Carbon Chapter 119 CD30

Carbon D 3

Immunity

Volume 13, Issue 1, 1 July 2000, Pages 107-115

Article

The NOD Idd9 Genetic Interval Influences the Pathogenicity of Insulitis and Contains Molecular Variants of Cd30, Tnfr2, and Cd137

Author links open overlay panelPaul ALyons4Linda SWicker1#

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Abstract

Previous analyses of NOD mice have shown that some genes control the development of both insulitis and diabetes, while other loci influence diabetes without reducing insulitis. Evidence for the existence of a gene only influencing diabetes, Idd9 on mouse chromosome 4, is provided here by the development of a novel congenic mouse strain, NOD.B10 Idd9. NOD.B10 Idd9 mice display profound resistance to diabetes even though nearly all develop insulitis. Subcongenic analysis has demonstrated that alleles of at least three B10 genes, Idd9.1, Idd9.2, and Idd9.3 are required to produce Idd9-mediated diabetes resistance. Candidate genes with amino acid differences between the NOD and B10 strains have been localized to the 5.6 cM Idd9.2 interval (Tnfr2, Cd30) and to the 2.0 cM Idd9.3 interval (Cd137).

Introduction

The NOD mouse spontaneously develops type 1 diabetes as well as other autoimmune syndromes such as inflammation of the thyroid, submandibular, and lacrimal glands (46, 33, 34, 11). Analysis of MHC congenic strains has shown that the NOD MHC (H2g7) is required for the development of insulin-dependent diabetes. However the non-MHC genes present on the NOD background are responsible for the breakdown of self-tolerance and the abnormal accumulation of inflammatory cells. For example, accumulation of T cells within the thyroid does not require the NOD MHC. In fact, the thyroid inflammation is more pronounced in the presence of the H2h4 MHC haplotype (33, 11). Thus, many of the non-MHC genes that contribute to diabetes are likely to be autoimmunity genes, and their identification will facilitate our understanding of the molecular basis of autoimmune disease.

Linkage analysis of the NOD genome has revealed the location of several of the non-MHC genes (42, 9, 17). They fall into two classes: genes with disease resistance alleles that reduce the development of both insulitis (T cell inflammation of pancreatic islets, evaluated by conventional histological staining with hematoxylin and eosin) and diabetes, and genes that prevent diabetes without reducing the amount of insulitis (Ghosh et al. 1993). The identification of these two patterns of protection suggests that genes of the latter class exert their effects despite the accumulation of inflammatory cells. NOD congenic strains containing specific segments of B6 chromosome 3 have been constructed and prove the existence of one of the former class of genes controlling both insulitis and diabetes (Wicker et al. 1994b). In addition, the strains show that the original linkage is due to at least four separate loci (Idd3, Idd10, Idd17, and Idd18) (45, 29,Podolin et al. 1998). In addition to reducing the frequency of spontaneous diabetes and insulitis, Idd3 also mediates protection from experimental autoimmune encephalomyelitis (EAE). Idd3 has now been mapped to a 0.15 cM interval encompassing the variant candidate gene Il2 (Lyons et al. 2000). Combined, these data support the hypothesis that Idd3 is an autoimmunity gene that contributes to the accumulation of self-reactive cells (43, 8).

In our initial genome scan, we obtained evidence for several genes that were only linked to diabetes, not to insulitis. We now provide evidence that one of these, Idd9 on chromosome 4, is a true locus. The B10 allele of Idd9 does not prevent the development of insulitis but does prevent the development of infiltrates expressing cytokines associated with β cell destruction such as IFNγ and TNFα. Insulitis in Idd9 mice is instead characterized by the presence of cells expressing CD30 and secreting IL-4. The Idd9 resistance phenotype contrasts sharply with that of NOD mice protected by a set of linked resistance alleles on chromosome 3 (Idd3, Idd17, Idd10, and Idd18), which severely restrict the accumulation of leukocytes in the islet, and defines a second mechanism of naturally occurring genetically programmed protection from autoimmune disease (Wicker et al. 1994b). We also provide evidence that the Idd9 locus is actually composed of at least three separate Idd loci: Idd9.1, Idd9.2, and Idd9.3. In the genetic interval containing Idd9.2 and Idd9.3, three variant TNFR family members have now been identified and represent functional candidate genes for mediating the diabetes-protective effects of this region.

Results

NOD.B10 Idd9 Mice Are Highly Protected from Spontaneous Diabetes

Earlier linkage studies with the B10 and B6 strains demonstrated that a gene, Idd9, located in the distal portion of chromosome 4 in the NOD strain, contributes to the development of spontaneous diabetes (9, 35). With the eventual goal of characterizing and fine-mapping Idd9, we developed a congenic strain on the NOD background containing approximately 48 cM of introgressed B10-derived genetic material (Table 1). Female and male NOD.B10 Idd9 mice were monitored for the development of diabetes and were found to be highly resistant (p < 10−4) to the development of disease as compared to the NOD parental strain (Figure 1). Thus, the existence of a diabetes resistance gene, Idd9, on chromosome 4 was confirmed by these observations.

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Figure 1. NOD.B10 Idd9 Mice Are Protected from Diabetes

Female (A) and male (B) NOD, NOD.B10 Idd9, and (NOD × NOD.B10 Idd9)F1 mice were monitored for the development of diabetes for 210 days. For females, comparisons of the 7 month cumulative diabetes frequencies of the two parental strains as well as the F1 strain versus both parental strains had p values of <10−4 (Fisher's exact test). For males, comparison of the two parental strains had a p value of <10−4. The F1 differed from NOD with a p value of 0.0005 and from the NOD.B10 Idd9 strain with a p value of 0.0031.

Table 1. Genetic Characterization of Idd9 Congenic Strains

MarkercMaGeneNOD.B10 Idd9NOD.B10 Idd9 R28NOD.B10 Idd9 R11NOD.B10 Idd9 R15NOD.B10 Idd9 R38NOD.B10 Idd9 R35NOD.B10 Idd9 R73D4Mit82NODNODNODNODNODNODNODD4Mit273.3bB10NODNODNODNODNODNODD4Mit2810.9bB10B10NODNODNODNODNODJak1c–––––––D4Mit316.3 (43/682)B10B10NODNODNODNODNODD4Mit728.7 (59/682)B10B10NODNODNODNODNODD4Nds201.0 (7/682)LckB10B10ndndndndndD4Mit694.7 (32/682)B10B10NODNODNODNODNODD4Mit2510 (0/368)ndB10NODndNODNODNODD4Nds234.1 (15/368)CD30B10B10B10B10B10NODNODD4Mit2330 (0/368)Tnfr2B10B10B10B10B10NODNODD4Mit2850.3 (2/682)ndB10B10B10B10NODNODD4Mit3100.6 (4/682)ndB10B10B10B10NODNODD4Mit1270.4 (3/682)B10B10B10B10B10NODNODD4Mit2260.2 (1/554)ndndndB10B10B10NODD4Mit330 (0/554)B10B10B10B10B10B10NODD4Mit1900 (0/554)ndndndB10B10B10NODD4Nds240 (0/554)Cd137B10B10B10B10B10B10NODD4Mit630 (0/554)ndndndB10B10B10NODWsl1d–––––––D4Mit421.8 (10/554)ndndndNODNODB10B10D4Mit1800.2 (1/554)B10B10B10NODNODB10B10D4Mit595.5bNODNODNDONODNODNODNODOx40d–––––––

nd, not determined; –, no variant marker available.

Intermarker distance in centimorgans (number of recombinants/total meioses typed).

Distance from MIT database.

Position determined from Mouse Genome Database.

Position determined by typing the T31 mouse radiation hybrid panel.

The level of diabetes resistance observed in the NOD.B10 Idd9 strain is profound and similar to that reported in the NOD.B6 Idd3 Idd10 Idd18 and NOD.B6 Idd3 Idd17 Idd10 Idd18 strains: 3%–7% in females and 0%–2% in males (45, 29). Resistance to diabetes was found to be a semidominant trait, since both male and female (NOD × NOD.B10 Idd9)F1 mice were partially protected from disease and were significantly different from both parental strains (Figure 1).

Insulitis in NOD.B10 Idd9 Mice

To determine if the protection from diabetes in the NOD.B10 Idd9 strain correlated with a reduction in insulitis, as was seen with the NOD.B6 Idd3 Idd17 Idd10 Idd18 strain (Wicker et al. 1994b), pancreata were examined histologically at various ages from both female and male NOD.B10 Idd9 mice. Almost all of the pancreata examined from young (3 months of age) and old (7–9 months of age) NOD.B10 Idd9 mice of both sexes had mild to extensive insulitis (Figure 2A). In fact, the insulitis seen with hematoxylin and eosin staining of NOD.B10 Idd9 pancreata could not be distinguished from that in NOD mice (Figure 2B). In contrast to the combined activities of Idd3, Idd17, Idd10, and Idd18, which reduce the occurrence and severity of insulitis (Figure 2A), diabetes resistance mediated by Idd9 must function on the pathogenicity of the insulitis or the resistance of the β cells to immune destruction.

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Figure 2. NOD.B10 Idd9 Mice Are Protected from Diabetes but Not from the Development of Insulitis

(A) Insulitis is more prevalent in NOD.B10 Idd9 mice than in NOD.B6 Idd3 Idd17 Idd10 Idd18 mice. Insulitis was assessed in nondiabetic mice. Each animal received one of the following scores: none (all islets observed are free of insulitis), mild (less than 10% of the islets are infiltrated), moderate (10%–50% of the islets are affected), or extensive (greater than 50% of the islets have insulitis). Data and observations for the NOD.B6 Idd3 Idd17 Idd10 Idd18 mice were previously described (Wicker et al. 1994b).

(B) Massive insulitis in an islet from a 7-month-old male NOD.B10 Idd9 mouse is shown after formalin fixation and hematoxylin and eosin staining (×100).

Qualitative Differences in the Inflammatory Cells within NOD and NOD.B10 Idd9 Islets

In order to distinguish between these possible mechanisms of action of Idd9-mediated protection in the islets, the expression of various cell surface receptors and cytokines was examined in the pancreatic infiltrates of NOD.B10 Idd9 and NOD mice. Pancreata from ten nondiabetic female mice of each strain ranging in age from 10 to 20 weeks were assessed. No significant differences between the two strains were observed in the numbers of CD4-, CD8-, or B220-positive cells present in the insulitic lesions (data not shown). CD4-positive cells constituted the most abundant cell type in both strains. Equivalent expression of several adhesion molecules was observed for the two strains. ICAM-1 was expressed by endothelial cells and leukocytes. ICAM-2 and PECAM-1 were observed on all endothelial cells, whereas VCAM-1 was expressed on a subset of endothelial cells. P-selectin and E-selectin were not observed (data not shown). In contrast, expression of CD30, IL-4, IFNγ, and TNFα (Figure 3), plus IL-13 and TGFβ (not shown), did differ between the two strains. IL-1, IL-2, IL-5, and IL-10 were not detected in either strain.

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Figure 3. Immunohistochemical Analysis of NOD and NOD.B10 Idd9 Pancreata

An infiltrated NOD islet (A, D, G, and J) and heavily (B, E, H, and K) and mildly (C, F, I, and L) infiltrated NOD.B10 Idd9 islets were assessed for the indicated cytokines and cell surface marker (×200). This figure is representative of ten nondiabetic female mice of each strain examined from 10 to 20 weeks of age. Within each strain there was no age-dependent variation of cytokine or CD30 expression observed.

Islet infiltrates from both a heavily (Figures 3B, 3E, 3H, 3K) and a mildly (Figures 3C, 3F, 3I, 3L) infiltrated NOD.B10 Idd9 islet were compared to an insulitis-positive NOD islet (Figures 3A, 3D, 3G, 3J). In the NOD islets IFNγ (Figure 3A–3C) and TNFα (Figure 3D–F) are preferentially expressed (Figure 3A and Figure 3D), while IL-4 (Figure 3G–3I) is the prominent cytokine in the infiltrating cells present in the NOD.B10 Idd9 islets (Figure 3H and Figure 3I). It is particularly striking that even when very few infiltrating cells are present in NOD.B10 Idd9 islets, IL-4 is still the prominent feature (Figure 3I). CD30 is a TNFR family member encoded within the Idd9 genetic region (Table 1) that is preferentially upregulated in cultured IL-4-secreting Th2 cells (26, 27) and appears to play a role in protection from diabetes mediated by CD8 T cells (Kurts et al. 1999). A comparison of CD30 expression (Figure 3J–3L) with IL-4- and IFNγ-producing cells within the islets showed a pattern nearly identical to that observed with IL-4: preferential expression in the NOD.B10 Idd9 islets (Figure 3K and Figure 3L). The differential expression of CD30 and IL-4 versus IFNγ and TNFα in the NOD and NOD.B10 Idd9 infiltrated islets was seen in all mice and was not affected by age. IL-13 and TGFβ were also observed in infiltrated islets of NOD.B10 Idd9 but not of NOD mice (data not shown). Thus, the protection afforded by the B10 Idd9 genetic region is consistent with an alteration of the pathogenicity of the insulitis.

Complete Protection from Spontaneous Diabetes in NOD Congenic Mice with Resistance Alleles on Both Chromosomes 3 and 4

Given the highly significant protection from diabetes afforded by introgression of non-NOD DNA from chromosome 3 (NOD.B6 Idd3 Idd17 Idd10 Idd18) or chromosome 4 (NOD.B10 Idd9), we developed a congenic strain that combines these two protective regions: NOD.B6 Idd3 Idd17 Idd10 Idd18 B10 Idd9 (triple congenic). There was no spontaneous diabetes observed in more than 250 female and male triple congenic mice followed for 7 months. A subgroup of mice (n = 133) aged to 12 months also did not develop diabetes. Despite the lack of diabetes, a small number of NOD.B6 Idd3 Idd17 Idd10 Idd18 B10 Idd9 mice developed insulitis (10%, n = 20).

The Original Idd9 Effect Is Due to the Combined Action of at Least Three Loci

To refine the map location of Idd9, we bred two series of subcongenic strains. The first set of subcongenic strains, NOD.B10 Idd9 R28 (R28) and NOD.B10 Idd9 R11 (R11), was derived from the original NOD.B10 Idd9 strain (Table 1). The R28 strain has a 44.7 cM congenic interval between but not including the microsatellite markers D4Mit27 and D4Mit59. The R11 strain has a 13.1 cM congenic interval between the markers D4Mit251 and D4Mit59 (Table 1). The development of diabetes in cohorts of female mice from each strain was assessed over a period of 7 months (Figure 4). The congenic intervals of strains R28 and R11 both conferred significant protection from diabetes (p < 0.0001 for both strains compared to the NOD parental strain). However, the R28 strain was significantly more protected than the R11 strain (p < 0.0001 for R28 versus R11), implying that the R28 congenic interval contains at least one additional protective locus as compared to the R11 interval. This locus, Idd9.1, must lie in the 35.7 cM interval between but not including the microsatellite markers D4Mit27 and D4Mit251 (Table 1).

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Figure 4. Localization of Idd9.1, Idd9.2, and Idd9.3

Subcongenic strains were developed and assessed for the development of diabetes.

A second set of Idd9 subcongenic mice was developed from the R11 strain. The strains NOD.B10 Idd9 R15 (R15) and NOD.B10 Idd9 R38 (R38) have congenic intervals of 7.4 cM between the microsatellite markers D4Mit251 and D4Mit42, the NOD.B10 Idd9 R35 (R35) strain has a congenic interval of 7.7 cM between the markers D4Mit127 and D4Mit59, and the NOD.B10 Idd9 R73 (R73) strain has a congenic interval of 7.5 cM between the markers D4Mit63 and D4Mit59 (Table 1). The development of diabetes in female mice from each of these strains is shown in Figure 4. The R15 and R38 strains fully recapitulate the diabetes protection seen in the parental R11 strain mapping the Idd locus contained within this congenic interval to proximal of D4Mit42. This mapping data is confirmed by the reciprocal R73 strain, which is NOD derived between the markers D4Mit251 and D4Mit42 and has an NOD-like frequency of diabetes (Figure 4). The R35 strain, while significantly protected from diabetes development (p = 0.0026 versus NOD), is significantly less protected than the R15, R38, or R11 strains (p < 0.0001, p = 0.0005, and p = 0.0024, respectively). Thus, the diabetes protection conferred by the R11 congenic interval is most likely due to the interaction of at least two loci: Idd9.2, which maps to the 5.6 cM interval between the markers D4Mit251 and D4Mit226, and Idd9.3, which maps to the 2.0 cM region between D4Mit127 and D4Mit42 (Table 1).

A Cluster of Genes Encoding TNFR Superfamily Members Maps to the Idd9.2/Idd9.3 Interval

Cd30, Tnfr2, Cd137 (4–1bb), Wsl1, and Ox40 are members of the TNFR superfamily that contribute to the activation and/or apoptosis of cells and have been mapped to distal chromosome 4. Genotyping the Idd9 subcongenic strains positions the CD30 and TNFR2 genes in the 5.6 cM Idd9.2 interval (Table 1). Both the CD30 (Siegmund et al. 2000) and TNFR2 (Powell et al. 1994) genes contain sequence variants that give rise to amino acid variation between the NOD and B10 forms of their respective proteins (Figure 5). This coding variation makes both genes candidates for Idd9.2. Sequencing of the CD137 gene in the present report identified three coding variants between B10 and NOD, a valine to alanine substitution at position 24, a leucine to proline substitution at position 211, and the insertion of an alanine in NOD between amino acids 174 and 175 (Figure 5). Analysis of the Idd9 congenic strains places the CD137 gene within the 2.0 cM Idd9.3 interval (Table 1), making it a candidate for this locus. The WSL1 gene maps to the distal boundary of the Idd9.3 interval between the microsatellite markers D4Mit63 and D4Mit42 (Table 1). Sequencing of WSL1 identified no nucleotide variation between NOD and B10 within either the coding region or the putative promoter (data not shown). The absence of any sequence variation between NOD and B10 makes Wsl1 an unlikely candidate for Idd9.3. The OX40 gene was excluded as a candidate for any of the Idd9 loci as it maps distal to D4Mit59 and therefore outside of the original long Idd9 congenic interval (data not shown).

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Figure 5. Variant Candidate Genes Located in the Idd9.2 and Idd9.3 Intervals

Variants of Cd30 (A), Tnfr2 (B), and Cd137 (C). B10 to NOD changes are noted below each diagram.

Cell Surface Expression of TNFR Superfamily Members

Since sequence variation has been observed between the NOD and B10 genes encoding CD30, TNFR2, and CD137, we compared the cell surface expression of these molecules on CD4 and CD8 splenic T cells following activation with anti-CD3. Spleen cells obtained from the NOD and R28 strains, which have the NOD and B10 alleles of Cd30, Tnfr2, and Cd137, respectively, were cultured with anti-CD3 using Th1 or Th2 conditions. After 72 hr of stimulation, cell surface expression of CD137 and CD30 was optimal with Th2 culture conditions in both strains. TNFR2 expression was equivalent using either Th1 or Th2 conditions in both strains. Representative profiles for CD137 and TNFR2 expression on anti-CD3-stimulated CD8 cells are shown in Figure 6A. A complete profile of CD30 expression on activated CD4 and CD8 cells is shown in Figure 6B. Interestingly, CD30 is highly expressed in infiltrated islets from diabetes-resistant NOD.B10 Idd9 mice (Figure 3K and Figure 3L) but not from NOD mice (Figure 3J). However, CD4 and CD8 Th2-polarized cells from both strains express CD30 (Figure 6B) and secrete IL-4 following recall at day 4 with anti-CD3 (data not shown). In contrast, CD4 and CD8 Th1-polarized cells from both strains express little or no CD30 (Figure 6B) and secrete no IL-4 upon recall challenge (data not shown). Significant levels of IL-4 are only observed in the islet infiltrates of NOD.B10 Idd9 mice (Figure 3H and Figure 3I). This suggests that the relatively low level of CD30 expression in the cellular infiltrates of NOD islets (Figure 3G) is due to insufficient levels of IL-4.

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Figure 6. Expression of Molecules Encoded by Variant Candidate Genes

(A) Cell surface expression of TNFR2 and CD137 on activated CD8 T cells. Spleen cells obtained from NOD and NOD.B10 Idd9 R28 mice were stimulated with anti-CD3 for 72 hr using Th2 conditions. Profiles display cell surface expression of TNFR2 and CD137 (versus isotype controls in each profile) on gated CD8 cells. The data are representative of two experiments.

(B) Cell surface expression of CD30 on CD4 and CD8 T cells. NOD and NOD.B10 Idd9 R28 spleen cells were stimulated under Th1 or Th2 conditions for 72 hr. The profiles shown are gated either on CD4 or CD8 cells as indicated and display cell surface expression of CD30 (versus an isotype control). The data are representative of two experiments.

Discussion

The NOD mouse model of human type 1 diabetes is complex, involving many genes and biochemical pathways. In order to analyze the natural disease process in NOD mice, congenic strains have been produced based on linkage mapping results. The observation of disease protection in such congenic strains provides evidence of the existence of individual Idd loci. Moreover, the development of congenic strains facilitates functional analyses of Idd loci, either in isolation or in specific combinations (36, 38, 45, 14, 48, 5, 29, 30, 20, 3). We have now constructed and analyzed the phenotypes of congenic strains carrying diabetes-resistance alleles on chromosomes 3 and 4 (45, 14, 5, 29, 30, 16). Results from these analyses clearly demonstrate that two mechanisms of protection from naturally occurring autoimmune β cell destruction can be defined. The first, which is a characteristic of the loci on chromosome 3, results in a severe reduction of inflammatory cells in islets. The second, observed with the loci on chromosome 4, does not reduce the number of infiltrating cells but causes a change in the cytokine profiles and pathogenic properties of the infiltrate.

The finding that the diabetes protection afforded by Idd9 is associated with the accumulation of IL-4-producing cells in the islets is reminiscent of other studies in which IL-4 is associated with a nonpathogenic outcome. For example, in orally tolerized NOD mice, disease suppression is accompanied by an increase in IL-4- and a decrease in IFNγ-producing cells (10, 28). In addition, long-term survival of allogeneic and xenogenic vascularized grafts induced by immunosuppression is associated with a Th2-like cellular response, whereas rejected grafts contain a Th1 cell infiltrate (24, 2). While it is likely that these models reflect different mechanisms of initiating immune regulation, it is clear that the ultimate accumulation of IL-4-producing cells is associated with disease protection. In light of the association of IL-4 production with Idd9-mediated diabetes protection, it is particularly striking that transgenic production of IL-4 in the islet β cell eliminates diabetes and insulitis in NOD mice (Mueller et al. 1996).

In the current study, subcongenic analysis of the Idd9 region has demonstrated that at least three loci are responsible for the B10 Idd9-mediated protection from diabetes: Idd9.1, Idd9.2 and Idd9.3. In addition, we have delineated the genetic intervals containing Idd9.2 and Idd9.3 to 5.6 and 2.0 cM, respectively. These intervals are now sufficiently small to sequence biologically relevant candidate genes contained within the regions (Lyons and Wicker 1999). To this end, we have found that the coding regions of the tightly linked genes Cd30 and Tnfr2 vary between NOD and B10. We have shown previously that the NOD allele of Tnfr2 varies at five amino acids from the B10 and B6 allele (Powell et al. 1994), and we have recently found that the CD30 gene has four amino acid differences between NOD and B10 (Siegmund et al. 2000). Some of the coding differences are not conservative and may influence trimerisation of these TNFR family members. Among other possibilities, suboptimal association of TNFR chains could alter cell surface stability, binding to the cognate TNF family ligand, or association of the cytoplasmic domain with TRAF proteins, which may in turn affect downstream signaling from the receptor. Thus, Cd30 and Tnfr2, whose protein products have been shown to mediate cell death (1, 47), are both candidates for Idd9.2.

Interestingly, a recent study demonstrated that CD30 is essential for controlling the diabetogenicity of CD8 T cells in a transgenic model of autoimmune diabetes (Kurts et al. 1999). CD30−/− CD8 cells were shown to have a much higher proliferative potential in vivo than wild-type CD8 cells. Since CD8 T cells are critical to the initiation and progression of diabetes in NOD mice (44, 37), the presence of a variant CD30 gene within the Idd9.2 interval is particularly intriguing. Given these recent observations by Kurts et al. 1999, it is possible that a major protective effect of IL-4 production in the pancreas is its ability to upregulate CD30 expression (Figure 3K and Figure 3L). Rapoport et al. 1993 have reported defective IL-4 secretion from concanavalin A–stimulated thymocytes in NOD mice, which could account for the reduced IL-4 levels seen in the parental strain.

Cd137, which is also a TNFR family member, also varies in the coding region between NOD and B10. Most noteworthy is a nonconservative leucine to proline alteration in the cytoplasmic domain. Such a change could alter the function of the trimeric receptor as outlined above, thereby making Cd137 a candidate gene for Idd9.3. In contrast to TNFR2 and CD30, CD137 signaling mediates positive stimulation in both CD4 and CD8 cells (39, 22). These three candidate genes along with the genes underlying Idd9.1 may represent autoimmunity loci, since other autoimmune diseases have been linked to this region (Vyse and Todd 1996). Loci controlling systemic lupus erythematosus (Nba1) and thymectomy-induced autoimmune gastritis (EAG, Gasa1) have been mapped to Idd9.1 (6, 41), and loci influencing autoimmune hemolytic anemia (Aia1) and EAG (Gasa2) have been mapped to the Idd9.2 and Idd9.3 intervals (12, 41). A locus influencing susceptibility to experimental autoimmune encephalomyelitis has also been localized to the distal region of chromosome 4 (Encinas et al. 1996). Idd11, defined originally in an outcross between the NOD and B6 strains, has been recently shown by congenic analysis to be localized to a 13 cM interval on chromosome 4 (Brodnicki et al. 2000). This interval is contained entirely within the Idd9.1 segment delineated in the current study. While these results are consistent with Idd11 and Idd9.1 being the same gene, B6 and B10 mice differ in more than one gene within this interval (19, 23), and hence the Idd9.1 segment could contain more than one Idd locus. Further refinement of the location of both loci is required to prove this hypothesis.

The NOD mouse genetic background predisposes to autoimmune inflammation and diabetes and appears to contain no major resistance alleles. Susceptibility alleles of many loci combine to exceed a disease threshold for which the introduction of only one resistance allele (in the homozygous state) can cause the majority of mice to fall below this threshold and become resistant to disease. Such a potent effect was observed when the B6 allele of Idd3 was introgressed onto the NOD genome (Lyons et al. 2000). However, when isolated within small congenic intervals, other alleles contributing to resistance such as Idd10 and Idd18 are much less potent (Podolin et al. 1998) than the resistance allele at Idd3. Although individually their effects are limited, the combination of Idd3, Idd10, and Idd18 provides almost complete protection from diabetes (Wicker et al. 1994b). A similar picture has emerged in the current study of the Idd9 region. At least three genes contribute to the nearly complete protection from disease seen when all three alleles are present in a homozygous state. The existence of two sets of linked resistance genes, one set on chromosome 3 and another set on chromosome 4, suggests that sets of linked alleles may have been selected to provide a particular advantage to some infectious agent. Interestingly, these two sets of resistance genes mediate protection in very different ways, suggesting that there may be several mechanisms that could be targeted in modulating the progression of autoimmunity. The molecular and cellular basis of such potent disease protection will contribute to the understanding of autoimmune disease pathogenesis and the development of therapeutic approaches. Such therapies may require the combined targeting of several genetically distinct pathways.

Experimental Procedures

Mice

NOD.B10 Idd9 mice were developed by backcrossing C57BL/10 mice purchased from the Jackson Laboratory (Bar Harbor, ME) to NOD/MrkTacfBR mice obtained from Taconic Farms (Germantown, NY) with selection for the described region on chromosome 4 (Table 1). Mice used in this study were from the N9F2–4 generations. Founders of the NOD.B10 Idd9 strain were tested for the presence of B10 chromosome segments throughout the genome using a panel of microsatellite markers that differentiate NOD and B10 genomic segments (Lord et al. 1995). All non–chromosome 4 markers examined were of NOD origin in the NOD.B10 Idd9 strain. The NOD.B10 Idd9 R11 (N12F2–4) and NOD.B10 Idd9 R28 (N12F2–4) subcongenic strains were developed from the NOD.B10 Idd9 strain, and the NOD.B10 Idd9 R15 (N14F2–4), NOD.B10 Idd9 R35 (N14F2–4), NOD.B10 Idd9 R38 (N14F2–4), and NOD.B10 Idd9 R73 (N14F2–4) strains were developed from the NOD.B10 Idd9 R11 strain, essentially as described previously (Podolin et al. 1997).

To produce the NOD.B10 Idd9 B6 Idd3 Idd10 strain, mice from the NOD.B6 Idd3 Idd10 (N6F9) strain (termed NOD.B6 Il2-Tshb in Wicker et al. 1994b), which has approximately 40 cM of B6 DNA from chromosome 3 introgressed onto the NOD background, were intercrossed with NOD.B10 Idd9 N9F4 mice. Progeny having the non-NOD regions on both chromosomes 3 and 4 were selected.

Assessment of Diabetes

Elevated urinary glucose was detected using Diastix (Miles, Elkhart, IN). Animals were classified as diabetic when urinary glucose was at least 500 mg/dl. Diabetic mice also exhibited polydipsia, polyuria, and weight loss.

Histology

Pancreata were fixed in 10% bufferred formalin and processed for paraffin embedding. Tissue sections (5 μm) were stained with hematoxylin and eosin and microscopically evaluated for the presence of mononuclear cell infiltrates. Two noncontiguous sections of each pancreas were examined. To evaluate islets for leukocyte infiltration and differential cytokine expression, pancreata were quick frozen in liquid nitrogen and processed as described for the following: cell markers—CD4, CD8, CD30, B220; adhesion molecules—P-selectin, E-selectin, ICAM-1, ICAM-2, PECAM-1, and VCAM-1; and cytokines—IL-1, IL-2, IL-4, IL-5, IL-10, IL-13, IFNγ, TNFα, and TGFβ (Hancock et al. 1995), using mAbs purchased from PharMingen (San Diego, CA).

Statistical Analysis

Fisher's exact test was used to compare the 7 month cumulative diabetes frequencies of the NOD congenic mice. Comparisons giving p values greater less than 0.05 were considered to be significant. To compare the frequency of diabetes in subcongenic strains, Kaplan-Meier survival analysis was performed using the Log-Rank test.

Candidate Gene Sequencing and Mapping

PCR primer pairs flanking each exon of Cd137 and Wsl1 were designed based on their genomic sequence. Following amplification, PCR products were sequenced directly using either a BigDye terminator cycle sequencing kit (PE Biosystems, Warrington, UK) or Bodipy Dye primers (Metzker et al. 1998). Cd137 was mapped by typing D4Nds24 on 277 F2 progeny of crosses between NOD and NOD.B10 Idd9 R28 or NOD.B10 Idd9 R11. Wsl1 and Ox40, for which no polymorphic markers exist, were mapped relative to the other markers using the T31 mouse hamster radiation hybrid panel (McCarthy et al. 1997).

Induction and Assessment of Cell Surface Expression of TNFR2 and CD137

Spleen cells (0.6 × 106/ml) were cultured for 72 hr using standard culture conditions (Chen et al. 1994) with anti-CD3 (500 ng/ml 2C11), 2 ng/ml IL2, and either 20 ng/ml IL4 (Th2 conditions) or 2 ng/ml IL12 and 2 μg/ml anti-IL4 (Th1 conditions). Cells were washed and incubated with FITC-conjugated anti-CD4 (RM4–5), APC-conjugated anti-CD8 (53–6.7), and one of the following PE conjugates: anti-CD137 (1AH2) or its isotype control (R3–34) followed by PE-anti-rat Ig (RG11/39.4) or PE-conjugated anti-TNFR2 (HM102, CALTAG, Burlingame, CA) or its PE-conjugated isotype control (R35–95). Unless otherwise noted, all cytokines and antibodies were obtained from PharMingen (San Diego, CA). Labeled cells were analyzed by flow cytometry (FACStar PLUS, BD Immunocytometry Systems, San Jose, CA).

Acknowledgements

We would like to thank Dr. Mike Owen for providing Wsl1 genomic sequence data prior to publication. This work was supported by grants from the Wellcome Trust, the Juvenile Diabetes Foundation, and Merck Research Laboratories.

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Chapter 119 - CD30

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