/801530; this version posted October 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. An integrated multi-omic single cell atlas to redefine human B cell memory David R. Glass,1,2,5 Albert G. Tsai,2,5 John Paul Oliveria,2,3 Felix J. Hartmann,2 Samuel C. Kimmey,2,4 Ariel A. Calderon,1,2 Luciene Borges,2 Sean C. Bendall1,2,6,* 1Immunology Graduate Program, Stanford University, Stanford, CA, 94305, USA 2Department of Pathology, Stanford University, Stanford, CA, 94305, USA 3Department of Medicine, Division of Respirology, McMaster University, Hamilton, ON, L8S4K1, Canada 4Department of Developmental Biology, Stanford, University, Stanford CA, 94305, USA 5Co-first author 6Lead Author *Correspondence: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/801530; this version posted October 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract: To evaluate the impact of heterogeneous B cells in health and disease, comprehensive profiling is needed at a single cell resolution. We developed a highly- multiplexed screen to quantify the co-expression of 351 surface molecules on low numbers of primary cells. We identified dozens of differentially expressed molecules and aligned their variance with B cell isotype usage, metabolism, biosynthesis activity, and signaling response.
    [Show full text]
  • A 39-Kda Protein on Activated Helper T Cells Binds CD40 and Transduces the Signal for Cognate Activation of B Cells RANDOLPH J
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6550-6554, July 1992 Immunology A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells RANDOLPH J. NOELLE*t, MEENAKSHI RoY*, DAVID M. SHEPHERD*, IVAN STAMENKOVICO, JEFFREY A. LEDBETTER§, AND ALEJANDRO ARUFFO§ *Department of Microbiology, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756; *Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121; and *Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 Communicated by Leon E. Rosenberg, April 22, 1992 (receivedfor review February 5, 1992) ABSTRACT CD40 is a B-cell surface molecule that has MATERIALS AND METHODS been shown to induce B-cell growth upon ligation with mono- clonal antibodies. This report shows that triggering via CD40 Mice. Female DBA/2J mice (The Jackson Laboratory) is essential for the activation of resting B cells by helper T cells were used for the preparation of filler cells to support the (Th). A soluble fusion protein of CD40 and human immuno- growth of Th clones and in the preparation of resting B cells. globulin, CD40-Ig, inhibited the induction ofB-cell cycle entry, Th Clones. D1.6, an I-Ad-restricted, rabbit Ig-specific ThO proliferation, and differentiation by activated Thi and Tb2. clone, and CDC35, an I-Ad-restricted, rabbit Ig-specific Th2 The ligand for CD40 was identified as a 39-kDa membrane clone, were obtained from David Parker (University of protein that was selectively expressed on activated Tb. A Massachusetts, Worcester). In this paper, D1.6 will be re- monoclonal antibody specific for the 39-kDa protein inhibited ferred to as Thl and CDC35 as Tb2 (7).
    [Show full text]
  • Double-Negative (CD27-Igd-) B Cells Are Expanded in NSCLC And
    Centuori et al. J Transl Med (2018) 16:30 https://doi.org/10.1186/s12967-018-1404-z Journal of Translational Medicine RESEARCH Open Access Double‑negative ­(CD27−IgD−) B cells are expanded in NSCLC and inversely correlate with afnity‑matured B cell populations Sara M. Centuori1, Cecil J. Gomes2, Samuel S. Kim3, Charles W. Putnam1,3, Brandon T. Larsen4, Linda L. Garland1,5, David W. Mount6 and Jesse D. Martinez1,7* Abstract Background: The presence of B cells in early stage non-small cell lung cancer (NSCLC) is associated with longer survival, however, the role these cells play in the generation and maintenance of anti-tumor immunity is unclear. B cells diferentiate into a variety of subsets with difering characteristics and functions. To date, there is limited informa- tion on the specifc B cell subsets found within NSCLC. To better understand the composition of the B cell populations found in NSCLC we have begun characterizing B cells in lung tumors and have detected a population of B cells that are ­CD79A+CD27−IgD−. These ­CD27−IgD− (double-negative) B cells have previously been characterized as uncon- ventional memory B cells and have been detected in some autoimmune diseases and in the elderly population but have not been detected previously in tumor tissue. Methods: A total of 15 fresh untreated NSCLC tumors and 15 matched adjacent lung control tissues were dissociated and analyzed by intracellular fow cytometry to detect the B cell-related markers CD79A, CD27 and IgD. All ­CD79A+ B cells subsets were classifed as either naïve ­(CD27−IgD+), afnity-matured ­(CD27+IgD−), early memory/germinal center cells ­(CD27+IgD+) or double-negative B cells ­(CD27−IgD−).
    [Show full text]
  • Contribution of CD30/CD153 but Not of CD27/CD70, CD134/OX40L, Or
    Contribution of CD30/CD153 but not of CD27/CD70, CD134/OX40L, or CD137/4-1BBL to the optimal induction of protective immunity to Mycobacterium avium Manuela Flo´ rido,* Margarida Borges,* Hideo Yagita,† and Rui Appelberg*,‡,1 *Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, and ‡ICBAS, Instituto de Cieˆncias Biome´dicas de Abel Salazar, University of Porto, Portugal; and †Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Abstract: A panel of monoclonal antibodies spe- the role played by the TNFRSF members CD27 (TNFRSF7), cific for CD27 ligand (CD70), CD30 ligand CD30 (TNFRSF8), CD134 (TNFRSF4 or OX40), and CD137 (CD153), CD134 ligand (OX40L), and CD137 li- (TNFRSF9 or 4-1BB) and their ligands CD70 (TNFSF7), gand (4-1BBL) were screened in vivo for their CD153 (TNFSF8), OX40L (TNFSF4), and 4-1BBL (TNFSF9), ability to affect the control of Mycobacterium respectively. avium infection in C57Bl/6 mice. Only the block- CD27/CD70 interactions participate in the early phases of T ing of CD153 led to increased mycobacterial bur- cell responses [2]. Engaging CD27 on T cells activates these dens. We then used CD30-deficient mice and found cells, leading to cytokine secretion and proliferation, and po- an increase in the proliferation of two strains of M. tentiates the activity of other accessory molecules [3]. Mice avium in these mice as compared with control an- deficient in CD27 have impaired responses to influenza virus imals. The increased mycobacterial growth was as- and reduced T cell memory generation [4]. Conversely, chronic sociated with decreased T cell expansion and re- in vivo triggering of CD27 by expression of a CD70 transgene duced interferon-␥ (IFN-␥) responses as a result of led to massive activation and proliferation of T cells with reduced polarization of the antigen-specific, IFN- conversion of naı¨ve cells into cells with an activated/memory ␥-producing T cells.
    [Show full text]
  • Fas/Fc Chimera (F8799)
    Fas/Fc Chimera mouse, recombinant expressed in Sf 21 cells Catalog Number F8799 Storage Temperature –20 °C Synonyms: Apo-1; CD95 The most studied receptors involved in apoptosis are CD95/Fas/Apo-1 (apoptosis inducing protein 1) and Product Description TNF receptor I (TNF RI). Apoptosis mediated by both Recombinant mouse Fas (CD95, Apo-1)/Fc Chimera is signaling cascades results in activation of a family of a transmembrane glycoprotein receptor expressed in cysteine proteases known as caspases. However, Fas- insect Sf 21 cells using a baculovirus expression mediated death occurs much more rapidly than that system. A cDNA sequence encoding the extracellular triggered by the TNF RI. Engagement of Fas by its domain (amino acids 1-169) of mouse Fas antigen1 is ligand (Fas ligand, FasL, CD95L), or by an appropriate fused to the carboxy-terminal 6´ histidine tagged Fc antibody, results in the rapid induction of PCD in region of human IgG1 by a linker peptide. The susceptible cell lines. This process bypasses the usual recombinant protein is a disulfide-linked homodimer long sequence of signaling enzymes and immediately with a blocked amino-terminus. The reduced monomer activates preexisting caspases.3 of mouse Fas/Fc has a calculated molecular mass of 46 kDa. However, due to glycosylation, recombinant The action of Fas is mediated via FADD (Fas- mouse Fas/Fc migrates as a 55 kDa protein in SDS- associated death domain)/MORT1, an adapter protein PAGE under reducing conditions. Mouse Fas cDNA that has a death domain at its C-terminus and binds encodes a 327 amino acid type 1 membrane protein to the cytoplasmic death domain of Fas.
    [Show full text]
  • Serum Proteomics Links Suppression of Tumor Immunity to Ancestry and Lethal Prostate Cancer
    Serum proteomics links suppression of tumor immunity to ancestry and lethal prostate cancer Tsion Minas Center for Cancer Research Julián Candia National Cancer Institute https://orcid.org/0000-0001-5793-8989 Tiffany Dorsey National Cancer Institute, NIH, Bethesda, MD Francine Baker Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute (NCI), National Institutes of Health (NIH) https://orcid.org/0000-0003-3133-3652 Wei Tang NCI/NIH https://orcid.org/0000-0002-7089-4391 Maeve Kiely Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH) Cheryl Smith Center for Cancer Research Symone Jordan Center for Cancer Research Obadi Obadi Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH) Anuoluwapo Ajao Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH) Yao Tettey University of Ghana Medical School Richard Biritwum University of Ghana Medical School Andrew Adjei University Of Ghana Medical School James Mensah University of Ghana Medical School Robert Hoover National Cancer Institute Frank Jenkins Hillman Cancer Center, University of Pittsburgh Rick Kittles City of Hope https://orcid.org/0000-0002-5004-4437 Ann Hsing Stanford Xin Wang Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health
    [Show full text]
  • Differential Signaling Via Tumor Necrosis Factor-Associated Factors (Trafs) by CD27 and CD40 in Mouse B Cells
    Differential Signaling via Tumor Necrosis Factor-Associated Factors (TRAFs) by CD27 and CD40 in Mouse B Cells So-Youn Woo1*, Hae-Kyung Park1 and Gail A. Bishop2,3,4 Department of Microbiology1, College of Medicine, Ewha Womans University, Seoul 158-710, Korea, Department of Microbiology2, and Department of Internal Medicine3, The University of Iowa, Veterans Affairs Medical Center4, Iowa City, IA 52242 ABSTRACT Background: CD27 is recently known as a memory B cell marker and is mainly ex- pressed in activated T cells, some B cell population and NK cells. CD27 is a member of tumor necrosis factor receptor family. Like CD40 molecule, CD27 has (P/S/T/A) X(Q/E)E motif for interacting with TNF receptor-associated factors (TRAFs), and TRAF2 and TRAF5 bindings to CD27 in 293T cells were reported. Methods: To investigate the CD27 signaling effect in B cells, human CD40 extracellular domain containing mouse CD27 cytoplamic domain construct (hCD40-mCD27) was transfected into mouse B cell line CH12.LX and M12.4.1. Results: Through the stimulation of hCD40-mCD27 molecule via anti-human CD40 antibody or CD154 ligation, expression of CD11a, CD23, CD54, CD70 and CD80 were increased and secretion of IgM was induced, which were comparable to the effect of CD40 stimulation. TRAF2 and TRAF3 were recruited into lipid-enriched membrane raft and were bound to CD27 in M12.4.1 cells. CD27 stimulation, however, did not increase TRAF2 or TRAF3 degradation. Conclusion: In contrast to CD40 signaling pathway, TRAF2 and TRAF3 degradation was not observed after CD27 stimulation and it might contribute to prolonged B cell activation through CD27 signaling.
    [Show full text]
  • Genieplex Quantitative Multiplex Immunoassays
    GeniePlex Quantitative Multiplex Immunoassays Quantitatively Measure 1-24 Analytes Per Sample! ELISA Genie: Maximum Support ELISA Genie is а proprietary range of ELISA kits & multiplex assays developed bу Reagent Genie, а global life science reagents company based in London and Dublin. Founded bу Colm Ryan PhD and Seán Mac Fhearraigh PhD, our goal is to provide you with premium quality ELISA kits along with excellent technical and logistics support, so you саn maximise your success. GeniePlex Multiplex Immunoassays GeniePlex: Quantitively Measure 1-24 Analytes Simultaneously! GeniePlex is a bead-based multiplex immunoassay technology. It enables the simultaneous & quantitative detection of up to 24 analytes in as little as 15 μl sample on almost any flow cytometer. It’s like doing 24 different ELISA in every well! Phenomenal Performance Very Sensitive: Measure as low as <10 pg/ml of each analyte Excellent Dynamic Range: Lower limit < 20 pg/mL | Upper limit > 5,000 pg/mL High Precision & Accuracy: Intra-assay CV: < 10% | Inter-assay CV: < 20% | Recovery: 70-130% Low Sample Volumes: Use as little as 15 µl of sample Validated: All assays fully tested for cross-reactivity in our lab Unbeatable Flexibility Comprehensive Choice of Targets: Up to 400 assays & custom formats available for human, mouse, rat, porcine, canine and primates Wide Range of Sample Types: Cell culture supernatants, saliva, plasma, cell/tissue lysates, serum, BALF, pleural and peritoneal fluids & more Multiple Formats: 32-well and 96-well sizes available Convenience Redefined Use Your Own Lab: Assays can be run on most validated flow cytometers (PE & either PE-Cy5 or APC detectors) Free Software: Analyse with commonly available software such as FCAP Software v3.0.
    [Show full text]
  • Mouse CD27 ELISA Kit (ARG81714)
    Product datasheet [email protected] ARG81714 Package: 96 wells Mouse CD27 ELISA Kit Store at: 4°C Component Cat. No. Component Name Package Temp ARG81714-001 Antibody-coated 8 X 12 strips 4°C. Unused strips microplate should be sealed tightly in the air-tight pouch. ARG81714-002 Standard 2 X 10 ng/vial 4°C ARG81714-003 Standard/Sample 30 ml (Ready to use) 4°C diluent ARG81714-004 Antibody conjugate 1 vial (100 µl) 4°C concentrate (100X) ARG81714-005 Antibody diluent 12 ml (Ready to use) 4°C buffer ARG81714-006 HRP-Streptavidin 1 vial (100 µl) 4°C concentrate (100X) ARG81714-007 HRP-Streptavidin 12 ml (Ready to use) 4°C diluent buffer ARG81714-008 25X Wash buffer 20 ml 4°C ARG81714-009 TMB substrate 10 ml (Ready to use) 4°C (Protect from light) ARG81714-010 STOP solution 10 ml (Ready to use) 4°C ARG81714-011 Plate sealer 4 strips Room temperature Summary Product Description ARG81714 Mouse CD27 ELISA Kit is an Enzyme Immunoassay kit for the quantification of Mouse CD27 in serum and cell culture supernatants. Tested Reactivity Ms Tested Application ELISA Specificity There is no detectable cross-reactivity with other relevant proteins. Target Name CD27 Conjugation HRP Conjugation Note Substrate: TMB and read at 450 nm. Sensitivity 31.25 pg/ml Sample Type Serum and cell culture supernatants. Standard Range 62.5 - 4000 pg/ml Sample Volume 100 µl www.arigobio.com 1/2 Precision Intra-Assay CV: 6.2%; Inter-Assay CV: 7.1% Alternate Names T-cell activation antigen CD27; Tp55; CD antigen CD27; CD27 antigen; S152.
    [Show full text]