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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gilmour, K. C. Articles by Gaspar, H. B. Search for Related Content PubMed PubMed Citation Articles by Gilmour, K. C. Articles by Gaspar, H. B. Related Collections Immunobiology Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 August 2001, Vol. 98, No. 3, pp. 877-879 BRIEF REPORT Defective expression of the interleukin-2/interleukin-15 receptor  subunit leads to a natural killer cell-deficient form of severe combined immunodeficiency Kimberly C. Gilmour, Hodaka Fujii, Treena Cranston, E. Graham Davies, Christine Kinnon, and Hubert B. Gaspar From the Molecular Immunology Unit, Institute of Child Health, University College London; the Departments of Clinical Molecular Genetics and Immunology, Great Ormond Street Hospital, London, United Kingdom; and the Department of Immunology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Japan.     Abstract Top Abstract Introduction Study design Results and discussion References Development of T and natural killer (NK) cells is critically dependent on cytokine signaling, and defects in cytokine receptor complex subunits have been shown to result in severe combined immunodeficiency (SCID) syndromes in humans and in murine models. An infant boy had typical clinical features of SCID and was found to lack NK cells in his peripheral circulation. Molecular analysis did not reveal abnormalities in his c or JAK-3 genes, and he was investigated for defects in the interleukin-15 (IL-15) receptor complex because functional IL-15 signaling is essential for NK cell development. Expression of the IL-2R/IL-15R chain was significantly reduced in the patient's peripheral blood mononuclear cells (PBMCs) by immunoblot, flow cytometry, and Northern blot analysis. Furthermore, IL-2 stimulation of PBMCs showed only minimal tyrosine phosphorylation of JAK-3. These data demonstrate that defects in IL-2R/1L-15R expression can lead to a unique NK-deficient SCID immunophenotype. (Blood. 2001;98:877-879) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Severe combined immunodeficiency (SCID) syndromes are a heterogeneous group of conditions arising from defects in the development and function of T-, B-, and natural killer (NK)-cell populations. The molecular basis of many of the immunologic phenotypes has now been identified1 but remains unclear in a significant number of patients. Cytokine receptor signaling pathways are essential in the early stages of lymphocyte development, and defects in these pathways have been shown to result in SCID phenotypes in murine models and in humans. The X-linked form of SCID is caused by mutations in the common -chain (c) subunit, which is a component of the high-affinity interleukin-2 (IL-2), -4, -7, -9, and -15 receptors.2,3 This abnormality results in the absence of T-cell and NK-cell development and an as yet uncharacterized defect of B cells (denoted TB+NK SCID). Other components of this signaling pathway are also affected in certain forms of SCID. The JAK-3 tyrosine kinase binds directly to and is activated by c after receptor stimulation and is defective in the autosomal recessive form of TB+NK SCID.4 IL-7 receptor  mutations result in a more lineage-restricted TB+NK+ form of SCID, thus illustrating the essential role of IL-7 signaling in T-lymphocyte development.5 Evidence suggests that the development and survival of NK cells is dependent on a functional IL-15/IL-15 receptor-signaling pathway. The IL-15 receptor consists of a unique IL-15R chain that combines with a  chain (which is shared with the IL-2R complex and is termed IL-2R/IL-15R) and c subunits to create a high-affinity receptor complex. Mice with homozygous mutations in any of these 3 receptor subunits,6-8 in the IL-15 cytokine itself,9 or in interferon regulatory factor-110 (IRF-1, a transcription factor necessary for IL-15 expression) show profound defects of NK cell development and survival. In contrast, mice deficient in IL-2, IL-2R, or IL-7R have normal numbers of NK cells, suggesting a specific requirement for IL-15 signaling in NK cell development.11-13 In this article, we describe a child in whom abnormalities in the IL-15R complex resulted in a unique NK-deficient form of SCID.     Study design Top Abstract Introduction Study design Results and discussion References Staining and fluorescence-activated cell-sorting analysis Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density centrifugation, washed, and resuspended in 1 mL RPMI 1640 (Life Technologies, Gaithersburg, MD). Then 5 µL of the indicated antibodies were added to 100 µL cell suspension and analyzed according to standardized protocols (Becton Dickinson, Oxford, United Kingdom). JAK-3 activation PBMCs from 5 mL EDTA blood were resuspended in 1 mL RPMI. One hundred units IL-2 (Chiron, Harefield, United Kingdom) was added, and a JAK-3 tyrosine phosphorylation assay was performed as previously described.14 Immunoblots PBMCs were isolated and lysed in NP40 lysis buffer. Immunoblotting was performed according to previously described protocols.15 The primary antibodies (anti-IL-2R/IL-15R, anti-IL-15R, anti-IL-15, and JAK-3) (Santa Cruz Biotechnology, Santa Cruz, CA) were used at a concentration of 10µL in 1 mL milk-PBS-T (phosphate-buffered saline with Tween). For all protein expression and functional assays, age-matched control samples were analyzed. Northern blot analysis Northern blot analysis of IL-2R  messenger RNA (mRNA) was performed as described previously.16 Analysis of genomic DNA for exon-intron boundary defects The exon-intron boundaries were investigated in genomic DNA by single-stranded conformational polymorphism analysis (SSCP) or sequencing of the coding exons.2-10 Primers were designed in-house from the genomic sequence (GenBank GI 4090209/AL022314) (primer sequences and polymerase chain reaction conditions available on application). SSCP conditions have been previously decribed,17 and sequencing used an ABI377 automated sequencer and Big Dye Chemistry (PE Applied Biosystems, Warrington, United Kingdom).     Results and discussion Top Abstract Introduction Study design Results and discussion References An infant boy, P1, the second child of nonconsanguineous parents, had severe viral and fungal infections. In the first year of life, he had recurrent episodes of respiratory syncytial virus (RSV) bronchiolitis, Candida enteritis with significant failure to thrive, hepatomegaly, and an episode of meningo-encephalitis for which no causative organism was found. Investigation of his immune system, detailed in Table 1, showed a TB+ NK immunophenotype with poor specific antibody production. His clinical condition stabilized after courses of antibacterial, antifungal, and antiviral therapy, and he underwent successful allogeneic unrelated donor bone marrow transplantation at 17 months of age with good immune reconstitution and resolution of clinical symptoms.                                View this table: [in this window] [in a new window]   Table 1. Immunological analysis In view of the low T-cell numbers and lack of NK cell development, the patient was suspected to have an atypical form of c- or JAK-3-deficient SCID that resulted in a small amount of T- cell development, consistent with previous reports.18,19 Flow cytometric analysis of PBMCs showed normal expression of c (Figure 1A). Screening of the gene by (SSCP) did not reveal any abnormalities, thus making a diagnosis of X-SCID highly unlikely. To investigate JAK-3 activation and expression, we examined tyrosine phosphorylation in response to IL-2 stimulation of PBMCs. A control sample showed normal JAK-3 tyrosine phosphorylation, but only minimal JAK-3 phosphorylation was detectable in PBMCs from P1 (Figure 1B, top panel). Immunoblotting with anti-JAK-3 antibody demonstrated that P1 expressed normal amounts of JAK-3 (Figure 1B, bottom panel). The JAK-3 gene was further screened by SSCP analysis and did not show any abnormalities. From these initial studies, we concluded that although there was no abnormality in either the c or the JAK-3 molecules, the lack of JAK-3 tyrosine phosphorylation indicated a significant abnormality in signaling through the IL-2R complex. View larger version (65K): [in this window] [in a new window]   Figure 1. IL-2 signaling and IL-2R/IL-15R expression abnormalities in TB+NK SCID. (A) Flow cytometric analysis of PBMCs for cell surface expression of c. Equivalent amounts of c expression are seen in both a control sample and sample from P1. (B) Tyrosine phosphorylation of JAK-3 after IL-2 stimulation. PBMCs from a control and P1 were stimulated with IL-2, lysed, and immunoprecipitated using an anti-JAK-3 antibody. JAK-3 immunoprecipitates were then immunoblotted using an antiphosphotyrosine antibody. Normal JAK-3 tyrosine phosphorylation is seen in the control sample, but only minimal tyrosine phosphorylation is seen in P1 (top panel). The filter was stripped and reblotted with an anti-JAK-3 antibody, and the blot shows equivalent amounts of JAK-3 expression in both samples (bottom panel). (C) Flow cytometric and Western blot analysis of IL-2R/IL-15R expression. PBMCs from a control and P1 were analyzed by flow cytometry for cell surface expression of IL-2R/IL-15R. Normal expression is seen in a control sample, but significantly decreased expression is seen in the sample from P1. Western blot analyses of whole cell lysates from a control and P1 also show decreased expression of IL-2R/IL-15R. Stripping the filter and reblotting with an anti--actin antibody shows equivalent protein loading in each lane. (D) Northern blot analysis of IL-2R/IL-15R expression. mRNA prepared from PBMCs from a control subject and from P1. Northern blot analysis using an IL-2R/IL-15R cDNA probe shows 7% of control IL-2R/IL-15R expression in the sample from P1. Equivalent mRNA loading was demonstrated after stripping and reblotting the filter with a probe for -actin. The most notable feature of the patient's immunophenotype was the complete absence of NK cells. Because IL-15 signaling is essential for NK-cell development, components of the IL-15 cytokine and receptor complex were analyzed. Normal expression of both IL-15 and the IL-15R chain was detected in P1, as demonstrated by immunoblot analysis of PBMCs (Figure 2). Having previously shown normal c expression, the third component of the IL-15 receptor complex, IL-2R/IL-15R, was analyzed. Flow cytometric analysis showed that less than 7% of PBMCs from P1 were positive for surface IL-2R/IL-15R expression. This was markedly low in comparison with a control sample (Figure 1C, top panel). Immunoblot analysis also showed a significant decrease in IL-2R/IL-15R expression in P1 compared with control samples, whereas expression of -actin was equivalent in both (Figure 1C, bottom panels). Similar results were found when mRNA was isolated from PBMCs and analyzed for IL-2R expression by Northern blot analysis. In comparison with a control sample, P1 expressed only 7% of the IL-2R/IL-15R message as quantitated by Phosphorimager (Molecular Dynamics, Sunnyvale, CA; Figure 1D). These results all suggested a significant abnormality in IL-2R/IL-15R expressiona protein that is normally constitutively expressed.20 Numerous genetic analyses of the IL-2R/IL-15R gene were undertaken. IL-2R/IL-15R gene complementary DNA (cDNA) was initially amplified and sequenced (the 2 alleles could be distinguished by the polymorphism c750c-> t (G250G)). In addition, genomic sequence containing 1.1 kbp promoter region of the human IL-2R/IL-15R gene was also amplified and sequenced. To exclude splice-site mutations, 8 of 9 coding exons including exon/intron boundaries were directly sequenced, and exon 5, which could not be sequenced for technical reasons, was analyzed by SSCP analysis. No abnormalities were detected in any of these assays. Furthermore, DNA from P1 was analyzed by Southern blot analysis, but there was no evidence of duplications or gross deletions (data not shown). After bone marrow transplantation, when the NK-cell lineage was fully reconstituted, expression of IL-2R/IL-15R on PBMCs by flow cytometry and immunoblot analysis was normal (data not shown). View larger version (59K): [in this window] [in a new window]   Figure 2. IL-15R and IL-15 expression. Western blot analysis of IL-15R and IL-15 expression in PBMCs of P1 shows expression equivalent to that seen in a normal control. Many different SCID immunophenotypes have now been described, but this is the first report of SCID in which the major developmental abnormality is in the NK-cell lineage. The experiments described suggest that the defect occurred as a result of abnormal IL-15 and IL-2 signaling caused by a marked decrease in IL-2R/IL-15R expression. Although no evidence of an abnormality in the IL-2R/IL-15R gene was identified, some explanations are possible. The sensitivity of the techniques used was not 100%, and it is possible that a mutation was not detected. It is also possible that the defect lay in an uncharacterized regulatory region of the gene. Transcription factors such as Egr-1 (early growth response protein 1) and the Sp family proteins have been shown to bind to the 170 to 139 IL-2R/IL-15R enhancer region,21 and it is conceivable that a defect in one of these molecules or another transcription factor was responsible for the lack of IL-2R/IL-15R expression. Although the most complete defect was in IL-15-mediated NK- cell development, it is important to note that the patient had abnormalities in T- and B-cell function (Table 1). Impairment of IL-15 signaling, which is known to have important effects on T cell function,6,9 might have contributed to the T cell defects, and IL-2 receptor mediated abnormalities would be predicted to affect both T and B lymphocyte function. This case study again highlights the critical nature of cytokine receptor signaling pathways to lymphocyte and NK-cell development and function.     Footnotes Submitted June 16, 2000; accepted March 27, 2001. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Hubert B. Gaspar, Molecular Immunology Unit, Institute of Child Health, 30, Guilford St, London WC1N 1EH, United Kingdom; e-mail: h.gaspar{at}ich.ucl.ac.uk.     References Top Abstract Introduction Study design Results and discussion References 1. Fischer A, Cavazzana-Calvo M, De Saint Basile G, et al. Naturally occurring primary deficiencies of the immune system. Annu Rev Immunol. 1997;15:93-124[CrossRef][Medline] [Order article via Infotrieve]. 2. Noguchi M, Yi H, Rosenblatt HM, et al. Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell. 1993;73:147-157[CrossRef][Medline] [Order article via Infotrieve]. 3. Leonard WJ, Noguchi M, Russell SM, McBride OW. The molecular basis of X-linked severe combined immunodeficiency: the role of the interleukin-2 receptor gamma chain as a common gamma chain, gamma c. Immunol Rev. 1994;138:61-86[CrossRef][Medline] [Order article via Infotrieve]. 4. Macchi P, Villa A, Giliani S, et al. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature. 1995;377:65-68[CrossRef][Medline] [Order article via Infotrieve]. 5. Puel A, Ziegler SF, Buckley RH, Leonard WJ. Defective IL7R expression in T(-)B(+)NK(+) severe combined immunodeficiency. Nat Genet. 1998;20:394-397[CrossRef][Medline] [Order article via Infotrieve]. 6. Lodolce JP, Boone DL, Chai S, et al. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity. 1998;9:669-676[CrossRef][Medline] [Order article via Infotrieve]. 7. Suzuki H, Duncan GS, Takimoto H, Mak TW. Abnormal development of intestinal intraepithelial lymphocytes and peripheral natural killer cells in mice lacking the IL-2 receptor beta chain. J Exp Med. 1997;185:499-505[Abstract/Free Full Text]. 8. DiSanto JP, Muller W, Guy-Grand D, Fischer A, Rajewsky K. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain. Proc Natl Acad Sci U S A. 1995;92:377-381[Abstract/Free Full Text]. 9. Kennedy MK, Glaccum M, Brown SN, et al. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med. 2000;191:771-780[Abstract/Free Full Text]. 10. Taki S, Sato T, Ogasawara K, et al. Multistage regulation of Th1-type immune responses by the transcription factor IRF-1. Immunity. 1997;6:673-679[CrossRef][Medline] [Order article via Infotrieve]. 11. Kundig TM, Schorle H, Bachmann MF, et al. Immune responses in interleukin-2-deficient mice. Science. 1993;262:1059-1061[Abstract/Free Full Text]. 12. Willerford DM, Chen J, Ferry JA, et al. Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity. 1995;3:521-530[CrossRef][Medline] [Order article via Infotrieve]. 13. Corcoran AE, Smart FM, Cowling RJ, et al. The interleukin-7 receptor alpha chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis. EMBO J. 1996;15:1924-1932[Medline] [Order article via Infotrieve]. 14. Gilmour KC, Reich NC. Receptor to nucleus signaling by prolactin and interleukin 2 via activation of latent DNA-binding factors. Proc Natl Acad Sci U S A. 1994;91:6850-6854[Abstract/Free Full Text]. 15. Gaspar HB, Lester T, Levinsky RJ, Kinnon C. Bruton's tyrosine kinase expression and activity in X-linked agammaglobulinaemia (XLA): the use of protein analysis as a diagnostic indicator of XLA. Clin Exp Immunol. 1998;111:334-338[CrossRef][Medline] [Order article via Infotrieve]. 16. Hatakeyama M, Tsudo M, Minamoto S, et al. Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNAs. Science. 1989;244:551-556[Abstract/Free Full Text]. 17. Gilmour KC, Cranston T, Jones A, et al. Diagnosis of X-linked lymphoproliferative disease by analysis of SLAM- associated protein expression [In Process Citation]. Eur J Immunol. 2000;30:1691-1697[CrossRef][Medline] [Order article via Infotrieve]. 18. Mella P, Imberti L, Brugnoni D, et al. Development of autologous T lymphocytes in two males with X-linked severe combined immune deficiency: molecular and cellular characterization. Clin Immunol. 2000;95:39-50[CrossRef][Medline] [Order article via Infotrieve]. 19. Brugnoni D, Notarangelo LD, Sottini A, et al. Development of autologous, oligoclonal, poorly functioning T lymphocytes in a patient with autosomal recessive severe combined immunodeficiency caused by defects of the JAK-3 tyrosine kinase. Blood. 1998;91:949-955[Abstract/Free Full Text]. 20. Dukovich M, Wano Y, Le Thi BT, et al. A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors. Nature. 1987;327:518-522[CrossRef][Medline] [Order article via Infotrieve]. 21. Lin JX, Leonard WJ. The immediate-early gene product Egr-1 regulates the human interleukin- 2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites. Mol Cell Biol. 1997;17:3714-3722[Abstract]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gilmour, K. C. Articles by Gaspar, H. B. Search for Related Content PubMed PubMed Citation Articles by Gilmour, K. C. Articles by Gaspar, H. B. Related Collections Immunobiology Brief Reports Social Bookmarking                   What's this?

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