Variation in the Cd3ζ (Cd247) 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! Every submission reviewed by practicing scientists

• 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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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 3 and the Ctex locus on vented by treatment with PMA plus ionomycin, supporting the notion 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 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). Cells were incubated for 48 h at 37˚C in 5% CO2 before analysis. manufacturer’s instructions. The following primers and probes were used to Flow cytometry analysis measure RNA expression: for the full-length CTLA-4, forward primer: 59- GGACGCAGATTTATGTCATTGATC-39, reverse primer: 59-CCAAGCTA- Abs used for FACS analysis included anti-CD4 FITC (clone H129.19), anti- ACTGCGACAAGGA-39,probe:59-(FAM)-AGAACCATGCCC-GGATTC- CD4PerCP(cloneRM4-5),anti-CD8PerCP(clone53-6.7),anti-CD69biotin TGACTTCC-(TAMRA)-39; acidic ribosomal phosphoprotein (36B4): for- (clone H1.2F3), anti-CD152 (CTLA-4) PE (clone UC10 4F10-11), and ICOS ward primer 59-CCCTGAAGTGCTCGACATCA-39, reverse primer 59-TGC- PE(clone7E.17G9).AllAbswerepurchasedfromBDBiosciences.After48h GGACACCCTCCAGAA-39, probe 59-(VIC)-AGAGCAGGCCCTGCACT- of culture, cells were harvested, stained, and analyzed by flow cytometry CTCGC-(TAMRA)-39;andb-actin: forward primer 59-GGACCTGACGG- (FACSCalibur, BD Biosciences). Forintracellular staining, cellswere stained ACTACCTCATG-39, reverse primer 59-TCTTTGATGTCACGCACGATTT- withanti-CD4FITC,anti-CD8PerCP,andanti-CD69biotin,andwashedonce 39, probe 59-(VIC)-CCTGACCG-AGCGTGGCTACAGCTTC-(TAMRA)-39. in FACS medium (PBS containing 3% FCS and 0.05% sodium azide), fol- Expression of the genes coding for 36B4 and b-actin was used as en- Downloaded from lowed by the addition of streptavidin-allophycocyanin conjugate. Cells were dogenous control. CTLA-4, 36B4, and b-actin primers and probes were fixated with 1% paraformaldehyde for 30 min at room temperature and designed using Primer Express (Applied Biosystems). TaqMan Mouse permeabilized with 0.5% saponin. After fixation and permeabilization, cells Assay-on-demand (NM031162, gene-expression product consisting of were stained with anti-CD152 PE Ab, for the detection of CTLA-4, or anti- amplification primers and a fluorescently labeled TaqMan probe formulated ICOS PE, for the detection of ICOS, in the cytoplasm. For CD3z intracellular into a single tube; Applied Biosystems) was used for the measurement of staining, extracellular staining was performed as above and then the cells total CD247 expression. Custom TaqMan Mouse Assay-on-demand was were fixated with 0.5% paraformaldehyde for 20 min. at room temperature, used to measure expression of the isoform CD3z. All reagents for real-time washed in 0.05% Tween/PBS, and incubated with 2.4G2 (CD16/CD32) to PCR analysis were purchased from Applied Biosystems. Relative expres- http://www.jimmunol.org/ avoid unspecific binding. For permeabilization, 10 g/ml digitonin solution sion of the transcripts was measured with the ABI Prism 7900HT Sequence- was used while adding anti-CD3z FITC (clone H146-968, Cedarlane Lab- detection system (Applied Biosystems) and determined by relative RNA oratories, Hornby, Ontario, Canada). For scoring regulatory cells, intra- quantification using the comparative cycle threshold (Ct) method, as de- cellular Foxp3 staining was performed according to the manufacturer’s scribed in the Applied Biosystems user’s bulletin (27). Briefly, the amount instructions (FITC anti-mouse/rat Foxp3 staining set, eBioscience). of target transcript normalized to an endogenous control gene and relative to a calibrator sample (total RNA prepared from naive B6 spleen cells) is given 2DDCt Cell proliferation by the formula: 2 , where ΔΔCt =(Cttarget 2 Ctendogenous control) sample in study 2 (Cttarget2 Ctendogenous control) calibrator. T cell proliferation was measured by flow cytometry using CFSE (CellTrace CFSE Cell Proliferation Kit, Invitrogen, Carlsbad, CA). Briefly, 4 3 106 Sequencing of the CD3z and CD3h isoforms cells/ml were incubated with 1 mM CFSE at 37˚C, washed with ice-cold by guest on September 25, 2021 3 6 culture medium (containing 50% FCS), and then added to the culture at 0.4 Splenocyte RNA was prepared from 5 10 cells using the RNeasy Mini Kit 3 106 cells/well in a 96-well plate. Forty-eight hours later, cells were (Qiagen). cDNA fragments were amplified by RT-PCR using the Super- stained with anti-CD4 allophycocyanin (clone RM4-5) or anti-CD8 allo- ScriptVilo cDNA Synthesis Kit (Invitrogen), with 1 mg total RNA as template. phycocyanin (clone 53-6.7). Dividing cells were detected by flow cy- The resulting cDNA samples were diluted 10 times to achieve the concentra- tometry (FACSCalibur, BD Biosciences), and the analysis was performed tion equivalent of starting with 100 ng RNA. Four PCR fragments were am- using FlowJo software (v. 8.8.4, Tree Star, Ashland, OR). plified and sequenced. Sequences were examined using Lasergene software (DNASTAR, Madison, WI). PCR fragments were amplified using the fol- Cytokine determination lowing primer pairs: forward primer 59-TGTCAGCCACAGAACAAAGC-39 and reverse primer 59-CTGGTAAAGGCCATCGTGC-39 (exons 1–7 of CD3z IL-2 and -4 and IFN-g cytokine production in cultured supernatants col- and CD3h); forward primer 59-CCTACAGTGAGATCGGCACA-39 and re- lected 48 h after stimulation was determined using commercially available verse primer 59-CTTCTTTGAGCCACCTCTGG-39 (exons 6–8 of CD3z);

FIGURE 1. Expression of CD3z is impaired in ac- tivated NOD T lymphocytes. MFI values of CD3z in- tracellular expression assessed by flow cytometry with gating on CD4+CD69+ (A) or CD8+CD69+ (C) T cells from B6 (n = 15), N.C1R11 (n = 14), and NOD (n = 15) mice. Horizontal bars represent the mean value within each group, and each symbol represents an individual mouse. Four independent experiments are combined and shown as relative values to the average of the MFI of activated B6 CD3z intracellular expression (93.4 6 3.7) (mean 6 SD from one representative experiment). Statistical significance was determined with the Mann- Whitney U nonparametric test (two-tailed). Represen- tative graphs of intracellular expression of CD3z in CD4+ (B) or CD8+ (D) T cells. Shaded graphs represent NOD mice, dashed-line graphs represent N.C1R11 mice, and open graphs represent B6 mice. The Journal of Immunology 5539

Table I. Physical location of the Ctex region in the congenic strains Results Defective expression of CD3z and CTLA-4 in NOD T cells NCBI after activation Distance Position Marker (cM) (bp) B.R1 N.C1R1 N.C1R11 As previously reported, the NOD allele in the Ctex locus on mouse D1Mit262 67 131165205 B6 NOD NOD chromosome 1 confers defective expression of CTLA-4 upon anti- D1Mit30 70 135259675 NOD B6 NOD CD3 activation of T cells in vitro (Supplemental Fig. 1) (11). The D1Mit194 71.5 148879591 NOD B6 NOD chromosomal region identified as Ctex is ∼30 Mb, including ∼375 D1Mit104 79 155528790 NOD B6 NOD genes. Among these, the Cd3z (Cd247) gene stands out as one D1Mit267 81.6 157938047 NOD B6 B6 D1Mit159 81.6 161592104 NOD B6 B6 plausible candidate gene in the Ctex region because it is involved in D1Mit15 87.9 170288805 NOD B6 B6 the TCR-mediated activation of T cells. In addition, Cd3z is located D1Mit353 92.3 172169777 NOD B6 B6 at 87.2 cM, close to marker D1Mit15, which also displayed the D1Mit403 100 177571382 NOD B6 B6 strongest linkage in a previous mapping study (11). Together, this D1Mit360 101.2 184353045 B6 NOD NOD makes Cd3z a strong candidate gene for the Ctex locus. CD3z is Boundaries of congenic regions on chromosome 1 are given in marker position essential for TCR complex signaling; therefore, the predicted effect and in bp, as retrieved using the NCBI database, version 37.1. The position of each marker in the linkage map is indicated in cM and was retrieved from the Mouse of a deficient CD3z would be T cell intrinsic. To directly address + Genome Informatics database. The congenic region for each strain is indicated by the this, we compared the induction of CTLA-4 in sorted CD4 and genotype at each marker. CD8+ T cells from NOD and B6 mice and found that the defective NCBI, National Center for Biotechnology Information. CTLA-4 expression upon activation is intrinsic to NOD T cells. In Downloaded from CD4+ T cells, the expression of CTLA-4 was 17.6% in B6 mice and forward primer 59-GCTCCTGCTGTAAATTTGGC-39 and reverse primer 59- 7.0% in NOD mice (p = 0.05); in CD8+ T cells, the expression of ATTGCTACCCCAGGCTTCAC-39 (exon 8 of CD3z); and forward primer 59- CTLA-4 was 33.4% in B6 mice and 10.2% in NOD mice (p = 0.05). CCTACAGTGAGATCGGCACA-39 and reverse primer 59-CTTCTCCGG- CCCTTTCAAC-39 (exons 6–9 of CD3h). Primers were designed based on To rule out the possibility that the observed difference in CTLA-4 sequence information of transcripts ENSMUST00000005907 (Cd3z)andEN- expression between NOD and B6 mice was a result of differences in SMUST00000027849 (Cd3h) of the gene ENSMUSG00000005763 (Cd247) activation through the TCR, we next analyzed ex vivo surface CD3ε available from Ensembl (www.ensembl.org/Mus_musculus/Info/Index). expression by flow cytometry. No difference in the expression of http://www.jimmunol.org/ Statistical analysis CD3ε was detected between T cells of NOD (32.8%) or B6 (28.5%) origin analyzed ex vivo. When T cells were stimulated in vitro with The Mann-Whitney U and Kruskal–Wallis H nonparametric (two-tailed) + tests were used to compare phenotypic differences between the groups of anti-CD3ε, CD4 T cells of NOD origin displayed a lower CD3z animals. The level of significance was considered p , 0.05. mean fluorescent intensity (MFI) compared with CD4+ T cells from by guest on September 25, 2021

FIGURE 2. CTLA-4 protein expression in B6, NOD, and different Ctex congenic mouse strains upon anti-CD3ε or PMA plus ionomycin stimulation in vitro. Total spleen cells were stimulated in vitro with anti-CD3ε or PMA plus ionomycin for 48 h. After culture, CTLA-4 extracellular protein expression in T cells activated by anti-CD3 (B6, n = 16; N.C1R1, n = 11; N.C1R11, n = 9; NOD, n = 16; and B.R1, n =6)orbyPMAplusionomycin (B6, n =9;N.C1R1, n =4;N.C1R11, n =8;NOD,n =8;andB.R1, n = 4). The T cells were analyzed by flow cytometry, and CTLA-4+ cells were gated as illustrated in Supplemental Fig. 1. CD8+CD69+ (A)andCD4+CD69+ (B)CD3ε-activated T cells. The values plotted are relative to the average percentage of B6 CTLA-4+ cells for CD8+CD69+ (50.0% 6 8.6%) and CD4+CD69+ (23.5% 6 8.0%) (mean 6 SD of one representative experiment). B6, N.C1R1,andN.C1R11 CD8+ T cells express significantly higher levels of CTLA-4 compared with NOD and B.R1 CD8+ T cells (p , 0.004). The expression of CTLA-4 in CD4+ T cells of B6, N.C1R1,N.C1R1,andB.R1 mice was significantly higher than that observed for NOD CD4+ Tcells(p , 0.001). N.C1R1,NOD,andB.R1 CD4+ T cells showed significantly lower expression of CTLA-4 compared with B6 CD4+ T cells (p , 0.001). Statistical significance was determined with the Kruskal–Wallis H and the Mann-Whitney U (two-tailed) nonparametric tests. CD8+CD69+ (C)and CD4+CD69+ (D) PMA plus ionomycin–activated T cells. The values provided are relative to the average percentage of B6 CTLA-4+ cells for CD8+ CD69+ (67.1% 6 2.1%) and CD4+CD69+ (23.9% 6 2.0%) (mean 6 SD of one representative experiment). The p values for Fig. 2 are given in Supplemental Table III. Horizontal bars indicate the mean value within each group, and each symbol represents an individual mouse. Data were compiled from three separate experiments. 5540 VARIATION IN Cd3z CORRELATES WITH DIABETES

B6 and N.C1R11 mice (Fig. 1A,1B). This difference in CD3z ex- pression was not observed in CD8+ T cells (Fig. 1C,1D). NOD allele of the Ctex/Cd3z confers defective induction of CTLA-4 expression We next constructed congenic mouse strains carrying different-sized chromosomal regions of the Ctex region containing the C57BL/6 (B6) allele on the NOD genetic background [ NOD.B6-(D1Mit262- D1Mit360)/Hmb (N.C1R1) and NOD.B6-(D1Mit104-D1Mit360)/ Hmb (N.C1R11)] or reciprocal congenic mice carrying the NOD allele on the C57BL/6 genetic background [B6.NOD-(D1Mit262- D1Mit360)/Hmb (B.R1)] (Table I). The congenic mice were ho- mozygous over the Ctex region in all markers tested (Table I). To further define and confirm that the Ctex region controls CTLA-4 expression, spleen cells from B6, N.C1R1,N.C1R11, NOD, and B. R1 mice were stimulated in vitro with anti-CD3ε. After 48 h of culture, cells were harvested and stained for CTLA-4 expression. As illustrated in Fig. 2A and 2B, CD3ε-activated T cells from N.

C1R1 and N.C1R11 mice expressed CTLA-4 at levels similar to FIGURE 4. Defective CTLA-4 protein expression in regulatory and Downloaded from T cells derived from the parental B6 strain, whereas the B.R1 strain effector CD4+ T cells from NOD mice. Spleen cells from B6 (A, n = 16; C, displayed a phenotype similar to the NOD mouse. The CTLA-4 n = 10), N.C1R11 (A, n = 10; C, n = 5), and NOD (A, n = 16; C, n = 10) reconstitution of the parental phenotype after anti-CD3ε activation mice were cultured with anti-CD3ε for 48 h and then harvested for flow was almost complete in CD8+ T cells but was only partial in CD4+ cytometry analysis. Individual percentages of surface CTLA-4 in regula- + T cells. This supports previous observations suggesting that addi- tory (A) or effector (C) CD4 T cells. Horizontal bars represent the mean value within each group, and each symbol represents one mouse. Statistical

tional loci contribute to the control of this phenotype (11). Fur- http://www.jimmunol.org/ thermore, the defect in CTLA-4 expression conferred by the NOD significance was determined with the Mann-Whitney U nonparametric test (two-tailed). Data were compiled from three independent experiments. B, allele was evident at the RNA level for the full-length CTLA-4 Representative dot plots of cell-surface expression of CTLA-4 on CD4+ isoform (Fig. 3) (11). These results confirmed that the Ctex/Cd3z + + 2 + + FoxP3 or CD4 FoxP3 T cells. Numbers indicate the percentage of locus controls CTLA-4 expression in CD4 and CD8 T cells and CTLA-4+ cells in each of these populations. restricted the region of interest to 28.8 Mb. CTLA-4 plays an important role in the function of CD4+CD25+ + Foxp3 regulatory T cells (28–30); therefore, we specifically an- levels of CTLA-4 expression compared with the parental strains alyzed the expression of CTLA-4 in this subset. A significantly B6 and NOD (Fig. 4). + lower frequency of Foxp3 T cells expressed CTLA-4 in NOD Activated T cells of NOD origin were reported to express higher by guest on September 25, 2021 compared with B6 mice (Fig. 4A,4B). Defective upregulation of levels of ICOS (32). Although Ctex constitutes the major locus + 2 CTLA-4 was also observed in CD4 Foxp3 T cells of NOD origin involved in the control of CTLA-4 expression, we observed that this (Fig. 4C,4D). This demonstrated that the impaired upregulation of locus does not influences ICOS protein expression in NOD mice CTLA-4 in NOD mice occurs to the same extent in regulatory and (Supplemental Fig. 2). Instead, as previously reported (11, 31), effector T cell subsets. Also, in this case, the allele-specific ex- higher ICOS levels in NOD mice seem to be controlled by gene(s) + + + 2 pression pattern was confirmed in CD4 Foxp3 and CD4 Foxp3 in the Idd5.1 region, possibly as a result of polymorphism(s) in T cells of N.C1R11 congenic origin, which displayed intermediate the Icos gene itself. Defective T cell activation conferred by the NOD allele of Ctex/Cd3z can be overcome by PMA plus ionomycin CD3z impairment would most probably lead to defects in CD3ε- mediated signaling, including effects at the level of proliferation and expression of activation-dependent genes. To test this, we an- alyzed IL-2 and -4 and IFN-g cytokine secretion and proliferation in B6, NOD.C1R11, and NOD spleen cells 48 h after stimulation with anti-CD3ε. As illustrated in Fig. 5A–C, although NOD spleen cells secreted lower amounts of IL-2 and -4 and IFN-g cytokines compared with B6 mice, N.C1R11 mice displayed IL-2 and IFN-g levels intermediate to B6 mice and IL-4 secretion exceeding that of B6 and NOD spleen cells. CD4+ T cells from B6 mice had a higher proliferation rate compared with CD4+ T cells from NOD mice, FIGURE 3. CTLA-4 RNA expression in B6, NOD, and various Ctex which was evident from CFSE dilution profiles and division plots congenic mouse strains upon anti-CD3 stimulation in vitro. CTLA-4 RNA (Fig. 5D,5E). This difference in proliferation between NOD and B6 expression was measured by real-time PCR. Total RNA was prepared from mice was not observed in CD8+ T cells (Fig. 5D,5E). As shown in stimulated spleen cells obtained from the various mouse strains (n $ 5 per + 2DDCt Fig. 4D and 4E, CD4 T cells from N.C1R11 mice displayed an mouse strain), and the amount of RNA expression was measured as 2 . + The percentages displayed are relative to the 22DDCt average of B6 mice intermediate proliferation rate compared with CD4 T cells from (22.4% 6 2.4%). Horizontal bars represent the mean value within each B6 and NOD mice. Together, these data support the hypothesis that group, and each symbol represents an individual mouse. Statistical signifi- the NOD allele of Cd3z [or other gene(s) in the Ctex chromosomal cance was determined with the Kruskal–Wallis H and Mann-Whitney U region] confers hypoproliferation and decreased production of (two-tailed) nonparametric tests. IFN-g and IL-2 and -4 in the NOD mouse. The Journal of Immunology 5541

FIGURE 5. The NOD allele of the Ctex/Cd3z region confers impaired proliferation and cytokine expression patterns. A–C, Spleen cells from 8-wk- old B6, N.C1R11, and NOD mice (n $ 3 per mouse strain) were cultured in the presence of anti-CD3ε or PMA plus ionomycin for 48 h. Levels of IL-2 (A), IFN-g (B), and IL-4 (C) in culture supernatant were determined by ELISA. Data were compiled from two independent experiments and are shown relative to the average values for cytokine release measured in B6 cultures (mean 6 SD of one representative experiment). For anti-CD3ε stimulation, this was 146.1 6 6.7 pg/ml for IL-2, 39.5 6 6.6 ng/ml for IFN-g, and 22.6 6 4.0 pg/ml for IL-4. For PMA plus ionomycin stimulation, this was 201.3 6 4.8 pg/ml for IL-2, 155.1 6 51.2 ng/ml for IFN-g, and 21.5 6 5.2 pg/ml for IL-4. D and E, CFSE-labeled spleen cells from 8-wk-old B6 (n = 6), N.C1R11 (n = 7), and NOD (n = 6) mice were stimulated in vitro with anti-CD3ε for 48 h or left unstimulated. D, Repre- sentative CFSE graphs of CD8+ T cells or CD4+ Downloaded from T cells stimulated with anti-CD3ε for 48 h (open graphs) or left unstimulated (shaded graphs). E, Percentages of divided cells of stimulated CD8+ or CD4+ T cells from two independent experiments. Horizontal bars represent the mean value within each group, and each symbol represents one mouse. http://www.jimmunol.org/ The Kruskal–Wallis H and Mann-Whitney U (two- tailed) nonparametric tests were used to identify statistical significance. There was no significant statistical difference in cytokine expression between the mouse strains after PMA activation.

The phorbol ester PMA and the calcium ionophore ionomycin The Cd3z splice variant uses exons 1–8, whereas the Cd3h variant fully trigger activation of T cells, bypassing the cross-linking of the uses exons 1–9 but skips exon 8 (Supplemental Fig. 3A). We se-

TCR/CD3 complex (32). Therefore, we reasoned that the addition quenced the message of the two splice variants of the NOD mouse by guest on September 25, 2021 of PMA plus ionomycin should improve the impaired T cell acti- and did not find polymorphisms compared with the B6 reference vation observed in NOD T cells if it was conferred by the NOD sequences (NM_001113391.1, NM_031162.3 [i.e., exonic varia- allele of Cd3z. To test this hypothesis, we examined IL-2 and -4 and tion was not found in the nine exons of the Cd247 gene comparing IFN-g cytokine secretion and CTLA-4 expression in B6, NOD, and the NOD and B6 sequences]) (Supplemental Fig. 3B,3C). A N.C1R11 congenic mice after PMA plus ionomycin activation. As search of the Mouse Phenome Database (www.jax.org/phenome) illustrated in Fig. 2C and 2D, PMA plus ionomycin stimulation identified 58 single-nucleotide polymorphisms (SNPs) in the Cd3z resulted in a comparable expression of CTLA-4 in CD8+ and CD4+ sequence that differed between the NOD and B6 mouse strains. T cells from B6, NOD, and N.C1R11 congenic mice. PMA plus Scrutinizing this genomic region in a set of other mouse strains, 23 ionomycin activation also restored the cytokine secretion in T cells SNPs located within the first intron of the gene were identified that carrying the NOD allele of Ctex/Cd3z (Fig. 5A–C). Together, these defined a SNP haplotype common to NOD and DBA/2 versus B6, observations demonstrated that defective CD3 signaling in NOD BALB/c and C3H/Hej. Based on this information, we selected T cells can be overcome by bypassing events in the TCR/CD3 DBA/2 carrying the NOD allele and C3H/Hej and BALB/c carrying signaling cascade that occur upstream of the induction of calcium the B6 allele of Cd3z for additional experimental work (Supple- fluxes. mental Table II). To test whether these allelic differences in Cd3z could underlie the observed defect of CTLA-4 induction observed The Ctex/Cd3z region constitutes a novel Idd affecting the in the NOD mouse, we stimulated NOD, B6, C3H/Hej, BALB/c, incidence of diabetes The defective T cell activation conferred by the NOD allele of the Ctex/Cd3z gene suggested that this locus could constitute a novel Idd diabetes-susceptibility locus. To directly test this hypothesis, we screened female NOD and N.C1R11 mice for the development of diabetes. Scoring of diabetes showed that N.C1R11 congenic mice developed clinical diabetes with a significantly lower frequency compared with NOD mice (Fig. 6). These data provide evidence that anovelIdd (Idd28) is located in this chromosomal region.

Defective CTLA-4 expression is conferred by the NOD allele of Cd3z (Cd247) in various genetic backgrounds FIGURE 6. Decreased incidence of diabetes in N.C1R11 mice. The cumulative incidence of diabetes is significantly higher in female NOD The murine Cd247 gene consists of nine exons covering ∼79 kb, mice (n = 40) compared with female N.C1R11 mice (n = 40). The log- with the first intron spanning .66 kb (www.informatics.jax.org). test was used to calculate the statistical significance. 5542 VARIATION IN Cd3z CORRELATES WITH DIABETES Downloaded from

FIGURE 7. Strains carrying the Cd3z (Cd247) alleles from B6 or NOD mice show phenotypic correlation. Spleen cells from B6 (n = 7), BALB/c (n = 4), C3H/HeJ (n = 4), NOD (n = 7), and DBA/2 (n = 4) mice were activated in vitro with anti-CD3ε or PMA plus ionomycin. Forty-eight hours later, cells were harvested and analyzed by flow cytometry, and CTLA-4+ cells were gated as illustrated in Supplemental Fig. 1. CD8+CD69+ (A) and CD4+CD69+ (B) CD3ε-stimulated T cells. The values are relative to the average percentage of CTLA-4+ cells from B6 CD8+CD69+ (42.3% 6 9.9%) and CD4+CD69+ (25.9% 6 8.3%) T cells (mean 6 SD). B6, BALB/c, and C3H/HeJ mice expressed significantly more CTLA-4 on CD3ε-activated CD8+ and CD4+ T cells

compared with NOD and DBA/2 CD3ε-activated T cells (p , 0.02). The Kruskal–Wallis H and Mann-Whitney U (two-tailed) nonparametric tests were http://www.jimmunol.org/ used to calculate statistical significance. CD8+CD69+ (C) and CD4+CD69+ (D) PMA plus ionomycin-activated T cells. The values are relative to the average percentage of B6 CTLA-4+ cells for CD8+CD69+ (67.1% 6 2.1%) and CD4+CD69+ (23.9% 6 2.0%). The p values are shown in Supplemental Table IV. The horizontal bars represent the mean value within each group, and each symbol represents an individual mouse. and DBA/2 spleen cells in vitro with anti-CD3 or PMA plus Reciprocal B6.R1 congenic mouse strain carrying the NOD Ctex ionomycin. As illustrated in Fig. 7, CD3-activated T cells from region on the B6 background displayed an impaired T cell acti- BALB/c and C3H/Hej mice expressed CTLA-4 at levels similar to vation similar to the NOD mouse, as demonstrated by the reduced

T cells derived from the B6 strain, whereas the DBA/2 strain CTLA-4 expression after anti-CD3 activation. This supported the by guest on September 25, 2021 displayed a phenotype similar to the NOD mouse. Together, these notion that the deficient T cell activation observed in NOD mice is observations suggest a phenotypic correlation between strains not the result of an ongoing autoimmune inflammatory process. carrying the 23 identified SNPs in the Cd3z alleles of NOD or B6 This was further underscored by the observation that the non- mice. autoimmune prone strain DBA/2, carrying the NOD allele of Cd3z gene, mediates defective CTLA-4 expression, whereas C3H/Hej Discussion and BALB/c, carrying the B6 allele, do not. One way to un- CD3z functions by amplifying the TCR signaling cascade and is equivocally determine whether the effect seen is due to poly- necessary for assembly of the TCR/CD3 complex on the surface of morphisms in the Cd3z gene is to generate a Cd3z gene knock-in T lymphocytes (14, 15). It is well established that defective CD3z mouse and include it in the study. function leads to impaired activation of T cells upon engagement of The fact that impaired cytokine secretion and defective CTLA-4 the TCR (33, 34). Moreover, mutations in different molecules of the expression could be overcome by PMA plus ionomycin stimulation TCR/CD3 signaling pathways affect the lineage-commitment de- suggested that the observed defect in T cell activation is mediated by cisions during T cell development (25), as well as the development a factor in the TCR signaling pathway, upstream of the protein kinase of autoimmunity (26). In line with this, defects in CD3z expression/ C, and is consistent with the notion of Cd3z mediating this effect. It function were suggested to have implications for the development of was reported that NOD mice have a defect in the TCR signaling human autoimmune phenotypes (24, 35, 36). Thus, several groups cascade mediated through the protein kinase C/Ras/MAPK path- reported altered expression and function of CD3z in patients with way (16–18). This NOD-specific trait, manifested by T cell hypo- SLE (19, 20) or rheumatoid arthritis (21, 22). Moreover, family- responsiveness with reduced IL-2 and -4 secretion, was genetically based association studies of SLE showed that polymorphisms in the mapped to gene(s) in the Idd4.2 region on chromosome 11 (39, 40). CD3z 39-untranslated region are associated with defective CD3z The fact that the B6 allele of the Ctex/Cd3z region only partially expression in SLE patients (37). These findings suggest that ab- restored the B6 IL-2 and IFN-g cytokine expression pattern and normal CD3z expression caused by variation in this gene can have proliferation profile is compatible with a complementary function functional consequences manifested by systemic autoimmunity. of the Idd4 and Ctex/Cd3z genes. In line with this, the less pro- The molecular mechanisms responsible for the defective expression nounced effects on cytokine expression and proliferation compared of CD3z in these diseases are diverse (reviewed in Refs. 23, 38). with CTLA-4 expression in the N.C1R11 mice may reflect different In the current study, we demonstrated that the NOD allele in the effects of CD3z expression on downstream signaling and in- Ctex/Cd3z locus confers reduced T cell activation, leading to re- teraction with other genetic factors, including the Idd4.2 locus. duced proliferative response, altered cytokine expression, and Direct analysis of CD3z protein expression demonstrated a dif- reduced expression of the CTLA-4 activation marker. This phe- ference between NOD and B6 mice after anti-CD3ε activation in notype could be restored, in part, by the B6 allele in this locus. CD4+, but not CD8+, T cells. This may be a reflection of disparate The Journal of Immunology 5543 function and activation requirements in CD4+ and CD8+ T cells (41, Acknowledgments 42). In contrast to protein expression, neither the expression of We thank Drs. A-M Wegener, A. Schmidt-Kristensen, and S. Gupta for help- specific CD3z RNA nor of total CD247 RNA differed between the ful discussions. strains. Because total anti-CD3–activated spleen cells were used in these experiments, further analyses of RNA expression in separate Disclosures cellular subsets needs to be performed to rule out defects at the The authors have no financial conflicts of interest. transcriptional level. Allele-specific differences in mRNA expres- z sion were reported for the human CD3 gene and suggested to be References the result of differential posttranscriptional gene regulation (37). 1. Castan˜o, L., and G. S. Eisenbarth. 1990. Type-I diabetes: a chronic autoimmune Therefore, similar effects of variants of the mouse homolog should disease of human, mouse, and rat. Annu. Rev. Immunol. 8: 647–679. also be investigated. 2. Vyse, T. J., and J. A. Todd. 1996. Genetic analysis of autoimmune disease. Cell 85: 311–318. Low expression of CD3z has been ascribed to a population of 3. Delovitch, T. L., and B. Singh. 1997. The nonobese diabetic mouse as a model of T cells associated with the promotion of a proinflammatory envi- autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7: 727–738. ronment, providing a possible mechanism for how deficient CD3z 4. Makino, S., K. Kunimoto, Y. Muraoka, Y. Mizushima, K. Katagiri, and Y. Tochino. 1980. Breeding of a non-obese, diabetic strain of mice. Jikken could promote inflammation and autoimmunity (37). Alternative Dobutsu 29: 1–13. possibilities could be inferred from the analysis of CD3z knockout 5. Ridgway, W. M., L. B. Peterson, J. A. Todd, D. B. Rainbow, B. Healy, mice (33, 34, 43, 44). In these mice, the interrupted TCR signaling O. S. Burren, and L. S. Wicker. 2008. Gene-gene interactions in the NOD mouse model of type 1 diabetes. Adv. Immunol. 100: 151–175. caused by the CD3z deficiency leads to a shift from negative to 6. Wicker, L. S., J. A. Todd, and L. B. Peterson. 1995. Genetic control of auto- positive selection of autoreactive T cells in the thymus (45). immune diabetes in the NOD mouse. Annu. Rev. Immunol. 13: 179–200. Downloaded from 7. Hill, N. J., P. A. Lyons, N. Armitage, J. A. Todd, L. S. Wicker, and Moreover, CD3z knockout mice display a low cell-surface expres- L. B. Peterson. 2000. NOD Idd5 locus controls insulitis and diabetes and sion of CTLA-4/CD28 and an altered cytokine-expression profile overlaps the orthologous CTLA4/IDDM12 and NRAMP1 loci in humans. Di- resembling the impairments observed in mice carrying the NOD abetes 49: 1744–1747. z 8. Ueda, H., J. M. Howson, L. Esposito, J. Heward, H. Snook, G. Chamberlain, allele of Cd3 (46). D. B. Rainbow, K. M. Hunter, A. N. Smith, G. Di Genova, et al. 2003. Asso- The incidence of T1D is lower in N.C1R11 mice compared with ciation of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune NOD mice, thus defining a novel diabetes-susceptibility locus disease. Nature 423: 506–511. http://www.jimmunol.org/ z 9. Wicker, L. S., G. Chamberlain, K. Hunter, D. Rainbow, S. Howlett, P. Tiffen, tentatively named Idd28. The fact that allelic variations in the Cd3 J. Clark, A. Gonzalez-Munoz, A. M. Cumiskey, R. L. Rosa, et al. 2004. Fine gene with the NOD/DBA allele conferred reduced CTLA-4 in- mapping, gene content, comparative sequencing, and expression analyses sup- duction compared with the B6/C3H/BALB allele upon anti-CD3 port Ctla4 and Nramp1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse. J. Immunol. 173: 164–173. stimulation makes the Cd3z gene a prime candidate. This would be 10. Colucci, F., M. L. Bergman, C. Penha-Gonc¸alves, C. M. Cilio, and D. Holmberg. in line with the observed effects of T cell signaling strength on the 1997. Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked development of autoimmunity. Evidence from the study of human to the Idd5 diabetes susceptibility region. Proc. Natl. Acad. Sci. USA 94: 8670– 8674. T1D and from the NOD mouse model of the disease has identified 11. Lundholm, M., V. Motta, A. Lo¨fgren-Burstro¨m, N. Duarte, M. L. Bergman, several common susceptibility genes that are involved in the control S. Mayans, and D. Holmberg. 2006. Defective induction of CTLA-4 in the NOD mouse is controlled by the NOD allele of Idd3/IL-2 and a novel locus (Ctex) by guest on September 25, 2021 of T cell activation, including Ctla4/CTLA-4, Il2/IL2R, and Ptpn8/ telomeric on chromosome 1. Diabetes 55: 538–544. PTPN22 (47). The observations in this study suggest that the Cd3z 12. Samelson, L. E., M. D. Patel, A. M. Weissman, J. B. Harford, and gene could be added to this set of susceptibility genes affecting R. D. Klausner. 1986. Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen . Cell control of T cell activity. It is known that subtle missense mutations 46: 1083–1090. in T cell signaling can lead to dysregulation and autoimmunity (48), 13. Baniyash, M., P. Garcia-Morales, E. Luong, L. E. Samelson, and R. D. Klausner. and it is likely that the alleles of these genes associated with disease 1988. The T cell antigen receptor zeta chain is tyrosine phosphorylated upon activation. J. Biol. Chem. 263: 18225–18230. susceptibility are of this type. Identifying and characterizing these 14. D’Oro, U., I. Munitic, G. Chacko, T. Karpova, J. McNally, and J. D. Ashwell. genes and biochemical pathways may prove to be important when 2002. Regulation of constitutive TCR internalization by the zeta-chain. J. Im- studying their functional contribution to pathogenesis and for munol. 169: 6269–6278. 15. Irving, B. A., and A. Weiss. 1991. The cytoplasmic domain of the T cell receptor identifying potential therapeutic targets, particularly when over- zeta chain is sufficient to couple to receptor-associated signal transduction lapping in humans and an animal model of disease. pathways. Cell 64: 891–901. 16. Zipris, D., A. H. Lazarus, A. R. Crow, M. Hadzija, and T. L. Delovitch. 1991. In conclusion, our data support a model in which the development Defective thymic T cell activation by concanavalin A and anti-CD3 in autoim- of autoimmune diabetes in the NOD mouse is dependent on a TCR mune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates signal mediated by a less efficient NOD allele of the Cd3z gene. This with the onset of insulitis. J. Immunol. 146: 3763–3771. 17. Rapoport, M. J., A. H. Lazarus, A. Jaramillo, E. Speck, and T. L. Delovitch. attenuated TCR/CD3 signaling could synergize with the 50% re- 1993. Thymic T cell anergy in autoimmune nonobese diabetic mice is mediated duction in IL-2 abundance and correlate with the reduced function by deficient T cell receptor regulation of the pathway of p21ras activation. J. Exp. Med. 177: 1221–1226. of regulatory T cells reported to be conferred by the NOD allele of 18. Zhang, J., K. V. Salojin, and T. L. Delovitch. 2001. CD28 co-stimulation restores the Idd3 locus (49). As recently discussed (50), such a scenario T cell responsiveness in NOD mice by overcoming deficiencies in Rac-1/p38 concurs with the quantal theory of immunity, stating that T cell mitogen-activated protein kinase signaling and IL-2 and IL-4 gene transcription. Int. Immunol. 13: 377–384. responses depend on cells receiving a critical number of TCR- and 19. Nambiar, M. P., J. P. Mitchell, R. P. Ceruti, M. A. Malloy, and G. C. Tsokos. IL-2R–mediated stimuli, which, in turn, may have consequences 2003. Prevalence of T cell receptor zeta chain deficiency in systemic lupus er- ythematosus. Lupus 12: 46–51. for the process of discrimination between self and nonself (51). The 20. Pang, M., Y. Setoyama, K. Tsuzaka, K. Yoshimoto, K. Amano, T. Abe, and results are also consistent with recently published observations that T. Takeuchi. 2002. Defective expression and tyrosine phosphorylation of the T cell receptor zeta chain in peripheral blood T cells from systemic lupus er- haplotypes in the human IL2RA gene associated with human T1D ythematosus patients. Clin. Exp. Immunol. 129: 160–168. and multiple sclerosis correlate with differences in the surface ex- 21. Berg, L., J. Ro¨nnelid, L. Klareskog, and A. Bucht. 2000. Down-regulation of the pression of IL-2Ra (50). Graded levels of TCR signaling are known T cell receptor CD3 zeta chain in rheumatoid arthritis (RA) and its influence on T cell responsiveness. Clin. Exp. Immunol. 120: 174–182. to affect the IL2-IL2R–mediated control of Foxp3 expression, 22. Romagnoli, P., D. Strahan, M. Pelosi, A. Cantagrel, and J. P. van Meerwijk. providing a potential route by which the observed disease associ- 2001. A potential role for protein tyrosine kinase p56() in rheumatoid arthritis synovial fluid T lymphocyte hyporesponsiveness. Int. Immunol. 13: 305–312. ations may affect the pathogenesis of autoimmunity through the 23. Baniyash, M. 2004. TCR zeta-chain downregulation: curtailing an excessive same biological pathway. inflammatory immune response. Nat. Rev. Immunol. 4: 675–687. 5544 VARIATION IN Cd3z CORRELATES WITH DIABETES

24. Cope, A. P. 2002. Studies of T-cell activation in chronic inflammation. Arthritis from patients with chronic inflammatory/autoimmune diseases. Arch. Immunol. Res. 4(Suppl. 3): S197–S211. Ther. Exp. (Warsz.) 55: 373–386. 25. Hayes, S. M., L. Li, and P. E. Love. 2005. TCR signal strength influences al- 39. Gill, B. M., A. Jaramillo, L. Ma, K. B. Laupland, and T. L. Delovitch. 1995. phabeta/gammadelta lineage fate. Immunity 22: 583–593. Genetic linkage of thymic T-cell proliferative unresponsiveness to mouse 26. Holst, J., H. Wang, K. D. Eder, C. J. Workman, K. L. Boyd, Z. Baquet, H. Singh, chromosome 11 in NOD mice. A possible role for chemokine genes. Diabetes K. Forbes, A. Chruscinski, R. Smeyne, et al. 2008. Scalable signaling mediated 44: 614–619. by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and 40. Grattan, M., Q. S. Mi, C. Meagher, and T. L. Delovitch. 2002. Congenic mapping prevents autoimmunity. Nat. Immunol. 9: 658–666. of the diabetogenic locus Idd4 to a 5.2-cM region of chromosome 11 in NOD 27. Pfaffl, M. W., G. W. Horgan, and L. Dempfle. 2002. Relative expression software mice: identification of two potential candidate subloci. Diabetes 51: 215–223. tool (REST) for group-wise comparison and statistical analysis of relative ex- 41. Foulds, K. E., L. A. Zenewicz, D. J. Shedlock, J. Jiang, A. E. Troy, and H. Shen. pression results in real-time PCR. Nucleic Acids Res. 30: e36. 2002. Cutting edge: CD4 and CD8 T cells are intrinsically different in their 28. Friedline, R. H., D. S. Brown, H. Nguyen, H. Kornfeld, J. Lee, Y. Zhang, proliferative responses. J. Immunol. 168: 1528–1532. M. Appleby, S. D. Der, J. Kang, and C. A. Chambers. 2009. CD4+ regulatory 42. Ravichandran, K. S., and S. J. Burakoff. 1994. Evidence for differential in- T cells require CTLA-4 for the maintenance of systemic tolerance. J. Exp. Med. tracellular signaling via CD4 and CD8 molecules. J. Exp. Med. 179: 727–732. 206: 421–434. 43. Love, P. E., E. W. Shores, M. D. Johnson, M. L. Tremblay, E. J. Lee, 29. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, A. Grinberg, S. P. Huang, A. Singer, and H. Westphal. 1993. T cell development T. W. Mak, and S. Sakaguchi. 2000. Immunologic self-tolerance maintained by in mice that lack the zeta chain of the T cell antigen receptor complex. Science CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte- 261: 918–921. associated antigen 4. J. Exp. Med. 192: 303–310. 44. Malissen, M., A. Gillet, B. Rocha, J. Trucy, E. Vivier, C. Boyer, F. Ko¨ntgen, 30. Wing, K., Y. Onishi, P. Prieto-Martin, T. Yamaguchi, M. Miyara, Z. Fehervari, N. Brun, G. Mazza, E. Spanopoulou, et al. 1993. T cell development in mice T. Nomura, and S. Sakaguchi. 2008. CTLA-4 control over Foxp3+ regulatory lacking the CD3-zeta/eta gene. EMBO J. 12: 4347–4355. 45. Yamazaki, T., H. Arase, S. Ono, H. Ohno, H. Watanabe, and T. Saito. 1997. A T cell function. Science 322: 271–275. shift from negative to positive selection of autoreactive T cells by the reduced 31. Greve, B., L. Vijayakrishnan, A. Kubal, R. A. Sobel, L. B. Peterson, level of TCR signal in TCR-transgenic CD3 zeta-deficient mice. J. Immunol. L. S. Wicker, and V. K. Kuchroo. 2004. The diabetes susceptibility locus Idd5.1 158: 1634–1640.

on mouse chromosome 1 regulates ICOS expression and modulates murine Downloaded from 46. She, J., M. C. Ruzek, P. Velupillai, I. de Aos, B. Wang, D. A. Harn, J. Sancho, experimental autoimmune encephalomyelitis. J. Immunol. 173: 157–163. C. A. Biron, and C. Terhorst. 1999. Generation of antigen-specific cytotoxic 32. Truneh, A., F. Albert, P. Golstein, and A. M. Schmitt-Verhulst. 1985. Early steps T lymphocytes and regulation of cytokine production takes place in the absence of lymphocyte activation bypassed by synergy between calcium ionophores and of CD3zeta. Int. Immunol. 11: 845–857. phorbol ester. Nature 313: 318–320. 47. Wicker, L. S., J. Clark, H. I. Fraser, V. E. Garner, A. Gonzalez-Munoz, B. Healy, 33. Ohno, H., T. Aoe, S. Taki, D. Kitamura, Y. Ishida, K. Rajewsky, and T. Saito. S. Howlett, K. Hunter, D. Rainbow, R. L. Rosa, et al. 2005. Type 1 diabetes 1993. Developmental and functional impairment of T cells in mice lacking CD3 genes and pathways shared by humans and NOD mice. J. Autoimmun. 25 zeta chains. EMBO J. 12: 4357–4366. (Suppl.): 29–33. 34. Liu, C. P., R. Ueda, J. She, J. Sancho, B. Wang, G. Weddell, J. Loring,

48. Siggs, O. M., L. A. Miosge, A. L. Yates, E. M. Kucharska, D. Sheahan, http://www.jimmunol.org/ C. Kurahara, E. C. Dudley, A. Hayday, et al. 1993. Abnormal T cell development -/- T. Brdicka, A. Weiss, A. Liston, and C. C. Goodnow. 2007. Opposing functions in CD3-zeta mutant mice and identification of a novel T cell population in the of the T cell receptor kinase ZAP-70 in immunity and tolerance differentially intestine. EMBO J. 12: 4863–4875. titrate in response to nucleotide substitutions. Immunity 27: 912–926. 35. Maurice, M. M., A. C. Lankester, A. C. Bezemer, M. F. Geertsma, P. P. Tak, 49. Yamanouchi, J., D. Rainbow, P. Serra, S. Howlett, K. Hunter, V. E. Garner, F. C. Breedveld, R. A. van Lier, and C. L. Verweij. 1997. Defective TCR- A. Gonzalez-Munoz, J. Clark, R. Veijola, R. Cubbon, et al. 2007. Interleukin-2 mediated signaling in synovial T cells in rheumatoid arthritis. J. Immunol. gene variation impairs regulatory T cell function and causes autoimmunity. Nat. 159: 2973–2978. Genet. 39: 329–337. 36. Tsokos, G. C., H. K. Wong, E. J. Enyedy, and M. P. Nambiar. 2000. Immune cell 50. Dendrou, C. A., V. Plagnol, E. Fung, J. H. Yang, K. Downes, J. D. Cooper, signaling in lupus. Curr. Opin. Rheumatol. 12: 355–363. S. Nutland, G. Coleman, M. Himsworth, M. Hardy, et al. 2009. Cell-specific 37. Gorman, C. L., A. I. Russell, Z. Zhang, D. Cunninghame Graham, A. P. Cope, protein phenotypes for the autoimmune locus IL2RA using a genotype-selectable and T. J. Vyse. 2008. Polymorphisms in the CD3Z gene influence TCRzeta human bioresource. Nat. Genet. 41: 1011–1015.

expression in systemic lupus erythematosus patients and healthy controls. J. 51. Hakonarson, H., H. Q. Qu, J. P. Bradfield, L. Marchand, C. E. Kim, by guest on September 25, 2021 Immunol. 180: 1060–1070. J. T. Glessner, R. Grabs, T. Casalunovo, S. P. Taback, E. C. Frackelton, et al. 38. Ciszak, L., E. Pawlak, A. Kosmaczewska, S. Potoczek, and I. Frydecka. 2007. 2008. A novel susceptibility locus for type 1 diabetes on Chr12q13 identified by Alterations in the expression of signal-transducing CD3 zeta chain in T cells a genome-wide association study. Diabetes 57: 1143–1146.