Genetic Disassociation of Autoimmunity and Resistance to Costimulation Blockade-Induced Transplantation Tolerance in Nonobese Diabetic Mice

Todd Pearson, Thomas G. Markees, David V. Serreze, Melissa A. Pierce, Michele P. Marron, Linda S. Wicker, Laurence B. Peterson, Leonard D. Shultz, John P. Mordes, Aldo A. Rossini and Dale L. Greiner
J Immunol July 1, 2003, 171 (1) 185-195; DOI: https://doi.org/10.4049/jimmunol.171.1.185

Abstract

Curing type 1 diabetes by islet transplantation requires overcoming both allorejection and recurrent autoimmunity. This has been achieved with systemic immunosuppression, but tolerance induction would be preferable. Most islet allotransplant tolerance induction protocols have been tested in nonobese diabetic (NOD) mice, and most have failed. Failure has been attributed to the underlying autoimmunity, assuming that autoimmunity and resistance to transplantation tolerance have a common basis. Out of concern that NOD biology could be misleading in this regard, we tested the hypothesis that autoimmunity and resistance to transplantation tolerance in NOD mice are distinct phenotypes. Unexpectedly, we observed that (NOD × C57BL/6)F1 mice, which have no diabetes, nonetheless resist prolongation of skin allografts by costimulation blockade. Further analyses revealed that the F1 mice shared the dendritic cell maturation defects and abnormal CD4+ T cell responses of the NOD but had lost its defects in macrophage maturation and NK cell activity. We conclude that resistance to allograft tolerance induction in the NOD mouse is not a direct consequence of overt autoimmunity and that autoimmunity and resistance to costimulation blockade-induced transplantation tolerance phenotypes in NOD mice can be dissociated genetically. The outcomes of tolerance induction protocols tested in NOD mice may not accurately predict outcomes in human subjects.

Nonobese diabetic (NOD)3 mice are used to model type 1 diabetes (1, 2, 3) and for the study of islet transplantation tolerance in the setting of autoimmunity (4). We and others have used them to test a costimulation blockade-based transplantation tolerance protocol (5, 6, 7). Surprisingly, this protocol, which readily prolongs skin and islet allograft survival in normal C57BL/6 mice, was largely unsuccessful in the NOD strain (8, 9). Given the well-recognized problem of recurrent autoimmunity in human islet transplantation (10), it initially seemed plausible to attribute the failure to induce transplantation tolerance to the underlying autoimmune process. These observations in the NOD mouse raised the possibility that transplantation tolerance may be extremely difficult to achieve in humans with autoimmune type 1 diabetes (9, 11).

However, we found on further study that this protocol also fails to prolong skin allograft survival in nondiabetic NOD male mice (8); skin is not a target of autoimmunity in these animals. In addition, NOD mice exhibit a large array of cellular and humoral immune abnormalities including defective macrophage and dendritic cell maturation (12, 13), defects in CD4+ T cell response to superantigens (14, 15), low levels of NK and NKT cell activity (16, 17, 18, 19), and deficiencies in regulatory CD4+CD25+ T cell function (20). This set of observations raises an important question: is the resistance of NOD mice to transplantation tolerance due to their expression of autoimmunity or to other abnormalities in their immune system? This is a critical distinction because humans with type 1 diabetes do not exhibit many of the immune abnormalities of the NOD mouse. Out of concern that NOD biology could be misleading with respect to the suitability of transplantation tolerance protocols for humans with autoimmune diabetes, we tested the hypothesis that autoimmunity and resistance to transplantation tolerance in NOD mice are distinct phenotypes.

In our initial test of this hypothesis, we observed that NOD congenic mice bearing strongly protective insulin-dependent diabetes (Idd) diabetes-resistant loci and expressing greatly reduced frequencies of diabetes and insulitis nonetheless resist prolongation of skin allograft survival using a costimulation blockade (21). Conversely, C57BL/6 mice congenic for NOD Idd diabetes-susceptibility loci were found to be fully responsive to costimulation blockade and exhibited prolonged skin allograft survival. These observations suggested that the expression of autoimmunity and the resistance to transplantation tolerance in NOD mice could be the result of distinct genetic mechanisms operating in one of two ways. First, autoimmunity and resistance to transplantation tolerance could be controlled by separate genetic loci that partially overlap. Second, induction of transplantation tolerance by costimulation blockade could require a higher genetic threshold than that required to retard or prevent autoimmunity.

To evaluate these two possibilities, we generated (NOD × C57BL/6)F1 mice. These hybrids are heterozygous at all NOD Idd loci; they have a C57BL/6-derived diabetes-resistant allele at each locus and are completely protected from autoimmune diabetes. We used these mice to test the hypothesis that the decrease in genetic susceptibility to autoimmunity would also decrease the genetic threshold for inducing transplantation tolerance. Unexpectedly, we found that that skin allograft survival was not markedly prolonged in (NOD × C57BL/6)F1 mice treated with costimulation blockade. Further analyses revealed that some, but not all, cellular immune abnormalities in the NOD mouse were corrected in the F1 mice.

Materials and Methods

Animals

C57BL/6 (H2b) and C3H/HeJ (H2k) mice were obtained from the National Cancer Institute (Frederick, MD). NOD/Lt (H2g7) mice are maintained in a breeding colony at our institution (University of Massachusetts Medical School, Worcester, MA). NOD.B6 Idd3 R450 B10 Idd5 R8 and NOD.B10 Idd9 R28 (Both H2g7) were purchased from Taconic Farms (Germantown, NY). C57BL/6.H2g7 (H2g7, official designation: C57BL/6.NODc17), developed by E. Wakeland (University of Texas Southwestern Medical Center, Dallas, TX), were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6.CD8a−/− (H2b, official designation: C57BL/6.129S2-Cd8atm1 Mak) were obtained from The Jackson Laboratory. The CD8a−/− targeted mutation was also congenically transferred to the NOD background. This N9 NOD.CD8a−/− stock (H2g7, official designation: NOD.129S2(B6)-Cd8atm1 Mak/Dvs) is homozygous for linkage markers delineating all known Idd loci of NOD origin (22, 23) and is strongly diabetes-resistant (1/17 females diabetic by 30 wk of age). In the course of developing this congenic stock, progeny carrying the CD8a−/− mutation were identified by PCR using the primer set 5′-GCTATTCGGCTATGACTGGG-3′ and 5′-GAAGGCGATAGAAGGCGATG-3′ which amplifies a 706-bp product from the neor insert used to disrupt the CD8α gene. All (NOD × C57BL/6)F1 animals were generated by a single intercross and are described with the following standard parental nomenclature: (female parent × male parent)F1. (NOD × C57BL/6)F1 CD8a−/− mice were generated by crossing NOD.CD8a−/− females with C57BL/6.CD8a−/− males.

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, given autoclaved food and acidified water, and maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School (Worcester, MA) 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).

Flow cytometry

Purified rabbit anti-asialo GM-1 (ASGM-1) polyclonal Ab was obtained from Wako Chemicals (Richmond, VA). FITC-conjugated goat anti-rabbit Ig polyclonal Ab obtained from BD PharMingen (San Diego, CA) was used to visualize cell bound anti-ASGM-1 Ab. Other Abs against mouse leukocyte Ags included: biotinylated anti-mouse pan-NK cell mAb (mAb, clone DX5), PE conjugated anti-CD25 mAb (clone PC61), PerCept-conjugated anti-CD4 mAb (clone RM4-5), FITC-conjugated anti-CD86 mAb (clone GL1), allophycocyanin-conjugated anti-CD11b mAb (clone M1/70), and PE-conjugated anti-CD11c mAb (clone HL3) were obtained from BD PharMingen. Allophycocyanin-conjugated streptavidin was used to visualize bound DX5 as well as any nonspecifically bound biotinylated rat IgM, κ (clone R4-22) isotype control. Additional isotype controls included PE-conjugated rat IgM, κ (clone R4-22), PerCept-conjugated rat IgG2a, κ (clone R35-95), FITC-conjugated rat IgG2a, λ (clone B39-4), allophycocyanin-conjugated rat IgG2b, κ (clone A95-1), and PE-conjugated hamster IgG1, λ (clone G235-2356). All isotype controls were obtained from BD PharMingen.

Multiparameter flow cytometry analyses of freshly isolated spleen cells were performed as described (24). Briefly, 1 × 106 viable cells were incubated for 5 min at 4°C with anti-FcγRIII/II mAb (clone 2.4G2) to eliminate nonspecific Fc binding of conjugated Abs. Cells were then washed and reacted with a mixture of conjugated mAbs for 20 min. In some cases, a third incubation with allophycocyanin-conjugated streptavidin was performed. Stained cells were washed, suspended in 1% paraformaldehyde-PBS, and analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA). Viable lymphoid cells were gated according to their light-scattering properties, and ∼2.5 × 104 events were acquired for each analysis.

Tolerance induction and allograft transplantation

MR1 hamster anti-mouse CD154 mAb was produced as tissue culture supernatant and purified by affinity chromatography (25, 26). Ab concentration was determined by measurement of OD and confirmed by ELISA (27). The concentration of contaminating endotoxin was determined commercially (Charles River Endosafe, Charleston, SC) and was uniformly <10 U/mg of mAb (25). Recipient mice were treated with a single donor-specific transfusion (DST) and anti-CD154 mAb and transplanted with skin allografts as described (5, 24, 28). Briefly, 107 spleen cells obtained from 5- to 10-wk-old female C3H/HeJ mice were injected i.v. in a volume of 0.5 ml. DST was given on day −7 relative to skin transplantation. Mice were injected i.p. with anti-CD154 mAb (0.5 mg/dose) on days −7, −4, 0, and +4 relative to skin transplantation.

Full thickness skin grafts 1–2 cm in diameter were transplanted onto the dorsal flanks of recipients as described (5). Grafts were examined three times weekly, and rejection was defined as the first day on which the entire graft surface appeared necrotic. Grafts adherent to the bandage or fully necrotic on day 6 were deemed technical failures and were excluded from analysis (28).

NK cell cytotoxicity assay

Cytotoxic activity of splenic NK cells was quantified using a previously described 51Cr-release microcytotoxicity assay (29). NK-sensitive YAC-1 virus-induced mouse T cell lymphoma (30) target cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in our laboratory in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal clone serum (HyClone Laboratories, Logan, Utah). YAC-1 target cells in growth phase were labeled with 51Cr as sodium chromate (100 μCi/million cells; New England Nuclear, Boston, MA), and labeled cells (1.0 × 104) were added to each well of a 96-well microtiter plate. Freshly isolated spleen cells obtained from animals injected i.p. with 100 μg of polyinosinic:polycytidylic acid (poly I:C) 24 h earlier were added at E:T cell ratios of 100, 50, 25, and 12.5:1 and incubated for 4 h at 37°C in a humidified atmosphere of 95% air-5% CO2 (31)

Total releasable radioactivity (cpmmaximal) was determined by incubating an aliquot of 51Cr-labeled target cells with 5% Triton X-100. After incubation, cells were pelleted by centrifugation, and 100 μl of aliquots of supernatant were transferred to a separate microtiter plate containing 100 μl of Optiphase Supermix β-scintillation fluid (Wallac, Gaithersburg, MD) and counted (cpmtest) using a 1450 Microbeta Trilux instrument (Wallac). Spontaneous release was uniformly <15% of maximal release. All assays were performed in triplicate and averaged and each assay was performed at least twice. Specific cytotoxicity was calculated as a percentage using the raw cpm and the formula: specific lysis (%) = ((cpmtest − cpmspontaneous)/(cpmmaximal − cpmspontaneous)) × 100%.

Macrophage IL-1β production assay

Bone marrow-derived macrophages were generated as previously described (13). Briefly, 5 × 106 bone marrow cells were cultured for 4 days at 37°C in complete medium plus 500 U/ml human M-CSF-1 (Sigma-Aldrich, St. Louis, MO) and 10 U/ml rat IFN-γ (R&D Systems, Minneapolis, MN). The cells were cultured an additional 16 h in fresh medium containing 10 μg/ml bacterial LPS (Sigma-Aldrich) in the absence of additional growth factors. Supernatants were harvested and levels of biologically active IL-1β were quantified in a C3H/HeJ thymocyte comitogenic assay as described (13).

Dendritic cell maturation assay

Bone marrow was flushed from the femurs and tibias of euthanized donors into RPMI 1640–10% FBS. Erythrocytes were lysed with 0.85% hypotonic NH4Cl lysis buffer and the mononuclear cells were washed twice more in RPMI 1640–10% FBS. Total mononuclear cells were counted using a Coulter counter (Coulter Z2; Miami, FL) and suspended at 2 × 106 cells/ml in RPMI 1640–10% FBS supplemented with 500 U/ml recombinant mouse GM-CSF and 1000 U/ml recombinant mouse IL-4 (R&D Systems). Bone marrow cells (3 × 106) were cultured in a six-well tissue culture plate in a total volume of 3 ml/well. The cultures for each strain consisted of pooled bone marrow from two mice. Cultures were incubated at 37°C in an atmosphere of 95% air-5% CO2. Forty-eight hours after initiation of culture, nonadherent cells were suspended by gentle swirling and half the medium was replaced with fresh medium supplemented with 500 U/ml GM-CSF and 1000 U/ml IL-4. On day 4, nonadherent cells were suspended by gentle swirling, and fresh medium supplemented with cytokines was added as on day 2. In selected wells, 5 μg/ml of an agonist anti-CD40 mAb (clone HM40-3; BD PharMingen) were added. Cultures were incubated an additional 48 h. On day 6 of culture, all cells (adherent and nonadherent) were harvested. Adherent cells were removed by gentle scraping, pooled with the nonadherent cells, and analyzed by flow cytometry.

In a different set of experiments, adherent and nonadherent cells from each well were collected separately. Each population was counted and then analyzed by flow cytometry. In all cases, cultured cells were washed twice in PBS-1% FBS in the presence of 0.1% sodium azide and prepared for flow cytometry.

Statistics

Average duration of graft survival is presented as the median. Graft survival among groups was compared using the method of Kaplan and Meier (32). The equality of allograft survival distributions for animals in different treatment groups was tested using the log rank statistic (32). Values of p <0.05 were considered statistically significant. Data is presented as the mean ± 1 SD. Comparisons of two means used the Student t test with separate variance estimates (33). Comparisons of three or more means used one-way ANOVA and the least significant difference procedure for a posteriori contrasts (33).

Results

Skin allograft survival is brief in (NOD × C57BL/6)F1 mice following treatment with costimulation blockade

NOD mice have a generalized defect in their response to costimulation blockade-based transplantation tolerance (8), and NOD congenic mice bearing strongly protective Idd diabetes-resistant loci that ameliorate autoimmunity also resist the induction of prolonged skin allograft survival (21, 34). To analyze the genetic basis for these observations, we generated (NOD × C57BL/6)F1 progeny and measured skin allograft survival after treatment with costimulation blockade.

Consistent with previous reports (8), survival of C3H/HeJ skin allografts on NOD/Lt mice treated with anti-CD154 mAb plus a single C3H/HeJ DST was brief (median survival time (MST) = 25 days; Fig. 1⇓), whereas on age-matched C57BL/6 mice it was, as expected (5, 8), prolonged (MST >98 days, p < 0.001). Unexpectedly, survival of C3H/HeJ skin grafts on age-matched (NOD × C57BL/6)F1 mice (MST = 34 days) was much shorter than on C57BL/6 mice (p < 0.001) and only slightly longer than on NOD/Lt mice (p < 0.001, Fig. 1⇓).

FIGURE 1.
FIGURE 1.

Life table analysis of skin allograft survival in mice treated with DST plus anti-CD154 mAb. Groups of 6- to 8-wk-old C57BL/6, NOD/Lt, (NOD × C57BL/6)F1, and (C57BL/6 × NOD)F1 mice were given a DST, anti-CD154 mAb, and C3H/HeJ skin allografts as described in Materials and Methods. DST (10 × 106 C3H/HeJ spleen cells) was given on day −7 relative to transplantation, and anti-CD154 mAb (0.5 mg/dose) was given on days −7, −4, 0, and +4. Graft survival was longer in the C57BL/6 group than in any other group (p < 0.001). Survival of allografts on (NOD × C57BL/6)F1 and (C57BL/6 × NOD)F1 mice was similar (p = NS) and in both cases modestly (∼10 days) longer than on NOD mice (p < 0.001). The experiment was terminated arbitrarily on day 120; a total of seven mice with intact grafts were removed from the study between days 98 and 112 for use in other experiments. Vertical bars indicate mice removed from the study with intact grafts or alive with intact grafts on day 120.

NOD mice are ancestrally related to ALR mice (4, 35), a strain that expresses a maternally inherited genetic resistance to β-cytotoxic cytokines and alloxan (36). To exclude the possibility that resistance to transplantation tolerance in (NOD × C57BL/6)F1 mice was dependent on the strain of the maternal parent, we generated and tested (C57BL/6 × NOD)F1 mice. The median survival time of skin allografts in (C57BL/6 × NOD)F1 mice (MST = 34 days) was not statistically different from that observed in (NOD × C57BL/6)F1 mice (MST = 34 days, p = NS, Fig. 1⇑).

Fixation of strong diabetes-resistant Idd loci to homozygosity does not increase skin allograft survival in (NOD × C57BL/6)F1 mice treated with costimulation blockade

We have demonstrated that NOD congenic mice bearing resistance alleles at various Idd loci remain resistant to transplantation tolerance, even though expression of autoimmunity is reduced (21, 34). In the (NOD × C57BL/6)F1 mice, all Idd loci are heterozygous. To determine whether prolongation of skin allografts by costimulation blockade would be restored in the presence of homozygous Idd diabetes-resistant loci, we generated (NOD.B6 Idd3 B10 Idd5 × C57BL/6.H2g7)F1 and (NOD.B10 Idd9 × C57BL/6.H2g7)F1 mice. Both F1 hybrids are homozygous for several diabetes-resistant alleles: Idd3, Idd5.1, and Idd5.2 in the case of (NOD.B6 Idd3 B10 Idd5 × C57BL/6.H2g7)F1 mice and Idd9.1, Idd9.2, and Idd9.3 for (NOD.B10 Idd9 × C57BL/6.H2g7)F1 mice. Both F1 strains, which are completely protected from autoimmune diabetes, are heterozygous for all other Idd diabetes-resistant loci.

As shown in Table I⇓, homozygous expression of Idd9 or Idd3 plus Idd5 resistance variants in (NOD × C57BL/6.H2g7)F1 mice does not lead to improved skin allograft survival following costimulation blockade. Skin allograft survival on both (NOD.B6 Idd3 B10 Idd5 × C57BL/6.H2g7)F1 (MST = 42 days) and (NOD.B10 Idd9 × C57BL/6.H2g7)F1 (MST = 37 days) mice was similar to that observed in (NOD × C57BL/6.H2g7)F1 mice (MST = 38 days, p = NS). Skin allograft survival was longer on C57BL/6.H2g7 mice (MST = 77 days) than on any F1 recipient group (p < 0.01, Table I⇓).

View this table:
Table I.

Duration of skin allograft survivala

(NOD × C57BL/6)F1 mice express abnormalities in dendritic cell maturation

To begin to understand the cellular basis for the inability of costimulation blockade to prolong skin allograft survival in (NOD × C57BL/6)F1 mice, we investigated dendritic cell maturation. Dendritic cells are regulators of immunity and self tolerance (37, 38) and play an important role in transplantation tolerance (39, 40, 41). Maturation of dendritic cells is dependent on CD40-CD154 interaction (42), and NOD mice reportedly exhibit abnormalities in dendritic cell maturation (12, 43, 44, 45, 46, 47, 48).

The proportion of total bone marrow-derived anti-CD40 mAb-stimulated and unstimulated CD86high dendritic cells is low in both (NOD × C57BL/6)F1 and NOD/Lt mice.

We cultured bone marrow in the presence of GM-CSF and IL-4 for 6 days, in some cases adding an agonist anti-CD40 mAb for the last 2 days. The total cell population (adherent and nonadherent) was recovered on day 6. The proportions of CD40 mAb-stimulated CD11b+CD11c+ dendritic cells expressing high levels of CD86 were high in C57BL/6 mice (36.3 ± 4.0%, n = 4) and significantly lower in both NOD (16.5 ± 8.0%, p < 0.01, n = 4) and (NOD × C57BL/6)F1 mice (23.7 ± 8.3%, p < 0.05, n = 3). Levels in the NOD and (NOD × C57BL/6)F1 mice were similar (p = NS). Representative histograms are shown in Fig. 2⇓.

FIGURE 2.
FIGURE 2.

Maturation of total dendritic cells recovered from bone marrow cultures of NOD, (NOD × C57BL/6)F1, and C57BL/6 mice. Bone marrow-derived dendritic cells from NOD (top panels), C57BL/6 (middle panels), and (NOD × C57BL/6)F1 (bottom panels) mice were incubated in the presence or absence of an agonist anti-CD40 mAb as described in Materials and Methods. Each culture consisted of a pool of combined adherent and nonadherent cells from two mice. Cells recovered after culture were analyzed by flow cytometry. Representative dot plots of CD11b+CD11c+ dendritic cells obtained from anti-CD40 mAb-stimulated cultures are shown in the left column. Gating was similar for plots for dendritic cells obtained from unstimulated cultures. Right column, Distribution of CD86 (B7.2) expression on stimulated (solid lines) and unstimulated (dotted lines) dendritic cells. The horizontal bars in the right column indicate the gate used for counting CD86high cells. The number above each horizontal bar indicates the percentage of CD86high dendritic cells in the anti-CD40 mAb-stimulated cultures. The number below each horizontal bar indicates the percentage of CD86high dendritic cells in the unstimulated cultures. Isotype controls are shown in the insets; levels of nonspecific CD86 staining were uniformly <0.3%. Shown are representative histograms; the experiment was repeated three times with similar results. Average percentages for all three trials are given in Results.

These overall results using pools of adherent and nonadherent cells suggest that both NOD and (NOD × C57BL/6)F1 dendritic cells fail to mature and are consistent with several previous analyses of NOD dendritic cells (12, 43, 44, 45, 46). However, others have reported that the abnormality of NOD dendritic cells is not immaturity but rather hyperactivation (47, 48). These discrepancies could be related both to the developing autoimmune state in the NOD mouse and/or to the particular subpopulation of cultured cells that was analyzed. To confirm that dendritic cells from (NOD × C57BL/6)F1 mice (which are free of autoimmunity) are truly NOD-like, we proceeded to evaluate comprehensively the phenotype of adherent (immature) and nonadherent (mature) dendritic cells generated in the presence and absence of anti-CD40 mAb stimulation.

The proportion of nonadherent stimulated CD86high dendritic cells in (NOD × C57BL/6)F1 mice is intermediate between that of NOD/Lt mice (low) and C57BL/6 mice (high).

Following incubation in the presence of an agonist anti-CD40 mAb, the percentage of nonadherent dendritic cells that expressed CD86 was higher in C57BL/6 (51.7 ± 13.3%, n = 3) than in NOD (26.5 ± 1.6%; n = 3, p < 0.005) or (NOD × C57BL/6)F1 (37.8 ± 4.4%; n = 3, p < 0.01) bone marrow cultures. Although trending toward higher levels, the proportion of CD86-positive nonadherent dendritic cells generated from (NOD × C57BL/6)F1 marrow was not significantly different from that of the NOD cultures. Representative histograms are shown in Fig. 3⇓ (right column, solid lines). The level of CD86 expression on the nonadherent cells was significantly higher in NOD (mean fluorescence intensity (MFI) = 1468 ± 174; p < 0.03) and in (NOD × C57BL/6)F1 (MFI = 1397 ± 260; p < 0.02) cultures than in C57BL/6 (MFI = 1011 ± 189).

FIGURE 3.
FIGURE 3.

Maturation of adherent and nonadherent dendritic cells recovered from bone marrow cultures of NOD, (NOD × C57BL/6)F1, and C57BL/6 mice. Bone marrow-derived dendritic cells from NOD (top panels), C57BL/6 (middle panels), and (NOD × C57BL/6)F1 (bottom panels) mice were incubated in the presence or absence of an agonist anti-CD40 mAb as described in Materials and Methods. Each culture consisted of a pool of cells from two mice. Cells recovered after culture were analyzed by flow cytometry. Histograms in the left column show the expression of CD86 on adherent CD11b+CD11c+ cells. Histograms in the right column show the expression of CD86 on nonadherent CD11b+CD11c+ cells. The horizontal bars in each histogram indicate the gate used for counting CD86high cells. The number above each horizontal bar indicates the percentage of CD86high dendritic cells in the anti-CD40 mAb-stimulated cultures. The number below each horizontal bar indicates the percentage of CD86high dendritic cells in the unstimulated cultures. Isotype controls are shown in the insets; levels of nonspecific CD86 staining were uniformly <0.3%. Shown are representative histograms; the experiment was repeated three times with similar results. Average percentages for all three trials are given in Results.

The proportion of nonadherent, unstimulated CD86high dendritic cells in (NOD × C57BL/6)F1 mice is intermediate between that of NOD/Lt mice (low) and C57BL/6 mice (high).

Consistent with the data from stimulated cultures, we observed a similar pattern in unstimulated cultures. The percentage of CD86high nonadherent cells in the C57BL/6 cultures (19.4 ± 3.7%) was higher than that in cultures of NOD (7.3 ± 0.4%; p < 0.03) or (NOD × C57BL/6)F1 bone marrow (11.4 ± 0.6%; p < 0.02, Fig. 3⇑). However, the level of CD86 expression on nonstimulated, nonadherent dendritic cells was significantly less in the C57BL/6 (MFI = 745 ± 177) than in NOD (MFI = 1158 ± 26; p < 0.01) or (NOD × C57BL/6)F1 cultures (MFI = 1015 ± 152; p < 0.01).

Bone marrow cultures of (NOD × C57BL/6)F1 and NOD mice generate larger numbers of immature adherent dendritic cells than do C57BL/6 mice.

We next determined the percentage and number of CD86high adherent dendritic cells in cultures stimulated with anti-CD40 mAb. The percentage of CD86high adherent dendritic cells was slightly higher in C57BL/6 (7.6 ± 1.0%) than in NOD (3.9 ± 1.1%; p < 0.01) or (NOD × C57BL/6)F1 (3.8 ± 0.5%; p < 0.004) bone marrow cultures. Representative histograms are shown in Fig. 3⇑ (left column). C57BL/6 bone marrow (4.3 ± 0.7 × 105 cells/well) generated fewer adherent CD11b+CD11c+ cells than did cultures of NOD (5.8 ± 0.6 × 105 cells/well, p < 0.05) or (NOD × C57BL/6)F1 (6.6 ± 0.8 × 105 cells/well; p < 0.02) origin (Fig. 3⇑). The number of adherent cells generated in cultures of NOD and (NOD × C57BL/6)F1 bone marrow was similar (p = NS).

This data set is consistent across CD11b+CD11c+ dendritic cell subpopulations and reveals that there is an overall increase in the number of adherent dendritic cells expressing low levels of CD86 in cultures of NOD and (NOD × C57BL/6)F1 bone marrow. This leads to a decrease in the percentage of CD86high dendritic cells that develop in the total culture (Fig. 2⇑). Although their percentages are decreased, it is important to note that the mature nonadherent dendritic cells that do develop from NOD and (NOD × C57BL/6)F1 bone marrow express higher levels of CD86 than those of C57BL/6 origin.

(NOD × C57BL/6)F1 mice genetically deficient in CD8+ T cells are resistant to transplantation tolerance

Allograft survival in recipients treated with DST and anti-CD154 mAb requires the deletion of alloreactive CD8+ T cells (24, 27). As part of the analysis of cellular defects that might be responsible for the resistance of (NOD × C57BL/6)F1 mice to costimulation blockade, we generated (NOD × C57BL/6)F1 CD8α−/− mice. These (NOD × C57BL/6)F1 CD8α−/− mice were used to determine whether the failure to delete alloreactive CD8+ T cells in response to costimulation blockade was responsible for their resistance to transplantation tolerance induced by costimulation blockade.

Three groups of CD8α−/− mice (NOD, C57BL/6, and (NOD × C57BL/6)F1) were given DST plus anti-CD154 mAb. Median duration of graft survival in C57BL/6 CD8α−/− mice was >101 days, and five of eight grafts were still intact at the end of the experiment (Table II⇓). Duration of skin allograft survival in NOD CD8α−/− mice (MST = 21 days) was significantly shorter than in C57BL/6 CD8α−/− mice (p < 0.003). Graft survival in (NOD × C57BL/6)F1 CD8α−/− mice (MST = 35 days) was also significantly shorter than that observed in C57BL/6 CD8α−/− mice (p < 0.01) but slightly longer than in NOD CD8α−/− mice (p < 0.03, Table II⇓). These data document that resistance of alloreactive CD8+ T cells to deletion by costimulation blockade cannot be the sole mechanism of resistance of NOD and (NOD × C57BL/6)F1 mice to transplantation tolerance.

View this table:
Table II.

Duration of skin allograft survivala

We also tested anti-CD154 mAb monotherapy in these animals because we have previously observed that it somewhat prolongs skin allograft survival in C57BL/6 CD8α−/− mice (24). Using anti-CD154 mAb monotherapy, we observed that uniformly brief skin allograft survival in all three groups: NOD CD8α−/− mice (MST = 17 days, n = 8), C57BL/6 CD8α−/− mice (MST = 23 days, n = 12), and (NOD × C57BL/6)F1 CD8α−/− mice (MST = 20 days, n = 7). None of these MSTs were statistically different.

NK cell number and cytotoxic activity are similar in (NOD × C57BL/6)F1 and C57BL/6 mice

NK cells are important in the rejection of allogeneic hemopoietic grafts (49), and NOD mice have deficient NK cell activity (16, 50). The role of NK cells in costimulation blockade-induced transplantation tolerance is unknown. Therefore, we compared NK cell number and activity in NOD and (NOD × C57BL/6)F1 mice to determine whether, like the NOD dendritic cell abnormality, it is a genetically dominant phenotype. Because NOD/Lt mice are NK1.1− (50), we quantified the number of NK cells by dual label analysis using the DX5 and anti-ASGM-1 anti-NK cell Abs. The percentage of DX5+ASGM-1+ cells in the spleen of C57BL/6 mice (2.18 ± 0.43%, n = 6) was higher than that in NOD/Lt mice (1.68 ± 0.31%, n = 6); this difference was not statistically significant (p < 0.06) but trended in the same direction as reported by others (50). The percentage of DX5+ASGM-1+ cells in the spleen of (NOD × C57BL/6)F1 mice (1.88 ± 0.28%, n = 6) was similar in C57BL/6 and NOD/Lt mice (p = NS). Representative dot plots are shown in Fig. 4⇓A.

FIGURE 4.
FIGURE 4.

Splenic NK cell percentages and function in NOD, (NOD × C57BL/6)F1, and C57BL/6 mice. A, Spleen cells were obtained from 6- to 12-wk-old NOD, C57BL/6, and (NOD × C57BL/6)F1 and stained with DX5 (vertical axis) and anti-ASGM-1 (horizontal axis) Abs as described in Materials and Methods. The circular gates indicate the percentage of total splenic lymphocytes that were DX5+ASGM-1+ NK cells. Shown are representative contour plots. Each analysis was performed six times with similar results. Average percentages for all trials are given in Results. Shown in the insets is staining of ASGM-1 and the isotype control for the DX-5 mAb. B, Cytotoxic activity directed against NK-sensitive YAC-1 target cells as determined by 51Cr release. Effector cells were spleen cells recovered from 6- to 12-wk-old NOD, (NOD × C57BL/6)F1, and C57BL/6 mice and were assayed as described in Materials and Methods. Mice were treated with a single injection of poly I:C 24 h before spleen cell recovery. Shown are the results of three independent experiments.

To assess NK killing activity in these same mice, we measured the cytotoxic activity of spleen cells against NK-sensitive YAC-1 targets. Surprisingly, the NK cell cytotoxic activity of spleen cells from (NOD × C57BL/6)F1 mice was similar to that of spleen cells from C57BL/6 mice, both of which were significantly more potent than that of spleen cells from NOD/Lt mice (Fig. 4⇑B).

Macrophage maturation in (NOD × C57BL/6)F1 mice is normal

NOD mice have defects in macrophage maturation, and it has been suggested that this defect could in part account for their resistance to transplantation tolerance (8). Macrophage maturation can be quantified by measuring the secretion of IL-1β by bone marrow-derived macrophages stimulated with LPS (13). Bone marrow from NOD/Lt, C57BL/6, and (NOD × C57BL/6)F1 mice was cultured in the presence of CSF-1 and IFN-γ to generate macrophages. Cultures were then stimulated with LPS to induce secretion of IL-1β, which was in turn measured in a thymocyte costimulation proliferation assay (13).

As expected, LPS-stimulated macrophages derived from NOD/Lt cultures produced less IL-1β than did LPS-stimulated macrophages derived from C57BL/6 cultures (Fig. 5⇓, p < 0.001). Surprisingly, (NOD × C57BL/6)F1-derived LPS-stimulated macrophages secreted high levels of IL-1β that were comparable to those of C57BL/6 macrophages (p = NS). Results of two independent trials were similar. These results indicate that, unlike the genetically dominant dendritic cell defect of the NOD mouse, the NOD macrophage maturation defect is not expressed in (NOD × C57BL/6)F1 mice.

FIGURE 5.
FIGURE 5.

Mitogen-stimulated production of IL-1β by cultures of bone marrow-derived macrophages from NOD, (NOD × C57BL/6)F1, and C57BL/6 mice. Pools of bone marrow-derived macrophages were obtained from NOD, (NOD × C57BL/6)F1, and C57BL/6 mice, stimulated with LPS, and assayed for production of IL-1β as described in Materials and Methods. Vertical bars indicates incorporation of [3H]thymidine by C3H/HeJ thymocytes in a comitogenic assay. Shown is a representative experiment of supernatants from a bone marrow-derived macrophage culture generated from a pool of two mice. The data represent the mean ± 1 SD of [3H]thymidine incorporated in six replicate wells for each strain. The experiment was repeated two times with similar results.

(NOD × C57BL/6)F1 mice have normal percentages of CD4+CD25+ T cells

The poor survival of skin allografts on NOD CD8α−/− and (NOD × C57BL/6)F1 CD8α−/− mice suggests that their resistance to transplantation tolerance could in part be due to an abnormal response of CD4+ T cells, in particular CD4+CD25+ regulatory T cells known to be important in transplantation tolerance (51, 52, 53, 54, 55). To begin to address this possibility, we measured the percentage of splenic CD4+CD25+ T cells. We found that the percentage of CD4+ cells that coexpressed CD25 in C57BL/6 (10.6 ± 2.0%, n = 6) was similar to that in both NOD (11.03 ± 1.67%, n = 6) and (NOD × C57BL/6)F1 (11.04 ± 3.14%, n = 6) mice. Representative dot plots are shown in Fig. 6⇓.

FIGURE 6.
FIGURE 6.

Splenic CD4+CD25+ T cells in NOD, (NOD × C57BL/6)F1, and C57BL/6 mice. Spleen cells from 6- to 8-wk-old NOD (top panel), C57BL/6 (middle panel), and (NOD × C57BL/6)F1 (bottom panel) mice were stained with anti-CD4 (horizontal axis) and anti-CD25 (vertical axis) mAbs and analyzed by flow cytometry. The percentage of CD4+ cells that are also CD25+ is indicated in the upper right of each plot. Shown are representative dot plots; the experiment was repeated two times using a total of six individual mice in each group mice with similar results. Average percentages for all mice are given in Results.

This result suggests that a deficiency in the percentage of CD4+CD25+ T cells is not responsible for the abnormal response of (NOD × C57BL/6)F1 mice to costimulation blockade. However, the data do not exclude the possibility of a functional abnormality in the CD4+CD25+ regulatory T cell population.

Discussion

These data unexpectedly reveal that poor skin allograft survival in response to costimulation blockade is a characteristic not only of autoimmune NOD mice (8, 9, 11) and NOD congenic mice that bear strongly protective Idd resistance loci (21, 34), but also of diabetes-free (NOD × C57BL/6)F1 mice. This dominant genetic resistance is not a maternally inherited trait and is not corrected by fixing to homozygosity strongly protective non-H2 Idd diabetes-resistant loci in the F1 NOD.Idd × C57BL/6 intercross mice.

These data further suggest possible dominant cellular phenotypes that could explain our genetic observations. First, we found that (NOD × C57BL/6)F1 mice exhibit abnormal dendritic cell maturation. Second, we observed an abnormal response of (NOD × C57BL/6)F1 CD8α−/− mice to costimulation blockade, suggesting a defect in the response of CD4+ T cells in these animals.

Most diabetes-susceptibility genes are recessive (56), but our results document that the resistance of (NOD × C57BL/6)F1 mice to transplantation tolerance is a dominant genetic trait. Interestingly, however, skin allograft survival in (NOD × C57BL/6)F1 mice was slightly but reproducibly longer than that observed in NOD mice. Speculatively, this difference could be the result of the unexpected macrophage phenotype observed in the (NOD × C57BL/6)F1 mice (Fig. 4⇑). The epistatic nature of the macrophage phenotype in F1 mice suggests complex genetic control of this phenotype that could in turn modify the penetrance of the genetic susceptibility of the F1 to transplantation tolerance. The data suggest that a complex set of genetic interactions involving several loci is needed for prolongation of allograft survival in response to costimulation blockade (57).

What is the cellular basis for the resistance of (NOD × C57BL/6)F1 mice to transplantation tolerance? An attractive candidate population is dendritic cells. Maturation of dendritic cells is abnormal in NOD mice (12, 43, 44, 45, 46, 47, 48, 58), and dendritic cells are the primary target of CD154 expressed by activated CD4+ T cells (42, 59, 60). We found that dendritic cells derived from NOD and (NOD × C57BL/6)F1 mouse bone marrow cultures mature abnormally, even when stimulated with an agonist anti-CD40 mAb. With respect to NOD dendritic cells, it is also interesting to note that, although reduced in number, the mature nonadherent dendritic cells generated by NOD bone marrow expressed higher levels of CD86 than did those from C57BL/6 controls. These apparently conflicting data on the maturation of NOD dendritic cells depending on the cell population analyzed may in part explain the differing reports in the literature that dendritic cells derived from NOD bone marrow cultures fail to mature normally (12, 43, 44, 45, 46) or are hyperactivated (47, 48).

We also observed that (NOD × C57BL/6)F1 CD8α−/− mice remain resistant to the induction of prolonged skin allograft survival by costimulation blockade. A requirement for prolongation of skin allograft survival in mice treated with costimulation blockade is the deletion of host alloreactive CD8+ T cells (24, 27, 61, 62, 63). The resistance of (NOD × C57BL/6)F1 CD8α−/− mice to transplantation tolerance suggests that their CD4+ T cells respond abnormally to costimulation blockade.

At least three explanations for the brief skin allograft survival are possible for the brief survival of skin allografts in (NOD × C57BL/6)F1 CD8α−/− mice treated with DST and anti-CD154 mAb. First, anti-CD154 mAb may fail to block alloreactive CD4+ T cell activity. Second, the absence of the CD8α+ dendritic cell subset in (NOD × C57BL/6)F1 CD8α−/− mice may be important (64). However, this possibility seems unlikely because we have shown that C57BL/6 CD8α−/− knockout mice are susceptible to transplantation tolerance. Third, there may be defects in the function of regulatory CD4+CD25+ T cells; these cells are important both for the induction of transplantation tolerance (51, 52, 53, 54, 55) and for the expression of autoimmunity in NOD mice (20). We found that NOD/Lt, C57BL/6, and (NOD × C57BL/6)F1 mice have comparable percentages of small resting CD4+CD25+ spleen cells, but we recognize that functional defects in CD4+CD25+ T cells could nonetheless be present in (NOD × C57BL/6)F1 mice. We are currently investigating this possibility.

We speculate that dendritic cell maturation abnormalities and abnormal response of CD4+ T cells to costimulation blockade may be causally related in (NOD × C57BL/6)F1 mice. Costimulatory molecule expression by dendritic cells is important for modulating CD4+ T cell responses in both autoimmunity (37) and transplantation (39, 40, 41), and dendritic cells are thought to control the generation of regulatory CD4+CD25+ T cells (65, 66, 67, 68, 69). It has also been suggested that low expression of CD86 on NOD dendritic cells leads to the failure of CD4+ T cells to up-regulate CTLA-4, contributing to impaired self-tolerance (12). CTLA-4 expression is also critical for the induction of peripheral transplantation tolerance and regulatory CD4+CD25+ T cells (5, 24, 70). Finally, NOD mice deficient in CD80 and CD86, the ligands for CTLA-4, rapidly develop diabetes, presumably due to deficiencies in regulatory CD4+CD25+ cells (20).

An additional cell population defective in NOD mice is the NK cell population. Our observation that NK cell number and cytotoxic activity is normal in (NOD × C57BL/6)F1 mice suggests that NK cell defects are unlikely to be responsible for the resistance of (NOD × C57BL/6)F1 mice to tolerance induction. However, we recognize that functions of NK cells not measured in these studies (e.g., cytokine production) could be defective.

Defective macrophage Ag presentation has previously been associated with resistance of NOD mice to transplantation tolerance (8, 71, 72). Our analyses of macrophages derived from bone marrow cultures of (NOD × C57BL/6)F1 mice showed that their macrophage maturation appears to be normal. These data permit us to separate defects in macrophage maturation from defects in dendritic cell maturation and resistance to transplantation tolerance.

Finally, it remains to be determined whether alloreactive CD8+ T cells in NOD and (NOD × C57BL/6)F1 mice resist deletion in response to costimulation blockade. Recent evidence suggests that NOD CD8+ T cells are resistant to deletion in response to peripheral tolerance induction to soluble Ags (73). In preliminary studies, we have obtained evidence that alloreactive CD8+ T cells in (NOD × C57BL/6)F1 mice are also relatively resistant to deletion following treatment with DST plus anti-CD154 mAb (T. Pearson, unpublished observations).

Our data documenting resistance to transplantation tolerance in (NOD × C57BL/6)F1 mice and in congenic NOD mice bearing strongly protective Idd diabetes-resistant loci (21, 34) could be due to two different but not mutually exclusive genetic mechanisms. First, the transplantation tolerance defect could be controlled by the same genetic loci that control autoimmune diabetes, but the “genetic threshold” for restoring susceptibility to transplantation tolerance could be higher than that required to prevent autoimmunity. Alternatively, the genes that control transplantation tolerance to skin allografts may be partially or even completely distinct from those that control autoimmunity.

Previous studies have lent support to the genetic threshold hypothesis for autoimmunity. These studies used NOD mice mated with various nonautoimmune strains and treated the F1 mice with cyclophosphamide to induce diabetes (74, 75). Approximately 30% of NOD female mice crossed with the closely related but diabetes-resistant MHC-compatible NOR/Lt strain developed diabetes using this treatment protocol (76). In another study, NOD mice were crossed with diabetes- and insulitis-free NOD.H2b mice. Approximately 50% of (NOD × NOD.H2b)F1 mice developed insulitis, a low percentage (3%) of female F1 mice spontaneously developed diabetes, and ∼20% become diabetic after treatment with cyclophosphamide (77). These data argue for a “genetic threshold” model for expression of autoimmunity.

We generated (NOD × C57BL/6)F1 mice to begin to test the genetic threshold hypothesis and the possible role of Idd loci in transplantation tolerance. (NOD × C57BL/6)F1 mice are heterozygous at all Idd loci distinguishing the two parental strains (78). Only three NOD Idd diabetes-susceptibility loci, Idd13, Idd14, and Idd15 (56, 79, 80, 81, 82) are dominant, the remainder are recessive. (NOD × C57BL/6)F1 mice treated with cyclophosphamide remain free of diabetes (83), suggesting that they have a high genetic threshold of resistance to autoimmunity. Therefore, if similar genes control autoimmunity and transplantation tolerance, (NOD × C57BL/6)F1 mice should have a higher “set-point” for the induction of transplantation tolerance. However, (NOD × C57BL/6)F1 mice clearly remain resistant to the induction of prolonged skin allograft survival by costimulation blockade.

We also tested the genetic threshold model and a role for Idd loci by generating (NOD.B6 Idd3 B10 Idd5 × C57BL/6.H2g7)F1 and (NOD.B10 Idd9 × C57BL/6.H2g7)F1 mice, which are homozygous for strongly protective diabetes-resistant Idd loci. We nonetheless observed skin allograft survival in response to costimulation blockade was no better in these congenic F1 progeny than that in (NOD × C57BL/6)F1 mice.

The exact genetic basis for resistance to prolonged skin allograft survival by costimulation blockade in NOD mice remains elusive, as does the exact genetic basis for their susceptibility to autoimmunity. Our observation that the autoimmune phenotype and the transplantation tolerance phenotype are differentially inherited in (NOD × C57BL/6)F1 hybrids suggests that an ongoing autoimmune process, even one with low penetrance as measured by diabetes, is not responsible for the failure of tolerance induction in NOD mice. Although we have separated the tolerance resistance phenotype from the autoimmunity phenotype, we recognize that our data could be used in support of either the genetic threshold hypothesis or the distinct or overlapping genes hypothesis. Additional studies are under way to adjudicate these two genetic hypotheses.

A final point to be made is that our genetic dissection of transplantation tolerance and autoimmune phenotypes in an animal model has clinical implications. Outcomes of tolerance induction protocols tested in NOD mice may not accurately predict outcomes in human subjects.

Acknowledgments

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

Footnotes

  • ↵1 This work was supported in part by Grants AR35506 and AI42669 and Institutional Diabetes Endocrinology Research Center 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. L.S.W. was supported by a joint grant from the Juvenile Diabetes Research Foundation and the Wellcome Trust. D.V.S. was supported by Grants DK46266 and DK51090 from the National Institutes of Health and by a grant from the Juvenile Diabetes Research Foundation. 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, and the Juvenile Diabetes Research Foundation. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

  • ↵2 Address correspondence and reprint requests to Dr. Dale L. Greiner, University of Massachusetts Medical School, 373 Plantation Street, Biotech 2, Suite 218, Worcester, MA 01605. E-mail address: dale.greiner{at}umassmed.edu

  • ↵3 Abbreviations used in this paper: NOD, nonobese diabetic; ASGM-1, asialo GM-1; DST, donor-specific transfusion; MST, median survival time; MFI, mean fluorescence intensity; Idd, insulin-dependent diabetes.

  • Received March 14, 2003.
  • Accepted April 22, 2003.

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