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Deoxynucleoside Kinases Development in Vitro by Inhibition Restoration of Adenosine Deaminase-Deficient Human Thymocyte Development In Vitro by Inhibition of Deoxynucleoside Kinases This information is current as of September 26, 2021. Michelle L. Joachims, Patrick A. Marble, Aletha B. Laurent, Peter Pastuszko, Marco Paliotta, Michael R. Blackburn and Linda F. Thompson J Immunol 2008; 181:8153-8161; ; doi: 10.4049/jimmunol.181.11.8153 Downloaded from http://www.jimmunol.org/content/181/11/8153 References This article cites 54 articles, 20 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/181/11/8153.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 26, 2021 • 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: 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 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Restoration of Adenosine Deaminase-Deficient Human Thymocyte Development In Vitro by Inhibition of Deoxynucleoside Kinases1,2 Michelle L. Joachims,* Patrick A. Marble,* Aletha B. Laurent,* Peter Pastuszko,3† Marco Paliotta,† Michael R. Blackburn,‡ and Linda F. Thompson4* Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an “in vivo-like” environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, Downloaded from largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients. The Journal of Immunology, 2008, 181: 8153–8161. http://www.jimmunol.org/ utations in the gene encoding adenosine deaminase deficient infants suggests a profound defect in thymocyte devel- (ADA),5 a purine metabolic enzyme that catalyzes the opment (4). With the demonstration of elevated dATP in the RBC M deamination of both adenosine (Ado) and deoxyade- of ADA-deficient patients (5, 6), the ADA substrate dAdo became nosine (dAdo), cause severe combined immunodeficiency in hu- the prime candidate for causing lymphoid toxicity. However, little mans (1). ADA-deficient humans have a pronounced deficiency of is known about the specific stage or stages of lymphocyte devel- T cells with variable numbers of B and NK cells, and they die early opment that might be sensitive to a lack of ADA or of specific in life of infections unless treated with enzyme replacement ther- mechanism(s) responsible for the lack of thymocyte differentiation by guest on September 26, 2021 apy, bone marrow transplantation, or gene therapy (reviewed in and/or proliferation in ADA deficiency because of the difficulty in Refs. 2, 3). The paucity of lymphoid cells in thymuses from ADA- obtaining thymus samples from these rare patients and the lack of convenient culture systems for studying human thymocyte *Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, development. Oklahoma City, OK 73104; †Department of Thoracic Surgery; University of Okla- The successive stages of human thymocyte maturation can be ‡ homa Health Sciences Center, Oklahoma City, OK 73104; and Department of Bio- categorized as double negative (DN), double positive (DP), or sin- chemistry and Molecular Biology, University of Texas-Houston Medical School, Houston, TX 77030 gle positive (SP) according to the presence or absence of the CD4 Received for publication November 16, 2007. Accepted for publication September 25, and CD8 coreceptors. Immature human DN thymocytes are clas- 2008. sified based on expression of CD34, CD38, and CD1a (reviewed in The costs of publication of this article were defrayed in part by the payment of page Ref. 7). The transition from the DN to DP stage occurs via a CD4 charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. immature single-positive (CD4 ISP) stage (8), accompanied by TCR gene rearrangements and the first developmental checkpoint, 1 This work was supported by National Institutes of Health grants F32 HD008709 (to M.L.J.), HD36044 (to L.F.T.), and AI43572 (to M.R.B.) and was conducted in a ␤-selection. ␤-selection involves the pairing of the TCR␤ protein facility constructed with support from Research Facilities Improvement Program with the pre-T␣ protein to produce a membrane-localized pre-TCR Grant C06 RR14570-01 from the National Center for Research Resources, National ␤ Institutes of Health. L.F.T. holds the Putnam City Schools Distinguished Chair in that signals survival, expansion, and allelic exclusion. -selection Cancer Research. occurs over a range of phenotypic stages from CD34ϩCD1aϩ cells 2 Parts of this work were presented in the Purine and Pyrimidine Society Meeting, to DP cells (9, 10). Successful ␤-selection leads to the expression July 2007, in Chicago, IL. of the ␣␤TCR, followed by positive and negative selection, and 3 Current address: Rady Children’s Hospital, San Diego, CA 92123. development into mature CD4 and CD8 SP T cells (7, 11). Thy- 4 Address correspondence and reprint requests to Dr. Linda F. Thompson, Oklahoma muses from ADA-deficient patients have not been analyzed for Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104. E-mail address: [email protected] their proportions of these normal human thymocyte subsets, nor 5 Abbreviations used in this paper: ADA, adenosine deaminase; Ado, adenosine; AK, has human thymocyte development been studied in vitro under adenosine kinase; dAdo, 2Ј-deoxyadenosine; 5ЈA5ЈdAdo, 5Ј-amino-5Ј-deoxyade- ADA-deficient conditions. nosine; dCF, 2Ј-deoxycoformycin; dCK, deoxycytidine kinase; dCyd, 2Ј-deoxycyti- Mouse models for ADA deficiency have, however, yielded im- dine; DN, double negative; DP, double positive; EDP, early double positive; FTOC, fetal thymic organ culture; hu/moFTOC, human/mouse chimeric fetal thymic organ portant clues to the pathogenesis of the immunodeficiency. In an in culture; ISP, immature single positive; SAH, S-adenosylhomocysteine; TCR␤ic, in- vitro model for murine thymocyte development, fetal thymic organ tracellular TCR␤. culture (FTOC) (12–14), inhibition of ADA led to a marked re- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 duction in lymphocyte development secondary to the accumulation www.jimmunol.org 8154 ADENOSINE DEAMINASE-DEFICIENT HUMAN THYMOCYTE DEVELOPMENT of intracellular dATP and mitochondria-dependent apoptosis. Few Human neonatal thymus was obtained from children (ages 1 day to 4 thymocytes survived past the point of ␤-selection, most likely be- years) undergoing cardiac surgery at Children’s Hospital in Oklahoma cause most thymocytes fail to pass this developmental checkpoint City, OK, under protocols approved by the Institutional Review Boards of both the University of Oklahoma Health Sciences Center and the Okla- and die by apoptosis providing a source of ADA substrates. Thy- homa Medical Research Foundation. Thymocyte cell suspensions were mocyte differentiation and proliferation could be rescued by the made by forcing the thymic tissue pieces through a nylon filter. Human adenosine kinase (AK) inhibitor 5Ј-amino-5Ј-deoxyadenosine CD34ϩ thymocytes were enriched with anti-CD34 magnetic beads (Dy- (5ЈA5ЈdAdo) (15), since the main route of dAdo phosphorylation nal Biotech) and sorted if needed to obtain Ն95% purity for use in hu/moFTOC. Staining for intracellular TCR␤ (TCR␤ic) expression was in murine cells is via AK (16, 17). performed by fixing cells with 1% formaldehyde and permeabilizing with It is well known that the pathway of dAdo phosphorylation in 0.5% saponin (10). Data were collected using an LSR II or FACSCalibur human thymocytes differs from that in the mouse (17, 18), making flow cytometer and analyzed with CellQuest software (BD Biosciences). it essential that models of human thymocyte development be uti- FITC annexin V (Caltag Laboratories) was used following the manufac- lized to understand the pathogenesis of ADA deficiency. Despite turer’s instructions after
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