Combined congenic mapping and nuclease-based gene targeting for studying allele-specific effects of Tnfrsf9 within the Idd9.3 autoimmune diabetes locus

Rodent complex trait genetic studies involving a cross between two inbred strains are usually followed by congenic mapping to refine the loci responsible for the phenotype. However, progressing from a chromosomal region to the actual causal gene remains challenging because multiple polymorphic genes are often closely linked. The goal of this study was to develop a strategy that allows candidate gene testing by allele-specific expression without prior knowledge of the credible causal variant. Tnfrsf9 (encoding CD137) is a candidate gene for the Idd9.3 type 1 diabetes (T1D) susceptibility locus in the nonobese diabetic (NOD) mouse model. A C57BL/10Sn (B10)-derived diabetes resistance Idd9.3 congenic region has been shown to enhance accumulation of CD137+ regulatory T cells and serum soluble CD137 in NOD mice. By combining the power of congenic mapping and nuclease-based gene targeting, we established a system where a pair of F1 hybrids expressed either the B10 or NOD Tnfrsf9 allele mimicking coisogenic strains. Using this approach, we demonstrated that the allelic difference in B10 and NOD Tnfrsf9 alone was sufficient to cause differential accumulation of CD137+ regulatory T cells and serum soluble CD137 levels. This strategy can be broadly applied to other rodent genetic mapping studies.

Rodent models have been widely used to study the genetics of human complex diseases 1,2 . Except for more complicated breeding methods, experimental strategies typically involve a cross between two inbred strains that are respectively resistant and susceptible to a trait to map genetic loci regulating the phenotype. Initial localization of the chromosomal regions is then followed by generation of congenic strains to physically map the underlying genes. While these approaches are effective to define a relatively small region harboring phenotype-modulating genetic variants, identification of the causal genes remains difficult. When several genes are closely linked within a small genetic region, it is not practical to further test them by a traditional backcross breeding scheme due to limited recombination. The underlying gene is often inferred by its expression level and function or the presence of deleterious polymorphisms. The causal variant could be conclusively determined if coisogenic strains respectively express only one of the two parental alleles of a candidate gene. However, this is practically challenging because multiple polymorphisms often exist in the candidate gene and precise genetic manipulation requires prior knowledge of the credible variant.
Nonobese diabetic (NOD) mice develop spontaneous type 1 diabetes (T1D) mimicking the human disease and have been an effective model for identifying pathogenic genetic determinants 3,4 . By outcrossing to other strains, more than 30 autoimmune T1D susceptibility (Idd) loci have been identified in NOD mice 3 . Among male mice were stimulated with anti-CD3 (2.5 μg/ml) overnight and analyzed for CD137 expression by flow cytometry the following day. Cells were stained with anti-CD8 (for detecting CD8 T cells) and anti-CD137 or the corresponding isotype control www.nature.com/scientificreports www.nature.com/scientificreports/ those, the Idd9.3 locus has been mapped to the distal end of chromosome 4 containing the CD137-encoding Tnfrsf9 gene 5,6 . Interestingly, NOD-derived CD137 is hypofunctional compared to the C57BL/10Sn (B10) counterpart, and three nonsynonymous polymorphisms are present between NOD and B10 Tnfrsf9 alleles 5,6 . NOD mice congenic for the B10-derived Idd9.3 region (NOD.Idd9.3 B10 ) are more resistant to T1D 7 . Compared to NOD mice, the NOD.Idd9.3 B10 congenic strain has significantly higher levels of CD137 + FOXP3 + regulatory CD4 T cells (Tregs) and serum soluble CD137 8 . CD137 + Tregs are more suppressive than the CD137subset in vitro and are the primary cellular source of soluble CD137 among T cells 8 . Importantly, recombinant soluble CD137 prevents NOD mice from developing T1D 9 . These observations provide a possible mechanism of B10 Idd9.3-mediated T1D resistance and support Tnfrsf9 as its underlying gene.
The purpose of this study was to develop a strategy that allows us to selectively express only the NOD or B10 Tnfrsf9 allelic variant in an identical genetic background. With the recent advance in nuclease-based genetic engineering 10 , we directly targeted Tnfrsf9 in both NOD and NOD.Idd9.3 B10 mice and generated a pair of F1 hybrids mimicking coisogenic strains. We conclusively demonstrate that Tnfrsf9 allelic difference directly controls the frequency of CD137 + Tregs and the level of serum soluble CD137. We also provide a general strategy for testing candidate genes in rodent genetic mapping studies.
Allelic difference in the Tnfrsf9 gene controls the accumulation of CD137 + tregs. Except the origin of the functional Tnfrsf9 allele, newly generated Tnfrsf9 NOD and Tnfrsf9 B10 F1 mice are considered genetically identical. These F1 mice allowed us to determine if selective expression of NOD or B10 Tnfrsf9 alone is sufficient to explain the phenotypic differences previously observed between NOD and NOD.Idd9.3 B10 mice. T1D suppression in NOD.Idd9.3 B10 mice is associated with a remarkably higher frequency of CD137 + Tregs 8 . Thus, we asked if this phenotype is controlled by the allelic difference between B10 and NOD Tnfrsf9. We first analyzed the frequencies of total Tregs and those that express CD137 in the spleen and pancreatic lymph node (PLN) of Tnfrsf9 NOD and Tnfrsf9 B10 F1 mice. We observed similar percentages of total FOXP3 + Tregs in the spleen and PLN of Tnfrsf9 NOD and Tnfrsf9 B10 F1 mice (Fig. 2a,b). Conversely, the frequency of CD137 + Tregs was higher in the spleen of Tnfrsf9 B10 than Tnfrsf9 NOD F1 mice (Fig. 2c), although the difference between Tnfrsf9 NOD and Tnfrsf9 B10 F1 hybrids was diminished relative to the comparison of NOD and NOD.Idd9.3 B10 mice (Fig. 2d). This was likely because only one copy of functional Tnfrsf9 was expressed in Tnfrsf9 NOD and Tnfrsf9 B10 F1 mice. Similarly, the frequency of CD137 + Tregs was also increased in the PLN of Tnfrsf9 B10 F1 progeny compared to that of Tnfrsf9 NOD F1 mice, but the difference was not as significant as what was observed between NOD and NOD.Idd9.3 B10 mice (Fig. 2e,f).
Allelic difference in the Tnfrsf9 gene controls the level of serum soluble CD137. CD137 is expressed as a membrane form or a soluble protein due to alternative splicing that removes the transmembrane domain 13 . Compared to NOD mice, the NOD.Idd9.3 B10 congenic strain has a higher level of serum soluble CD137 8 . Thus, we asked if the level of serum soluble CD137 is also controlled by the allelic difference of Tnfrsf9. Sera were collected from Tnfrsf9 NOD and Tnfrsf9 B10 F1 mice and analyzed for soluble CD137 by enzyme-linked immunosorbent assay (ELISA). We observed a significant increase of serum soluble CD137 in Tnfrsf9 B10 compared to Tnfrsf9 NOD mice (Fig. 3a), albeit the difference was considerably reduced compared to that between NOD and NOD.Idd9.3 B10 (Fig. 3b).
T1D incidence in Tnfrsf9 NOD and Tnfrsf9 B10 F1 mice. Compared to NOD mice, T1D development is partially suppressed in the NOD.Idd9.3 B10 congenic strain 5,7 . The Tnfrsf9 NOD and Tnfrsf9 B10 F1 "coisogenic" strains provided an opportunity to directly test if Tnfrsf9 variants are responsible for the T1D modulatory effect of the Idd9.3 locus. To address this question, we monitored Tnfrsf9 NOD and Tnfrsf9 B10 F1 females for T1D development. While there was a trend of reduced T1D incidence in Tnfrsf9 B10 compared to Tnfrsf9 NOD F1 mice (p The percentage of CD137 + Tregs is increased in the spleen of Tnfrsf9 B10 F1 mice. Representative flow cytometry profiles of CD137 staining in splenic Tregs of 9-11 weekold Tnfrsf9 NOD and Tnfrsf9 B10 F1 female mice are shown on the left. Cells from a NOD.Tnfrsf9 −/− mouse was used as the negative control. Summarized results from three independent experiments are shown on the right. As indicated, the average frequency of CD137 + Tregs in the spleen of Tnfrsf9 B10 F1 is 2.6% higher than that of Tnfrsf9 NOD F1 mice. *p < 0.05 by unpaired t test. (d) NOD.Idd9.3 B10 mice have a higher frequency of splenic CD137 + Tregs than that of NOD mice. The results are summarized from 2 experiments using 9-12 week-old female mice. As indicated, the average frequency of splenic CD137 + Tregs in NOD.Idd9.3 B10 is 7.2% higher than that of NOD mice. ***p < 0.0005 by unpaired t test. www.nature.com/scientificreports www.nature.com/scientificreports/ Log-rank test), the difference did not reach statistical significance (Fig. 4). This could be due to the overall reduction of CD137 expression from a single Tnfrsf9 copy in Tnfrsf9 B10 and Tnfrsf9 NOD F1 mice that minimized the allele-specific functional differences important for regulating T1D development. This possibility is supported by the comparative analyses for the frequency of CD137 + Tregs and the level of serum soluble CD137 between Tnfrsf9 B10 and Tnfrsf9 NOD F1 hybrids and between NOD and NOD.Idd9.3 B10 mice as shown in Figs 2 and 3.

Discussion
In this study we combined the power of congenic mapping and nuclease-based mutagenesis to establish a genetic strategy to directly compare two different alleles of the gene of interest in genetically identical strains. We tested this approach using the Idd9.3 candidate gene Tnfrsf9 in the NOD mouse model of T1D. NOD Tnfrsf9 encodes a hypofunctional CD137 compared to the B10 variant 6 . The functional difference has been associated with an increased frequency of CD137 + Tregs in the NOD.Idd9.3 B10 strain compared to standard NOD mice 8 . In the present study, we also detected a significant increase in the frequency of CD137 + Tregs in the spleen and PLN of our F1 mice expressing the B10 allele of Tnfrsf9. These results indicate that a more functional CD137 molecule alone leads to the accumulation of CD137 + Tregs, independent of other genes within the Idd9.3 region or the differential state of diabetes progression in NOD and NOD.Idd9.3 B10 mice. CD137 + Tregs exhibit superior function than their negative counterpart, possibly due to the production of soluble CD137 8 . Consistent with the previous study 8 , we also found a higher level of serum soluble CD137 in NOD.Idd9.3 B10 than in NOD mice. We further show that a higher concentration of soluble CD137 was found in the serum of F1 mice expressing the B10 Tnfrsf9 allele compared to those possessing the NOD allele. These results indicate that the Tnfrsf9 allelic difference also directly controls the level of circulating soluble CD137. It is currently not known how alternative splicing of Tnfrsf9 is regulated to give rise to membrane and soluble forms of CD137. Whether an increased level of serum soluble CD137 in mice carrying the B10 Tnfrsf9 allele is a direct consequence of elevated CD137 + Tregs or involves regulation by other non-coding regions remains to be determined.
Tnfrsf9 has been implicated as an Idd9.3 underlying gene 6 . The Tnfrsf9 NOD and Tnfrsf9 B10 F1 "coisogenic" strains allowed us to directly test if allelic difference in Tnfrsf9 alone is sufficient to modulate T1D progression. Different from the results of the frequency of CD137 + Tregs and the level of serum soluble CD137, we did not observe significant T1D suppression in Tnfrsf9 B10 compared to Tnfrsf9 NOD F1 mice. The lack of diabetes inhibition conferred by the B10 Tnfrsf9 allele in our F1 mice could be that the functional difference conferred by one copy of B10 and NOD Tnfrsf9 is not sufficient to impact T1D development. This possibility is supported by the diminished differences in the frequency of CD137 + Tregs and the level of serum soluble CD137 between Tnfrsf9 NOD and Tnfrsf9 B10 F1 hybrids relative to the comparison between NOD and NOD.Idd9.3 B10 mice (Figs 2 and 3). Another non-mutually exclusive explanation to the lack of T1D inhibition in Tnfrsf9 B10 F1 mice is that other polymorphic gene(s) in the Idd9.3 region also impact T1D development. One possible candidate is the non-coding micro RNA Mir34a. In a recent study it was found that the NOD.Idd9.3 congenic strain had a significantly lower number of B cells than that in NOD mice 14 . The reduction of B cells in NOD.Idd9.3 congenic strain correlated with a higher level of Mir34a that was thought to impair normal B cell development by negatively regulating FOXP1 expression 14 . However, we did not observe a reduction of B cells in the NOD.Idd9.3 B10 congenic strain compared to NOD mice ( Supplementary Fig. S1). The reason for the discrepancy is currently not known but could be due to differences in the housing environment. Immune cells are under the circadian control 15 . The circadian gene Per3 represents another possible candidate in the Idd9.3 region. A similar genetic approach reported here can be used to test additional genes within the Idd9.3 region for their role in T1D development.
In conclusion, we demonstrate the feasibility to establish a "coisogenic" system by using a pair of F1 hybrids that selectively express an allele of the gene of interest for direct comparison of its functional consequence. The limitation of our approach is that only one copy of the gene is expressed, and the phenotypic difference may be quantitatively reduced if there is a dose-dependent effect. This possibility needs to be considered when testing candidate genes using our approach. Nevertheless, our strategy has the potential to be broadly applied to other experimental models to facilitate genetic mapping studies.

Materials and Methods
Mouse strains. NOD/ShiLtDvs (hereafter NOD) and NOD.B10Sn-Idd9.3 C57BL/10SnJ /1106MrkTacJ (hereafter NOD.Idd9.3 B10 ) were obtained from The Jackson Laboratory and subsequently maintained at the Medical College of Wisconsin MCW). NOD.Tnfrsf9 −/− mice (line-6), currently maintained at the N4 generation, have been previously reported 11 . NOD.Idd9.3 B10 .Tnfrsf9 −/− mice were generated using the previously described Tnfrsf9 specific zinc-finger nuclease pairs designed, assembled, and validated by Sigma-Aldrich 11 . The condition of microinjection and methods of founder screening and genotyping have been described in detail 11 . A founder (line-24) with a two base-pair insertion at the target site, resulting in a premature stop codon (Fig. 1a), was backcrossed to NOD.Idd9.3 B10 for 4 generations followed by intercrossing to fix the mutation to homozygosity. All mouse experimental protocols were carried out in accordance with the MCW Institutional Animal Care and Use Committee guidelines and approved by the committee. was used as the negative control. Summarized results from three independent experiments are shown on the right. As indicated, the average frequency of CD137 + Tregs in the PLN of Tnfrsf9 B10 F1 is 3.7% higher than that of Tnfrsf9 NOD F1 mice. *p < 0.05 by unpaired t test. (f) NOD.Idd9.3 B10 mice have a higher frequency of PLN CD137 + Tregs than that of NOD mice. The results are summarized from 2 independent experiments using 9-12 week-old female mice. As indicated, the average frequency of PLN CD137 + Tregs in NOD.Idd9.3 B10 is 10% higher than that of NOD mice. ***p < 0.0005 by unpaired t test.
Soluble CD137 enzyme-linked immunosorbent assay (ELISA). Sera were collected from 10-week-old females of the indicated strains. The concentration of soluble CD137 was determined using the Mouse 4-1BB/TNFRSF9 DuoSet ELISA kit (R&D Systems). Soluble CD137 was not detectable in serum from NOD.Tnfrsf9 −/− or NOD.Idd9.3 B10 .Tnfrsf9 −/− mice, validating the specificity of the ELISA kit. (a) Serum soluble CD137 is higher in Tnfrsf9 B10 than in Tnfrsf9 NOD F1 mice. Sera were collected from 10-week-old female mice and analyzed by ELISA for CD137. As indicated, the average level of serum soluble CD137 in Tnfrsf9 B10 F1 is 28.3 pg/ml higher than that of Tnfrsf9 NOD F1 mice. *p < 0.05 by unpaired t test. (b) Serum soluble CD137 is higher in NOD.Idd9.3 B10 than in NOD mice. Sera were collected from 10-week-old female mice and analyzed by ELISA for CD137. As indicated, the average level of serum soluble CD137 in NOD.Idd9.3 B10 is 332.2 pg/ml higher than that of NOD mice. **p < 0.005 by unpaired t test. . T1D incidence in Tnfrsf9 NOD and Tnfrsf9 B10 F1 mice. Tnfrsf9 B10 and Tnfrsf9 NOD F1 female mice were monitored for T1D development for 30 weeks. The incidence of T1D is not significantly different between Tnfrsf9 B10 and Tnfrsf9 NOD F1 mice (p = 0.13 by Log-rank test).