NOD Congenic Mice Genetically Protected From Autoimmune Diabetes Remain Resistant to Transplantation Tolerance Induction Free

C57BL/6 (H2^b), C3H/HeJ (H2^k), and BALB/c (H2^d) mice were obtained from the National Cancer Institute (Frederick, MD). NOD/MrkTacfBR, NOD.B6 Idd3R450, NOD.B10 Idd5R444, NOD.B6 Idd3R450 B10 Idd5R8, NOD.B10 Idd9R28, NOD.B6 Idd10Idd18R, and NOD.B6 Idd3Idd10Idd18R323 (all H2^g7) were obtained from Taconic Farms (Germantown, NY). NOD/Lt (H2^g7) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6.NODc1c, C57BL/6.NODc1t, C57BL/6.NODc3, and C57BL/6.NODc6 mice (H2^b; developed by Edward Wakeland, University of Texas Southwestern Medical Center, Dallas, TX) were the gift of Dr. Edward Leiter (The Jackson Laboratory). B10.D2-HC⁰ (genetic designation: B10.D2-H2^d H2-T18^c Hc⁰/oSnJ) and B10D2-HC¹ mice (genetic designation: B10.D2-H2^d H2-T18^c Hc¹/nSnJ) were obtained from The Jackson Laboratory. Table 1 summarizes the congenic interval(s) and diabetes incidence of each of the congenic mouse strains listed above that were used in these experiments.

All animals were certified to be free of Sendai virus, pneumonia virus of mice, murine hepatitis virus, minute virus of mice, ectromelia, lactate dehydrogenase–elevating virus, mouse poliovirus, Reo-3 virus, mouse adenovirus, lymphocytic choriomeningitis virus, polyoma, Mycoplasma pulmonis, and Encephalitozoon cuniculi. They were housed in a specific pathogen-free facility in microisolator cages and given autoclaved food and acidified water ad libitum. All animal use was in accordance with the guidelines of the institutional animal care and use committee of the University of Massachusetts Medical School and recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996).

Graft recipient mice were treated with DST and anti-CD154 mAb and transplanted with skin allografts as described (22,31). Briefly, the DST consisted of a single intravenous injection of 10⁷ C3H/HeJ or BALB/c female spleen cells obtained from 5- to 10-week-old donors. The DST was injected on day −7 relative to skin transplantation. Clone MR1 hamster anti-mouse CD154 mAb (32) was produced as ascites in scid mice and purified by protein A affinity chromatography (Amersham Pharmacia Biotech, Uppsala, Sweden). Antibody concentration was determined by measurement of optical density and confirmed by enzyme-linked immunosorbent assay (ELISA) as described previously (33). The concentration of contaminating endotoxin was determined commercially (Charles River Endosafe, Charleston, SC) and was uniformly <10 endotoxin units per milligram of mAb. Mice were injected intraperitoneally with anti-CD154 mAb (0.25 or 0.5 mg/dose as indicated in text) on days −7, −4, 0, and +4 relative to skin transplantation. Full-thickness skin grafts 1–2 cm in diameter were obtained from the flanks of donor mice and transplanted onto the dorsal flanks of recipients as described (22). Grafts were examined three times weekly, and rejection was defined as the first day on which the entire graft surface appeared necrotic (22). Grafts adherent to the bandage or fully necrotic on day 7 were deemed technical failures and were excluded from analysis (22).

Clearance kinetics of the hamster MR1 anti-CD154 mAb was determined in groups of NOD and C57BL/6 mice that were given a single intraperitoneal injection of 2.0 mg of antibody. Serum was collected from blood obtained on days 13, 17, 20, 24, 32, and 40 after antibody injection. MR1 hamster IgG levels were quantified in the serum samples by ELISA (33).

The average duration of graft survival is presented as the median. Graft survival among groups was compared using the method of Kaplan and Meier (34). The equality of allograft survival distributions for animals in different treatment groups was tested using the log rank statistic (34). P values <0.05 were considered statistically significant. Comparisons of two means used unpaired t tests (35). MR1 clearance rates were calculated by single-phase exponential decay curves fitted by regression analysis using Prism (Version 3.0; Graphpad Software, San Diego, CA).

The NOD mouse has numerous immune defects that may be involved in their resistance to the induction of transplantation tolerance. One defect that could impact this strain’s response to in vivo antibody therapy is the absence of hemolytic complement caused by their genetic deficiency in C5a (8). To test this, we attempted to tolerize B10.D2 congenic mice that genetically differ in the presence or absence of C5a (36). These congenic mice were treated with DST, anti-CD154 mAb, and C3H/HeJ skin allografts. As shown in Fig. 1, the genetic absence of C5a in B10.D2-HC⁰ mice did not impair prolongation of skin allograft survival induced by costimulation blockade (median survival time [MST] = 54 days), and in fact, it slightly prolonged skin allograft survival as compared with similarly treated B10.D2-HC¹ mice in which C5a was present (MST = 39 days, P < 0.01).

In a related experiment, we sought to determine whether NOD mice are resistant to tolerance induction because of an accelerated clearance rate of anti-CD154 mAb from the circulation. We have previously documented that skin allograft rejection in tolerized mice is inversely correlated with the level of circulating anti-CD154 mAb (33). Groups of NOD and C57BL/6 mice were given a single intraperitoneal injection of 2.0 mg of anti-CD154 mAb. The concentration of MR1 in their serum was determined periodically between days 13 and 40 after injection to calculate the clearance rate of the antibody. The serum concentration of antibody on day 13 in NOD and C57BL/6 mice was ∼300 mcg/ml and declined to ∼100 mcg/ml in both groups by day 40. The calculated half life of MR1 in NOD (10.7 days, n = 6) and C57BL/6 (10.4 days, n = 5) mice was similar. The serum concentrations at these time points after antibody injection and clearance rates of antibody are comparable to those we observed in CBA mice (33). These data document that neither the absence of hemolytic complement nor an accelerated clearance of anti-CD154 mAb in NOD mice accounts for the inability of costimulation blockade to prolong skin graft survival.

We next tested the hypothesis that selected NOD-derived Idd susceptibility loci are responsible for the defect in peripheral transplantation tolerance induction by costimulation blockade. This was first assessed by determining whether the ability to prolong skin allograft survival was abrogated in C57BL/6 mice congenic for various NOD-derived Idd loci (22). None of the C57BL/6 congenic mice developed insulitis or diabetes (Table 1). We tested four congenic strains. C57BL/6.NODc1c mice carry the NOD-derived Idd5.1 region, which includes genes encoding CD152 (CTLA4) and CD28, costimulatory molecules that are important for tolerance induction and immune activation (21). C57BL/6.NODc1t mice carry the NOD-derived Idd5.1 as well as the Idd5.2 region, which includes the CXCR2 and interleukin-8 receptor genes. C57BL/6.NODc3 mice carry the NOD-derived Idd3, Idd17, Idd10, and Idd18 loci, which include genes that control cell activation. C57BL/6.NODc6 mice carry the NOD Idd6 locus, which includes genes contained within the NK receptor protein-1a (NKR-P1) complex (Table 1).

The median skin allograft survival time of each of these C57BL/6 congenic strains ranged from 62 to >93 days and were statistically similar to that observed in C57BL/6 wild-type mice (MST = 81 to >94 days) (Table 2). Skin allograft survival in the C57BL/6 congenic mice was significantly greater than that observed in similarly treated NOD mice (MST = 20–24 days) (Tables 3 and 4).

We next determined whether NOD stocks congenic for selected C57BL/6- or C57BL/10-derived Idd loci that mediate protection from insulitis and diabetes would also exhibit prolonged skin allograft survival after treatment with DST and anti-CD154 mAb. We tested three groups of NOD congenic mice with intervals that encompass the non–major histocompatibility complex (MHC) Idd loci with the greatest effect on diabetes expression (18,37–39). The first of these were NOD congenic stocks carrying a C57BL/6-derived Idd3 or C57BL/10-derived Idd5 locus alone or in combination. These NOD single-Idd congenic mice each have a reduced frequency of diabetes, and when the Idd3 and Idd5 resistance loci are combined, the frequency of diabetes is reduced to 1%, and insulitis is absent in most mice (18) (Table 1). The median survival time of C3H/HeJ skin allografts in these NOD congenic mice treated with DST plus anti-CD154 mAb (24–31 days) was statistically significantly shorter than that of C57BL/6 mice (>115 days) (Table 3). Skin allograft survival in these NOD congenic mice was similar to that of NOD/Lt (MST = 20 days) or NOD/MrkTac (MST = 23 days) mice (Table 3).

We next tested NOD congenic mice that carried various combinations of C57BL/6-derived Idd3, Idd10, and Idd18 resistance loci. The frequency of diabetes in these NOD congenic mice varies from 9 to 50% (Table 1). A significant reduction in the frequency of insulitis was also observed in NOD.B6 Idd3/10/18 triple congenic mice (40,41). Again, all of the NOD congenic strains with various combinations of these genetic intervals were resistant to tolerance induction and had median skin allograft survival times of 17–29 days (Table 4).

Finally, we tested an NOD stock congenic for the C57BL/10-derived Idd9 locus. This locus contains molecular variants of Cd30, Tnfr2, and Cd137 (39), and these mice have a frequency of diabetes of only 3% (Table 1). The MST of skin allografts in NOD.B10 Idd9 mice was 20 days, similar to that observed in NOD/Lt mice (MST = 24 days, Table 4).

In the present study, we have investigated the relationship between genes that control autoimmune diabetes expression in NOD mice with those controlling their resistance to transplantation tolerance induced by costimulation blockade. We document that single or small combinations of the evaluated C57BL/6-derived Idd loci that dramatically alter diabetes expression are not able to correct the response of NOD mice to costimulation blockade and, conversely, that NOD-derived Idd loci do not shorten skin allograft survival in C57BL/6 mice. We further eliminate two other potential explanations: the genetic absence of hemolytic complement or an accelerated clearance rate of anti-CD154 mAb from the circulation.

Peripheral transplantation tolerance induction by DST and anti-CD154 mAb involves the deletion of alloreactive CD8⁺ T-cells (31,33). Because CD8⁺ T-cells in NOD mice appear to be resistant to tolerance induction (42), this may be one mechanism by which NOD mice are resistant to costimulation blockade–induced tolerance. However, we have recently determined that the majority of high-affinity alloreactive CD4⁺ T-cells are also deleted by treatment with DST and anti-CD154 mAb (D.L.G., unpublished observations). CD4⁺ T-cells express CD154 when activated (32). This suggests that one mechanism by which DST and anti-CD154 mAb induces tolerance could involve deletion of alloreactive CD4⁺ T-cells by antibody-mediated, complement-dependent lysis. However, the ability to prolong skin allograft survival in congenic B10.D2 mice that lacked C5a and hemolytic complement argues that this defect does not prevent tolerance induction in NOD mice. We have also documented that the circulating level of anti-CD154 mAb is inversely correlated with skin allograft survival in recipients treated with DST and anti-CD154 mAb (33). However, the clearance rate of anti-CD154 mAb from the circulation of NOD mice was similar to that of C57BL/6 mice. This data suggests that rapid clearance of anti-CD154 mAb, hence potentially lowering anti-CD154 mAb concentrations below effective tolerizing levels (∼100 mcg/ml) (33), was not the basis for the resistance of NOD mice to tolerance induction.

We then tested the hypothesis that some of the genetic loci associated with development of autoimmune diabetes in NOD mice would also be important in their resistance to induction of peripheral transplantation tolerance. Prolonged allograft survival was not abrogated in C57BL/6 stocks congenic for any of the analyzed NOD-derived Idd susceptibility loci. We note, however, that no combination of Idd susceptibility loci introgressed into C57BL/6 mice to date has rendered them susceptible to the spontaneous development of insulitis or autoimmune diabetes.

Similarly, none of the analyzed C57BL/6- or C57BL/10-derived Idd congenic intervals that confer various degrees of diabetes resistance to NOD mice restored their ability to be tolerized by DST and anti-CD154 mAb treatment. This observation was surprising because congenic introgression of even a few of the Idd resistance loci into NOD mice profoundly reduces the frequency of insulitis and diabetes (17,18,38,39) (Table 1). Furthermore, many of the tested Idd congenic intervals are characterized by polymorphisms in genes important for costimulation and immune activation.

There is evidence that autoimmune diabetes in NOD mice is due primarily to defects in central tolerance (43). Bone marrow chimerism is known to prevent autoimmunity in NOD mice by this mechanism (44). In humans, bone marrow cells from diabetic donors have been documented to adoptively transfer disease to nondiabetic recipients, suggesting that central tolerance defects are also important in type 1 diabetes in humans (45). Additionally, there is data to suggest that manipulation of the peripheral immune system can affect self-tolerance and the expression of autoimmune diabetes in NOD mice (46–48). Mechanisms that control central and peripheral tolerance are different. Central tolerance is primarily mediated by intrathymic deletion of autoreactive T-cells during thymic development, whereas peripheral tolerance is mediated by multiple mechanisms, including deletion, anergy, and regulatory processes (21).

It is currently unknown whether improved central or peripheral tolerance is the mechanism by which the NOD congenic mice we studied in these experiments were rendered resistant to autoimmune diabetes. Our data suggest, however, that if the mechanism of protection from diabetes is due to restoration of the factors that permit peripheral regulation of autoimmunity, these mechanisms are not sufficient for the induction of peripheral allogeneic transplantation tolerance by costimulation blockade. Alternatively, it is possible that the results from the current study reflect the fact that more Idd resistance loci are required to genetically alter the phenotype of abnormal transplantation tolerance induction in NOD mice than are required to decrease the incidence of spontaneous autoimmune diabetes. Specifically, peripheral transplantation tolerance induction by costimulation blockade may be under the control of a complex combination of Idd loci not yet tested. Future experiments examining C57BL/6.H2^g7 congenic mice bearing NOD diabetes-susceptibility loci will be required to identify potential interactions of the unique NOD H2^g7 MHC with these genetic loci and their effect on tolerance induction. However, these data raise the possibility that the loss of self-tolerance leading to autoimmunity in NOD mice may be mediated by mechanisms that differ, in part, from their resistance to peripheral transplantation tolerance, a hypothesis we are currently testing.

This study was supported in part by grants AR35506 and AI42669 and an institutional Diabetes Endocrinology Research Center (DERC) grant DK52530 from the National Institutes of Health and by grant DK53006 jointly funded by the National Institutes of Health and the Juvenile Diabetes Research Foundation (JDRF). L.S.W. is supported by a joint grant from the JDRF and the Wellcome Trust. D.V.S. is supported by grants DK46266 and DK51090 from the National Institutes of Health and by a grant from the JDRF. The availability of NOD congenic mice through the Taconic Farms Emerging Models Program has been supported by grants from the Merck Genome Research Institute, National Institute of Allergy and Infectious Diseases (NIAID), and the JDRF.

We thank Linda Paquin, Stephanie Gibbons, Linda Leehy, and Jean Leif for technical assistance.