The Journal of Immunology Genome-Wide Microarray Expression Analysis of CD4؉ T Cells from Nonobese Diabetic Congenic Mice Identifies Cd55 (Daf1) and Acadl as Candidate Genes for Type 1 Diabetes1 Junichiro Irie,* Brian Reck,† Yuehong Wu,* Linda S. Wicker,‡ Sarah Howlett,‡ Daniel Rainbow,‡ Eleanor Feingold,† and William M. Ridgway2* NOD.Idd3/5 congenic mice have insulin-dependent diabetes (Idd) regions on chromosomes 1 (Idd5)and3(Idd3) derived from the nondiabetic strains B10 and B6, respectively. NOD.Idd3/5 mice are almost completely protected from type 1 diabetes (T1D) but the genes within Idd3 and Idd5 responsible for the disease-altering phenotype have been only partially characterized. To test the hypothesis that candidate Idd genes can be identified by differential gene expression between activated CD4؉ T cells from the diabetes-susceptible NOD strain and the diabetes-resistant NOD.Idd3/5 congenic strain, genome-wide microarray expression analysis was performed using an empirical Bayes method. Remarkably, 16 of the 20 most differentially expressed genes were located in the introgressed regions on chromosomes 1 and 3, validating our initial hypothesis. The two genes with the greatest differential RNA expression on chromosome 1 were those encoding decay-accelerating factor (DAF, also known as CD55) and acyl-coenzyme A dehydrogenase, long chain, which are located in the Idd5.4 and Idd5.3 regions, respectively. Neither gene has been implicated previously in the pathogenesis of T1D. In the case of DAF, differential expression of mRNA was extended to the protein level; NOD CD4؉ T cells expressed higher levels of cell surface DAF compared with NOD.Idd3/5 CD4؉ T cells following activation with anti-CD3 and -CD28. DAF up-regulation was IL-4 dependent and blocked under Th1 conditions. These results validate the approach of using congenic mice together with genome-wide analysis of tissue-specific gene expression to identify novel candidate genes in T1D. The Journal of Immunology, 2008, 180: 1071–1079. n complex genetic diseases such as type 1 diabetes (T1D),3 cessful in excluding candidate genes and narrowing the list of pos- dozens of allelic variants interact to promote or discourage sible candidate genes (5–14). I disease manifestation (1–3). Similar findings have been made There is a practical limit, however, to fine-mapping disease in animal models of complex genetic disease such as the NOD genes using congenic strains of mice since recombination in the mouse, which spontaneously develops T1D (4, 5). Positional clon- genome is not random and often the smallest congenic interval that ing to discover genes causing T1D in NOD mice has involved the can be isolated can contain up to 50 genes (4). Expression analysis generation of congenic mice and this approach has been very suc- of genes within the interval can contribute evidence of genetic variation and thereby greatly assist candidate gene prioritization in a manner unbiased by previous biological knowledge of any of the *Division of Rheumatology and Immunology, School of Medicine, University of genes. The use of genome-wide microarray expression analysis Pittsburgh, Pittsburgh, PA 15261; †Department of Biostatistics, Graduate School of (15, 16) not only allows simultaneous assessment of all of the Public Health, University of Pittsburgh, Pittsburgh, PA 15261; and ‡Juvenile Diabetes genes within the interval (given microarray chips with sufficient Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, De- partment of Medical Genetics, Cambridge Institute for Medical Research, University gene substrate to recognize splice variants), but also highlights of Cambridge, Cambridge, United Kingdom any genes controlled in trans by a candidate causal gene. Received for publication September 13, 2006. Accepted for publication October The combination of microarray analysis with congenic strain 31, 2007. fine-mapping has had variable success in genetic mouse models of The costs of publication of this article were defrayed in part by the payment of page autoimmunity. In two different lupus mouse models, microarray charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. analysis identified strong genetic candidates (17, 18). In T1D, 1 W.M.R. was supported by National Institutes of Health (NIH) National Institute of however, an earlier attempt at this analysis was not successful Diabetes and Digestive and Kidney Diseases 60714 and NIH RFA A102-006. L.S.W. when applied to expression in whole, naive spleen (19). The au- is a Juvenile Diabetes Research Foundation (JDRF)/Wellcome Trust Principal Research Fellow and the research in the laboratory of L.S.W. for this study was also thors concluded that analyzing expression in a noninduced whole supported by NIH P01 AI039671. The availability of NOD congenic mice through the organ was not informative, implying that selecting specific im- Taconic Farms Emerging Models Program has been supported by grants from the mune cell subsets may be more productive. Therefore in this re- Merck Genome Research Institute, National Institute of Allergy and Infectious Dis- eases, and the JDRF. port, we have chosen to analyze differential gene expression in ϩ 2 Address correspondence and reprint requests to Dr. William M. Ridgway, Division purified, activated CD4 T cells because a large amount of liter- of Rheumatology and Immunology, School of Medicine, University of Pittsburgh, ature supports a pathogenic role for this cell type in NOD mice S725 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. E-mail address: [email protected] (20–22), suggesting that some genes causing T1D, known as Idd (for insulin dependent diabetes) genes, are expressed in the CD4ϩ 3 Abbreviations used in this paper: T1D, type 1 diabetes; Idd, insulin-dependent di- abetes; SNP, single nucleotide polymorphism; DAF, decay-accelerating factor; Ct, T cell subset. cycle threshold; ACADL, acyl-coenzyme A dehydrogenase, long chain; SAPE, The NOD.Idd3/5 congenic mouse, with the B6-derived Idd3 in- streptavidin-PE. terval on chromosome 3, and the B10-derived Idd5 interval on Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 chromosome 1, is almost completely protected from diabetes www.jimmunol.org 1072 MICROARRAY ANALYSIS OF NOD CONGENIC CD4ϩ T CELLS (1–2% incidence at 7 mo of age for NOD.Idd3/5 females compared with 80% for NOD females) (23). The genetic basis of T1D pro- tection from diabetes in NOD.Idd3/5 mice has been partially char- acterized. The Idd3 region is a 650-kb interval containing five known genes (Tenr, Il2, Il21, Centrin4, and Fgf2), two predicted genes of unknown function (KIAA1109 and KIAA1371), and three pseudogenes. The prime candidate gene encodes IL-2 and the causative single nucleotide polymorphisms are hypothesized to subtly alter its mRNA expression levels (24). As detailed in the accompanying article (25), there are four subregions within the FIGURE 1. Dot plot representation of the whole gene chip data set from ϩ larger Idd5 region: Idd5.1 (2.0 Mb), Idd5.2 (1.5 Mb), Idd5.3 (3.5 NOD-, NOD.Idd3/5-, and B6.G7-activated CD4 T cells. Shown is the Mb), and Idd5.4 (78 Mb). The genes accounting for Idd5.1 and distribution of probe sets representing the entire data set from the NOD, NOD.Idd3/5, and B6.G7 gene chips, analyzed by intersecting probe sets Idd5.2 are most likely Ctla4 (6–8) and Nramp1 (4, 8), respec- from different strains represented on the x and y axes (see Results). Each tively. The molecular basis of these two candidate genes has been circle represents ϳ10 probe sets. Probe sets in the top 7% of differential attributed to single nucleotide polymorphisms in the coding re- expression between NOD- and NOD.Idd3/5-activated CD4ϩ T cells (ver- gions which alter splicing in case of Ctla4 and the primary amino tical line) intersected with probe sets in top 7% of differential expression acid sequence for Nramp1. between NOD- and B6.G7-activated CD4ϩ T cells (horizontal line). The To further characterize the genetic basis of T1D resistance in upper right quadrant shows the probe sets that were in the top 7% in both NOD.Idd3/5 mice, we formulated the hypothesis that the geneti- the NOD vs NOD.Idd3/5 and the NOD vs B6.G7 comparisons.; the full list cally controlled resistance to diabetes would be correlated to al- of these genes is found in supplemental table 1. tered gene expression patterns in candidate genes on chromosomes 1 and 3 of activated NOD.Idd3/5 CD4ϩ T cells. Thus, activated CD4ϩ T cells from NOD and NOD.Idd3/5 should show differ- within the B10-derived introgressed region of chromosome 1 in ential expression primarily in the congenic intervals on chro- NOD.Idd3/5 mice. mosomes 1 and 3; conversely, the common NOD genome out- side the congenic region on these two chromosomes should be Preparation, purification, and stimulation of splenocytes equivalently expressed. Exceptions to this hypothesis, i.e., dif- ϩ The spleen from each mouse was removed aseptically and minced. After ferential expression of genes from NOD and NOD.Idd3/5 CD4 lysing RBC, the cells were washed three times with PBS. To purify CD4- T cells outside the congenic intervals, could result from down- positive splenocytes, splenocytes were prepared by magnetic separation stream or trans effects of genes in the congenic intervals. As a using a MiniMACS system (Miltenyi Biotec) according to the manufac- ϩ turer’s instructions. Purified CD4-positive splenocytes were suspended in control, we also compared gene expression between CD4 T RPMI 1640 medium (Invitrogen Life Technologies) supplemented with cells purified from NOD and B6.G7 mice, two strains differing 10% heat-inactivated FBS (Invitrogen Life Technologies) and 1 mM L- throughout the genome except that they share the NOD MHC alanyl-glutamine (Invitrogen Life Technologies), 100 U/ml penicillin, 100 region on chromosome 17.