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656.Full.Pdf Distinct T Cell Developmental Consequences in Humans and Mice Expressing Identical Mutations in the DLAARN Motif of ZAP-70 This information is current as Melissa E. Elder, Suzanne Skoda-Smith, Theresa A. of September 29, 2021. Kadlecek, Fengling Wang, Jun Wu and Arthur Weiss J Immunol 2001; 166:656-661; ; doi: 10.4049/jimmunol.166.1.656 http://www.jimmunol.org/content/166/1/656 Downloaded from References This article cites 32 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/166/1/656.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Distinct T Cell Developmental Consequences in Humans and Mice Expressing Identical Mutations in the DLAARN Motif of ZAP-701 Melissa E. Elder,2* Suzanne Skoda-Smith,¶ Theresa A. Kadlecek,§ Fengling Wang,* Jun Wu,† and Arthur Weiss‡§¶ The protein tyrosine kinase, ZAP-70, is pivotally involved in transduction of Ag-binding signals from the TCR required for T cell activation and development. Defects in ZAP-70 result in SCID in humans and mice. We describe an infant with SCID due to a novel ZAP-70 mutation, comparable with that which arose spontaneously in an inbred mouse colony. The patient inherited a homozygous missense mutation within the highly conserved DLAARN motif in the ZAP-70 kinase domain. Although the mutation only modestly affected protein stability, catalytic function was absent. Despite identical changes in the amino acid sequence of Downloaded from ZAP-70, the peripheral T cell phenotypes of our patient and affected mice are distinct. ZAP-70 deficiency in this patient, as in other humans, is characterized by abundant nonfunctional CD4؉ T cells and absent CD8؉ T cells. In contrast, ZAP-70-deficient mice lack both major T cell subsets. Although levels of the ZAP-70-related protein tyrosine kinase, Syk, may be sufficiently increased in human thymocytes to rescue CD4 development, survival of ZAP-70-deficient T cells in the periphery does not appear to be dependent on persistent up-regulation of Syk expression. The Journal of Immunology, 2001, 166: 656–661. http://www.jimmunol.org/ inding of Ag by the TCR initiates intracellular signaling ylates SLP-76 and LAT, triggering distal pathways that bring pathways that lead to transcription of genes required for about T cell activation (12–15). Studies in humans and gene-tar- B T cell activation. Propagation of Ag-binding signals from geted mouse models have confirmed the importance of ZAP-70 to the TCR to the nucleus is dependent on activation of the nonre- TCR signaling (16–20). An autosomal recessive form of SCID in ceptor cytoplasmic protein tyrosine kinases (PTKs),3 Lck and humans has been described due to mutations within the kinase ZAP-70 (reviewed in Refs. 1–3). PTK activation is a rapid and domain of ZAP-70 that abolish protein expression (16–18). In hu- critical event that results in phosphorylation of numerous down- mans, ZAP-70 deficiency is characterized by absent CD8ϩ T cells stream molecules, including phospholipase C␥1 (PLC␥1), linker and normal numbers of nonfunctional CD4ϩ T cells in the periph- of activated T cells (LAT), Src homology 2 domain-containing eral blood. This unusual phenotype suggests that ZAP-70 is critical by guest on September 29, 2021 76-kDa leukocyte protein (SLP-76), and Vav (reviewed in Refs. for CD8ϩ T cell development but may be dispensable for selection 2–7). These tyrosine phosphorylation reactions are required for of CD4ϩ T lymphocytes in the thymus. The presence of peripheral 2ϩ ϩ mobilization of intracellular free calcium ([Ca ]i) and activation CD4 T cells in ZAP-70-deficient patients contrasts with their of the Ras/mitogen-activated protein kinase (MAPK) and phos- absence in ZAP-70 knockout mice, which are blocked at the phatidylinositol 3-kinase pathways, and culminate in T cell acti- CD4ϩCD8ϩ double-positive (DP) stage of thymocyte differentia- vation and initiation of T cell-specific responses (reviewed in Refs. tion (20). Recently, a point mutation within the highly conserved 1 and 3–6). DLAARN motif in the zap70 gene that results in the amino acid One PTK critical for induction of Ag-specific T cell responses is conversion R464C was described in inbred Strange (ST) mice (21). ZAP-70. Recruitment of ZAP-70 to the TCR and its subsequent Although R464C affects ZAP-70 expression, its principal effect is phosphorylation and activation, largely by Lck, is essential for all to abrogate ZAP-70 catalytic activity (21). Like zap70 gene-tar- downstream signaling events (8–11). Activated ZAP-70 phosphor- geted animals, the affected ST mice are devoid of mature T cells. In this report, we describe an infant with SCID who is homozy- gous for the comparable DLAARN mutation (R465C) but who has Departments of *Pediatrics, †Microbiology and Immunology, and ‡Medicine, and normal numbers of circulating CD4ϩ T cells. The biochemical §Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; and ¶Department of Pediatrics, University of Florida, Gainesville, FL 32610 consequences of this ZAP-70 mutation on T cell activation are examined, and the role of Syk expression in survival of human Received for publication July 17, 2000. Accepted for publication September 27, 2000. ZAP-70-deficient T cells in the periphery is assessed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported in part by National Institutes of Health Grant M01 Patient RR01271, UCSF Pediatric Clinical Research Center, and National Institutes of Health Grants 1 P01 AI45865-01 and 5 P60 AR20684. The patient was a 10-mo-old Caucasian male, the only child of second- 2 Address correspondence and reprint requests to Dr. Melissa E. Elder, Box 0105, degree cousins, who presented for immunologic evaluation after develop- University of California, San Francisco, CA 94143. E-mail address: elderm@peds. ing Pneumocystis carinii pneumonia at the age of 7 mo. Serum IgG was 63 ucsf.edu mg/dl (normal range, 250–690 mg/dl) with normal IgM and IgA for age. Initial blood count revealed 10,600 leukocytes/mm3 and 80% lymphocytes. 3 Abbreviations used in this paper: PTK, protein tyrosine kinase; PLC␥1, phospho- lipase C␥1; LAT, linker of activated T cells; SLP-76, Src homology 2 domain-con- In vitro lymphocyte-proliferative responses were absent to PHA, PWM, 2ϩ and Con A. At 1 year of age, the patient received a T cell-depleted bone taining 76-kDa leukocyte protein; [Ca ]i, intracellular free calcium; MAPK, mito- gen-activated protein kinase; DP, double-positive; ST mice, Strange mice; TCL , T marrow transplant from his mother. He later developed a non-EBV-asso- cell line. ciated large B cell lymphoma that responded to treatment with combination Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 657 chemotherapy. Subsequently, the patient underwent successful retransplan- an excitation wavelength of 355 nm and emission wavelengths of 400 and tation with mobilized peripheral blood stem cells from his father. 500 nm. To ensure loading of Indo-1, 1 mM ionomycin was added to the cell suspensions. T cell culture and in vitro proliferative assays DNA isolation, PCR amplification, and sequencing Because the availability of the patient’s PBMC for study was limited, many experiments were performed with patient and normal T cell lines (TCL). Genomic DNA was isolated from PBMC using standard SDS/proteinase K For production of TCL, PBMC were grown for 2–4 wk in RPMI-C media digestion and phenol/chloroform extraction. Total RNA was isolated from (RPMI plus 20% human serum; NABI, Boca Raton, FL) with 0.5 ng/ml PBMC using TRIzol reagent (Life Technologies, Grand Island, NY). PMA (Sigma, St. Louis, MO), 1 ␮M ionomycin (Calbiochem, La Jolla, cDNA was synthesized from total RNA using oligo(dT) or random prim- CA), and 20 IU/ml IL-2 (Boehringer Mannheim, Indianapolis, IN). Immu- ers. RT-PCR was used to amplify ZAP-70 cDNAs. PCR primers used to ϩ nofluorescence analysis confirmed that the TCL were CD3 . For some generate nearly full length cDNAs (bp 33–1996; stop codon at bp 2067) ϩ experiments, CD8 T cells were depleted from normal TCL using mag- were 5Ј-GGAGCTCAGCAGACACCAG-3Ј (primer A) and 5Ј-GTTACTA netic beads (Dynal Biotech, Lake Success, NY). In vitro lymphocyte pro- CAGCCTGGCCAGCAA-3Ј (primer B), respectively. Nucleotides 1554– liferative assays were done as previously described (22). 2178 of ZAP-70 were amplified separately using primers C (5Ј-GGGAT GAAGTACCTGGAGGAGAAG-3Ј)andD(5Ј-GTTGTCTCCACACA Immunofluorescence analyses CAGCTG-3Ј). ZAP-70 sequence from nucleotides 1554–1877 was Cell surface expression of CD3, CD4, CD8, and CD69 was determined amplified from genomic DNA using primers C and E (5Ј-GCCTTCATC using a FACScalibur (Becton Dickinson, San Jose, CA).
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