Proc Natl Acad Sci U S A 103(44): 16400-16405

Modeling <em>CTLA4</em>-linked autoimmunity with RNA interference in mice

Section on Immunology and Immunogenetics, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215

*To whom correspondence may be addressed at: Section on Immunology and Immunogenetics, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215., E-mail: ude.dravrah.nilsoj@mdbc

Contributed by Christophe Benoist, September 13, 2006

Author contributions: Z.C., D.M., and C.B. designed research; Z.C. and J.S. performed research; Z.C., J.S., D.M., and C.B. analyzed data; and Z.C., J.S., D.M., and C.B. wrote the paper.

Abstract

The CTLA4 gene is important for T lymphocyte-mediated immunoregulation and has been associated with several autoimmune diseases, in particular, type 1 diabetes. To model the impact of natural genetic variants of CTLA4, we constructed RNA interference (RNAi) “knockdown” mice through lentiviral transgenesis. Variegation of expression was observed in founders but proved surmountable because it reflected parental imprinting, with derepression by transmission from male lentigenics. Unlike the indiscriminate multiorgan autoimmune phenotype of the corresponding knockout mice, Ctla4 knockdown animals had a disease primarily focused on the pancreas, with rapid progression to diabetes. As with the human disease, the knockdown phenotype was tempered by genetic-modifier loci. RNAi should be more pertinent than gene ablation in modeling disease pathogenesis linked to a gene-dosage variation.

Keywords: autoimmune diabetes, imprinting, lentiviral transgene, RNAi, variegation

Abstract

Common human diseases are thought to be determined by complex genetic variations. Advances in genetics and genomics, such as whole-genome scans of single-nucleotide polymorphisms (SNP) and genome-wide expression profiling, have led to the identification of many potential disease-susceptibility alleles, but definitively establishing that a candidate gene is truly causal remains a challenge. Often, the genetic linkage of an allele to a disease condition is manifested as a modest change in mRNA and/or protein expression. For instance, many studies have linked the CTLA4 locus to human autoimmune disorders, including type 1 diabetes (1), a disease caused by T lymphocyte-mediated destruction of pancreatic-islet β cells (2). No variation in the regions coding for mature CTLA4 protein has been implicated, but several polymorphisms in the promoter or 3′ untranslated region (UTR) have been shown to affect promoter efficacy (3 –6) and/or the ratio of alternatively spliced forms (7), thus reducing the level of functional CTLA4 protein. It is these polymorphisms that most closely associate with autoimmune risk (5, 7).

The Ctla4 gene also has been linked to disease susceptibility in the nonobese diabetic (NOD) mouse model of type 1 diabetes. Some reports suggested a defect in overall expression or in an alternative splice form (liCtla4) unable to bind B7.1/B7.2, the conventional ligands of Ctla4 (7 –10). However, for both humans and mice, definitive assignment of susceptibility to an individual SNP (or even to Ctla4 proper, as opposed to combinatorial haplotypes involving Ctla4 and the immediately adjacent genes encoding members of the costimulatory family) is still lacking.

Consistent with its function as an inhibitory receptor (11), recombinational inactivation of Ctla4 results in a devastating lymphoproliferative and autoimmune phenotype, with inflammatory infiltration of multiple organs and death of the homozygous knockout mice within a few weeks (12 –14). However, such a complete abrogation of gene function is a gross overrepresentation of most natural variation. In this study, we generated “knockdown” mice with a partial reduction in Ctla4 expression, employing RNAi derived from short hairpin RNAs (shRNA), delivered into the mouse germ line by lentiviral transgenesis (15, 16). RNAi has been harnessed extensively, by using shRNA precursors of interfering RNAs, to manipulate gene expression in vitro (17, 18), and it seemed most appropriate to mimic the variation of expression levels associated with the CTLA4 polymorphisms.

Litters by the same parent were separated with a semicolon. ND, not determined.

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Acknowledgments

We thank C. Dillon for advice on lentiviral shRNA, and E. Hyatt, K. Hattori, G. Buruzula, J. Lavecchia, A. Pinkhasov, and C. Laplace for assistance. This work was supported by Juvenile Diabetes Research Foundation Grant 4-2004-368, National Institutes of Health Grant P01 AI56299-04, grants from the William T. Young Chair (to D.M. and C.B.), and the core services of Joslin's National Institute of Diabetes and Digestive and Kidney Diseases-funded Diabetes Endocrinology Research Center. Z.C. has been supported by postdoctoral fellowships from the Juvenile Diabetes Research Foundation and the Program in AIDS Research (Dana–Farber Cancer Institute/National Institutes of Health).

Acknowledgments

Abbreviations

2′5′-OAS	2′5′-oligoadenylate synthetase
B6	C57BL/6
KD	knockdown
CT4KD	cytotoxic T lymphocyte antigen 4 KD
NOD	nonobese diabetic
PLN	pancreatic lymph nodes
lentigene	lentiviral transgene

Abbreviations

Footnotes

The authors declare no conflict of interest.

Footnotes

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