Variation in the Cd3ζ (Cd247) Gene Correlates with Altered T Cell Activation and Is Associated with Autoimmune Diabetes This information is current as Marie Lundholm, Sofia Mayans, Vinicius Motta, Anna of September 25, 2021. Löfgren-Burström, Jayne Danska and Dan Holmberg J Immunol 2010; 184:5537-5544; Prepublished online 16 April 2010; doi: 10.4049/jimmunol.0904012 http://www.jimmunol.org/content/184/10/5537 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/04/16/jimmunol.090401 Material 2.DC1 http://www.jimmunol.org/ References This article cites 51 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/184/10/5537.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! 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The Journal of Immunology Variation in the Cd3z (Cd247) Gene Correlates with Altered T Cell Activation and Is Associated with Autoimmune Diabetes Marie Lundholm,* Sofia Mayans,*,† Vinicius Motta,*,‡,x Anna Lo¨fgren-Burstro¨m,* Jayne Danska,‡,x and Dan Holmberg*,† Tuning of TCR-mediated activation was demonstrated to be critical for lineage fate in T cell development, as well as in the control of autoimmunity. In this study, we identify a novel diabetes susceptibility gene, Idd28, in the NOD mouse and provide evidence that Cd3z (Cd247) constitutes a prime candidate gene for this locus. Moreover, we show that the allele of the Cd3z gene expressed in NOD and DBA/2 mouse strains confers lower levels of T cell activation compared with the allele expressed by C57BL/6 (B6), BALB/c, and C3H/HeJ mice. These results support a model in which the development of autoimmune diabetes is dependent on a TCR signal mediated by a less-efficient NOD allele of the Cd3z gene. The Journal of Immunology, 2010, 184: 5537–5544. Downloaded from ype 1 diabetes (T1D) is a complex disease caused by genetic has been associated with autoimmune diseases, including systemic and environmental factors (1, 2). The NOD mouse is one of lupus erythematosus (SLE) (19, 20) and rheumatoid arthritis (21, 22), T the most extensively studied animal models for T1D, with T cells from these patients displaying a functional impairment. spontaneously developing the disease through a process that re- Together, these data suggest that downregulation of CD3z has func- sembles the pathogenesis in humans (3, 4). Several insulin-dependent tional consequences in systemic autoimmunity (23, 24). This also (Idd) susceptibility loci have been identified in the NOD mouse; agrees with the notion that the strength of the TCR signal is critical http://www.jimmunol.org/ however, the contribution of various factors to the pathogenesis is for cell lineage fate in T cell development (25), as well as in con- largely unknown (5, 6). CTLA-4, a negative regulator of T cell ac- trolling autoimmunity (26). In this article, we demonstrate that the tivity, constitutes one of the genes that has repeatedly been associated NOD allele of Cd3z confers impaired T cell activation, resulting in with susceptibility to human and murine T1D (7–9). NOD T cells deficient CTLA-4 expression, altered cytokine expression patterns, display a defect in CTLA-4 expression after anti-CD3 activation and reduced proliferation. This activation defect can be circum- in vitro; the Idd3/Il-2 locus on chromosome 3 and the Ctex locus on vented by treatment with PMA plus ionomycin, supporting the notion chromosome 1 contribute to the control of this phenotype (10, 11). that this phenotype is conferred by variations in the Cd3z gene. NOD One candidate gene in the Ctex region is Cd3z (Cd247), encoding congenic mice carrying the B6 allele of the Ctex/Cd3z gene display a transmembrane protein in the TCR/CD3 complex containing three a significantly reduced incidence of diabetes compared with litter- by guest on September 25, 2021 ITAMs. CD3z is involved in the TCR signaling cascade, and phos- mates carrying the NOD allele at this locus. This defines a novel di- phorylation of CD3z at tyrosine residues that are present in ITAMs is abetes susceptibility locus denoted Idd28. Moreover, the congenic one of the earliest detectable events that occurs after TCR engage- mice display reduced and/or delayed development of insulitis, sug- ment (12, 13). Moreover, CD3z is important for the expression and gesting that the observed effect on diabetes pathogenesis is mediated assembly of the TCR/CD3 complex on the surface of T lymphocytes by effects on the autoimmune-induced inflammation process. (14, 15). It was reported that NOD mice have a defect in TCR- mediated signaling, and this defect is associated with a block in Ras Materials and Methods activation (16–18). Ras is a GTPase upstream of the MAPK cascade Mice pathway, and its activity is dependent on phosphorylation of CD3z and ZAP-70. In humans, a defective expression of the CD3z-chain C57BL/6 (B6) and NOD mice were originally obtained from Bomholt- gaard (Ry, Denmark). C3H/Hej, BALB/c, and DBA/2 mice were purchased from Taconic Mo¨llegaard and Bomholtgaard. Establishment of NOD.B6- (D1Mit262-D1Mit360)/Hmb (N.C1R1), NOD.B6-(D1Mit104-D1Mit360)/ *Department of Medical Biosciences, Medical and Clinical Genetics, Umea˚ Univer- Hmb (N.C1R11), and B6.NOD-(D1Mit262-D1Mit360)/Hmb (B.R1) mice sity, Umea˚, Sweden; †Department of Disease Biology, Facility of Life Sciences, used for this study have been described previously (11). B6-(D1Mit3- Copenhagen University, Copenhagen, Denmark; and ‡Department of Immunology x D1Mit117)/Hmb (NOD.C1), was obtained by crossing the B.R1 strain to B6 and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, and screening for new recombinations. N.C1R1 mice were backcrossed 10 Canada times to NOD and intercrossed 3 to 8 times, N.C1R11 mice were backcrossed Received for publication December 17, 2009. Accepted for publication March 16, 12 times to NOD and intercrossed 2 to 3 times, and B.R1 mice were back- 2010. crossed 8 times to B6 and intercrossed 5 to 6 times. When backcrossing, This work was supported by grants from the NOVO-Nordisk Fonden, the Swedish offspring were screened using mouse low density linkage (Illumina, San Diabetes Foundation, the Juvenile Diabetes Foundation, the Swedish Research Diego, CA), with markers spread over the genome (Supplemental Table I). Council, and the Danish Research Council. Sex- and age-matched animals were used at 7–9 wk of age. The incidence of ∼ Address correspondence and reprint requests to Dr. Dan Holmberg, Department of diabetes in our NOD colony reaches 50% in females at 25 wk of age. Di- Disease Biology, Life Sciences Faculty, Copenhagen University, Ridebanevej 9, 1870 abetes was analyzed weekly from 10 wk of age, using colorimetric test strips Frederiksberg C, Copenhagen, Denmark. E-mail address: [email protected] for glucosuria. All animals were maintained in a specific pathogen-free en- vironment at the animal facilities of Umea˚ University. The online version of this article contains supplemental material. Abbreviations used in this paper: Ct, cycle threshold; MFI, mean fluorescent inten- Isolating and culturing cells sity; NCBI, National Center for Biotechnology Information; SLE, systemic lupus erythematosus; SNP, single-nucleotide polymorphism; T1D, type 1 diabetes. Cell suspensions were prepared from spleens of 7–9-wk-old mice in HBSS. Erythrocytes were depleted with Gey’s lysis buffer. Cells were cultured at 6 Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 2 3 10 /ml in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, www.jimmunol.org/cgi/doi/10.4049/jimmunol.0904012 5538 VARIATION IN Cd3z CORRELATES WITH DIABETES 1 mM sodium pyruvate, penicillin (100 U/ml), streptomycin (100 mg/ml), ELISA kits (Mouse ELISA Max Deluxe, BioLegend, San Diego, CA), and 50 mM/l 2-ME. Total spleen cells were stimulated with soluble anti- according to the manufacturer’s instructions. CD3 (4 mg/ml clone; 145 2C11, BD Pharmingen, San Diego, CA) or PMA (10 ng/ml; Sigma-Aldrich, St. Louis, MO) together with ionomycin (1 mg/ Reverse transcription followed by quantitative PCR ml; Sigma-Aldrich). For sorting, total spleen cells were erythrocyte depleted Cultured spleen cells were washed once with PBS and stored at 280˚C. RNA with Gey’s lysis buffer and then stained with anti-mouse CD8-Alexa (clone was prepared from the cells using RNeasy Mini Kit (Qiagen, Valencia, CA), 53-6.7) and anti-mouse CD4-PerCP-Cy5.5 (clone RM4-5) from BD Bio- according to the manufacturer’s instructions, and dissolved in 40 ml RNase- sciences (San Jose, CA). Cells were then sorted by flow cytometry in . free water, followed by treatment with DNase I (Ambion, Austin, TX). RNA a FACSDiva (BD Biosciences); the purity of sorted populations was 95%. concentrations were measured using a Nanodrop spectrophotometer, and Sorted CD4+ or CD8+ T cells were activated by plate bound anti-CD3 (10 cDNAwas prepared from 300 ng total RNA using the Reverse Transcription mg/ml; clone 145 2C11) and anti-CD28 (10 mg/ml; clone 37.51, BD Phar- Reagents (TaqMan, Applied Biosystems, Foster City, CA), according to the mingen).