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International Immunology, Vol. 13, No. 10, 1321-1333, October 2001
© 2001 Japanese Society for Immunology

Relative resistance to nasally induced tolerance in non-obese diabetic mice but not other I-Ag7-expressing mouse strains

Anthony Quinn, Marco Melo2,3,, Douglas Ethell1, and Eli. E. Sercarz

La Jolla Institute for Allergy and Immunology, Division of Immune Regulation, and
1 Division of Cellular Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA
2 Department of Microbiology and Molecular Genetics, University of California, Los Angeles,CA 90095-1489, USA

Correspondence to: Correspondence to: A. Quinn


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
I-Ag7 is a unique class II MHC molecule that is clearly associated with autoimmune diabetes in non-obese diabetic (NOD) mice. To determine if I-Ag7 is defective in its ability to deliver tolerogenic signals in vivo, H-2g7 mice were nasally pretreated with antigen, prior to immunization, to induce antigen-specific regulation. Nasally pretreated NOR (H-2g7) and (NON).NOD (H-2g7) congenic mice showed responses similar to those of NON (H-2nb1), BALB/c (H-2d) and B10.PL (H-2u) mice—a reduced recall response and a deviated Th cytokine profile. However, we found that NOD (H-2g7) mice are comparatively resistant to immunological tolerance induced by nasal pretreatment, such that at the usually effective dose no significant reduction was seen in the proliferative recall responses to nominal antigen after immunization. (NOD x BALB/c)F1 (H-2g7/d) and (NOD x NOR)F1 (H-2g7) mice were similarly resistant to nasal-induced tolerance, although significantly higher nasal doses of antigen were able to overcome the resistance in NOD and F1 mice. Interestingly, activated NOD T cells were resistant to cell death induced by re-stimulation with plate-bound anti-CD3. These results demonstrate that activated T cells in NOD mice are defective in their ability to respond to regulatory signals delivered in vivo or in vitro. Furthermore, NOD T cells have an increased resistance to tolerance induced by I-Ag7-dependent (antigen) or I-Ag7-independent (anti-CD3) mechanisms. Thus, while I-Ag7 may contribute to insulin-dependent diabetes mellitus by selecting a particular repertoire of self-reactive T cell clones, additional defects in the peripheral T cells themselves are required to allow the expansion of diabetogenic clones and the development of autoimmune disease.

Keywords: apoptosis, immune deviation, hen egg lysozyme, mucosal, T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Insulin-dependent diabetes mellitus (IDDM) in the non- obese diabetic mouse (NOD) is an autoimmune disease characterized by a T cell-mediated destruction of the insulin-producing ß cells of the pancreas (1). NOD mice spontaneously develop insulitis and autoimmune diabetes, as well as lymphocytic infiltration into a number of other organs (2). It has been suggested that the mechanisms that are normally responsible for the induction and maintenance of self-tolerance are defective in these mice, and are unable to provide adequate protection from autoreactivity and autoimmune disease (3,4). The spontaneous appearance of cellular and humoral immune responses to islet antigens, prior to the onset of diabetes, demonstrates that NOD mice lack the necessary components to maintain immunological homeostasis in the pancreas. A loss of regulation in a self-reactive repertoire may contribute to the organ-specific pathogenesis in type I diabetes and in other autoimmune diseases as well.

To establish and maintain self-tolerance, distinct regulatory mechanisms such as clonal deletion, clonal anergy, activation-induced cell death (AICD) and active suppression are thought to play significant roles in controlling the in vivo expansion of autoreactive, pathogenic T lymphocytes. T cells bearing antigen receptors with a high affinity for self-determinants displayed in the context of MHC are thought to be clonally deleted during thymic education, leaving T cells with low avidity to self to form the peripheral repertoire (5,6). Therefore the efficient presentation of `self-determinants' bound to class II MHC molecules is critical to the establishment of a self-tolerant immune system. Due to a mutation in the promoter region of the NOD E{alpha} gene, the only class II MHC molecule expressed on the surface of antigen-presenting cells in the NOD mouse is I-Ag7 (7,8). I-Ag7 expression has been shown to be required for the development of diabetes in the NOD mouse (9), as are the allele-specific residues at positions 56 and 57 (9). A similar mutation to a non-Asp residue at position 57 of HLA-DQß may be associated with human type I diabetes (10). It has been reported that I-Ag7 is a relatively unstable MHC molecule with a short half-life and a low peptide affinity (4). Such a defect would be expected to hamper I-Ag7 in its ability to deliver tolerogenic or regulatory signals to CD4+ T cells (4,11,12) and its efficacy in maintaining peripheral tolerance (3).

Mucosal sensitization is an established method for inducing peripheral immune tolerance to exogenous antigens in naive mice (13–15). Animals exposed to antigens via oral feeding or nasal installation (NI) demonstrate an antigen-specific loss of responsiveness upon subsequent exposure to homologous antigen (13). Using hen egg lysozyme (HEL) as a model antigen, we compared the efficacy of NI in the induction of antigen-specific tolerance in I-Ag7+ mouse strains. Although NOD mice were relatively resistant to the nasal pretreatment, compared to BALB/c and B10.PL mice, a similar defect was not observed in other mice that express the I-Ag7molecule. Furthermore, NOD-derived T cells were less sensitive to tolerance induction via MHC-independent signals. Collectively, the data suggest that the observed defects in peripheral tolerance in the NOD mouse are not solely mediated by I-Ag7, but rather require another NOD-derived element that is dominantly inherited and may allow autoreactive T cells to persist in the periphery.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Female BALB/c, NOD.Lt (non-obese diabetic), NOR1/Lt (non-obese diabetes resistant), NON.NOD (non-obese nondiabetic) and B10.PL mice were purchased from the Jackson Laboratory (Bar Harbor, ME), and female NOD mice were purchased from Taconic Farms (Germantown, NJ). (NODxBALB/c)F1 were bred at the animal facility at the University of California (Los Angeles) and (NODxNOR)F1 mice were bred in our facilities at the La Jolla Institute for Allergy and Immunology. The mice were age- and sex-matched in all experiments.

Antigens
HEL and ovalbumin (OVA) were purchased from Sigma (St Louis, MO). The HEL was further purified by chromatography on a weak ion-exchange column of Bio-Rex 70 (BioRad, Richmond, CA), at pH 7.18.

Nasal installation of antigen
Animals (6–12 weeks old) were nasally instilled with antigen in 20 µl of saline upon recovery from light anesthesia (Halothane; Halocarbon Laboratories, River Edge, NJ). Seven days later, the mice were immunized in the footpad with antigen/complete Freund's adjuvant (CFA).

T cell proliferation assay
Popliteal and inguinal lymph nodes, and spleens were removed 9–14 days after s.c. immunization to prepare single-cell suspensions. Lymph node cells (LNC) and spleen cells (SPC) were plated in 96-well microtiter plates at 5x105 and 8x105 cells/well respectively in serum-free medium (X-VIVO-10 or HL-1; Biowhittaker, Walkersville, MD) supplemented with 2x10–5 M 2-mercaptoethanol. Multiple concentrations of HEL (100, 50, 25 and 12.5 µg/ml) or OVA (100, 33.3, 11.1, 3.7 and 1.2 µg/ml) were used to induce proliferative recall responses. The 50 µg/ml concentration of HEL and the 33.3 or 11.1 µg/ml concentrations of OVA were usually selected to compare responses since they were on the linear portion of the dose–response curve. Tuberculin, purified protein derivative (PPD), was used as a positive control for proliferation. [3H] Thymidine (ICN, Irvine, CA), 1 µCi/well, was added for the last 16 h of a 4-day culture. The cells were harvested from microtiter plates using a Micro Cell Harvester (Skatron, Sterling, VA) and incorporation of label was measured by liquid scintillation counting in an LKB 1205 Betaplate counter. The results were read as mean c.p.m. of triplicate wells and the SD was <15% in all experiments. For comparisons, the results were expressed as {delta}c.p.m. (experimental mean c.p.m. – media control c.p.m.) to minimize the influence that minor differences in media control wells can have (relatively small changes in background can have dramatic effects on the stimulation index).

Based on data accumulated in experiments with BALB/c, C57BL/6, B10.A, C3H/HeJ and CBA/J mice (data not shown), nasal treatments which resulted in a >50% reduction in the {delta}c.p.m. in the proliferation assay (c.p.m. from the corresponding saline-treated mice were considered as 100%) were judged to be tolerogenic. We observed that while the magnitude of the suppressive effect of NI with antigen varied among experiments (55–95% reduction in c.p.m.), in no experiment was there <50% reduction in mice previously determined to be susceptible.

Lymphokine measurements
IFN-{gamma}- and IL-5-producing cells were each enumerated using the ELISPOT cellular assay as described previously (16). The spots were enumerated visually with a stereo microscope and with a computer image analysis system (Lightools Research, Encinitas, CA) using the image analyzer program, NIH Image 1.61. The few background spots (LNC cultured in ELISPOT plates without antigen) were subtracted from all reported results.

Religation of the TCR
Susceptibility to cell death induced by religation of the TCR was determined via a modified version of a previously described method (17). Splenic suspensions, 5x106/ml, from untreated mice were stimulated in vitro with concanavalin A (Con A; 2 µg/ml) for 72 h. Viable blasts were recovered by Histopaque (Sigma) separation, washed and resuspended to 5x105 cells/ml in complete medium supplemented with rIL-2 (20 U/ml). The blasts were then plated at 5x104 cells/well in 96-well plates coated with anti-CD3{varepsilon} (clone 145-2C11; PharMingen, San Diego, CA) or hamster IgG. After 30 h, the wells were pulsed with [3H]thymidine, incubated for an additional 16 h and then harvested as per the proliferation assay described above. Reactivation of recently activated T cell blasts has been reported to engage an AICD pathway (17–19).

RNase protection assay (RPA)
The expression of apoptosis specific mRNA species was analyzed in spleen cells 48 h after activation with Con A or anti-CD3, using a RPA (PharMingen, San Diego, CA) according to the manufacturer's instructions. Briefly, Trizol (Gibco/BRL, Grand Island, NY) was used to extract total RNA from T cell blasts. Ten micrograms of RNA was then hybridized with 32P-labeled probes, treated with RNase and resolved in a 5% acrylamide gel. The protected probes were visualized by autoradiography.

Western blot methods
Activated T cell blasts were produced by stimulating spleen cells with Con A or anti-CD3 for 48 h. The cells were then pelleted and resuspended in lysis buffer, on ice, for 20 min with mixing every 2 min. Nuclei and other non-soluble debris were pelleted at 15,000 g for 15 min at 4°C. Supernatants were placed into new tubes and protein concentrations determined by spectrophotometry. Twenty micrograms of each sample was mixed with 10 µl of 4xsample buffer and boiled for 5 min, after which the samples were resolved on 4–15% gradient gels and blotted to Hybond-ECL overnight (equal loading and transfer of protein was confirmed by Ponceau staining of the blots). After blocking with Blotto (3% BSA, 3% powdered milk in PBS and 0.1% Tween 20) for 1 h, blots were probed with anti-FLIP (N-terminus specific; Aleyis, San Diego, CA), anti-caspase 3 (Cell Signaling Technologies, Beverly, MA) or anti-Bcl-2 (PharMingen) antibodies at 1:1000 in Blotto overnight at 4°C, with rocking. Blots were then washed 3 times with PBS/Tween and probed with anti-rabbit horseradish peroxidase conjugate (1:2000; Amersham, Piscataway, NJ) for 1 h at room temperature. After washing the blot 3 times with PBS/Tween, bands were resolved with chemiluminescent detection (Pierce, Rockford, IL).

Apoptotic nuclei methods
Con A-activated T cell blasts, described above, were fixed with 2% formalin and 0.05% glutaraldehyde overnight at 4°C. The cells were then rinsed with PBS and stained with 2.5 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) in PBS for 30 min at room temperature, washed twice with PBS, placed onto chambered glass slides, and allowed to settle and adhere to the glass overnight. Six images of each condition were taken with an inverted video-microscope, with fluorescence capabilities. The nuclei from each image were scored for apoptotic (fragmented or condensed) or non-apoptotic morphologies. The numbers from each condition were converted to percentages. The mean and SEM were calculated for each condition.

Statistical analysis
Proliferative recall responses from antigen-pretreated and control groups were compared by Student's t-test (two-tailed), using the StatView software package (Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
NOD (H-2g7) mice are resistant to nasally induced tolerance
Previously, we found that NI with 50 µg of HEL was effective in suppressing antigen-specific proliferative recall responses in adult BALB/c (H-2d) mice (20). To determine whether the effects of NI on NOD mice were comparable, mice were NI with 50 µg of HEL or saline and then challenged in vivo 7 days later with HEL/CFA. While pretreatment with HEL resulted in 60–70% reductions in the proliferative recall responses of BALB/c (H-2d) and B10.PL (H-2u) mice (Table 1Go), NOD (H-2g7) mice treated in the same fashion showed no significant reduction in their recall responses (17.5%) (Table 1Go). NI with 50 µg of HEL also failed to reduce the recall response in (NODxBALB/c)F1 mice (Table 1Go). Moreover, in some instances we observed an enhanced immune response in HEL nasally treated NOD and (NODxBALB/c)F1 mice, when compared to saline-pretreated subjects (Table 1Go). These experiments were repeated numerous times (>10) with similar results. Since the nasal pretreatment consistently suppressed the recall response by >50% in BALB/c mice, they were used as a positive control strain throughout the study.


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  		Table 1. NOD (H-2g7) mice are resistant to nasally induced tolerance with HEL
 
To characterize the Th phenotype of the antigen-responsive cells, we used the ELISPOT cellular assay to enumerate the HEL-specific IFN-{gamma}- or IL-5-producing cells recovered from the draining lymph nodes of NOD, BALB/c and (NODxBALB/c)F1 mice that had NI with 50 µg of HEL prior to immunization with HEL/CFA. In titration experiments, 50 µg of HEL was shown to be optimal for the induction of immune deviation and suppression of the proliferative response in BALB/c mice (data not shown). Here, as expected, pretreatment with HEL in BALB/c mice resulted in a reduction in IFN-{gamma} spot-forming cells (SFC) (>50%) and an increase in IL-5 SFC (Fig. 1Go); however, no substantial changes in the number of SFC for either cytokine were observed in NOD or (NODxBALB/c)F1 mice receiving the same nasal pretreatment (Fig. 1Go). There was also <5% reduction in the proliferative recall response in NOD or (NODxBALB/c)F1 mice in this set of experiments (data not shown). Thus, in contrast to BALB/c and B10.PL, mice the nasal treatment was ineffective in inducing antigen-specific tolerance in NOD mice and (NODxBALB/c)F1 mice.



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  		Fig. 1. NOD (H-2g7) and (NODxBALB/c)F1 (H-2g7/d) mice are resistant to NI-induced immune deviation. Female NOD, BALB/c and (NODxBALB/c)F1 mice (8–12 weeks old) were NI with 50 µg of HEL (20 µl) or PBS (three mice per group), 1 week prior to s.c. immunization with 100 µg of HEL in CFA. Ten days later, the draining LNC were collected, pooled and stimulated in vitro with HEL (100–2.5 µg/ml). Analysis was compared at the 50 µg/ml dose (see Methods). The antigen-responsive IL-5- and IFN-{gamma}-producing cells were enumerated using the ELISPOT cellular assay as described in Methods. The results are expressed as the mean number of SFC in duplicate wells. The mean of number of spots in media control wells was 0–4/well.

 
NON (H-2nb1) and NON.NOD (H-2g7) mice are susceptible to nasally induced tolerance
The MHC region of the NOD mouse contains several genes that could contribute to abnormal immune responses and susceptibility to IDDM in NOD mice. To determine if the expression of the H-2g7 allele was sufficient to confer resistance to nasally induced tolerance, we studied the influence of NI on the recall response in NON mice (H-2nb1) and congenic NON.NOD (H-2g7) mice. Although NON and NOD mice were derived from the same JCl:ICR stock (21,22) and share several non-MHC genes, they are nonetheless distinct haplotypes with the NON-derived MHC complex, H-2nb1, asserting a protective influence in the diabetes-resistant NON and (NODxNON)F1 strains (21). NON (H-2nb1) and congenic NON.NOD (H-2g7) mice had NI with saline or 50 µg of HEL, challenged with HEL/CFA, and 10 days later their proliferative recall response to HEL was determined. A greater than 50% reduction in proliferation was observed in NON and NON.NOD mice NI with HEL, as compared to saline-treated controls (Fig. 2Go). The clear demonstration that both strains were susceptible to the regulatory effect of the antigen-pretreatment indicates that the expression of the H-2g7 in NON.NOD mice was not sufficient to confer resistance to NI tolerance. Groups of NOD mice were tested in parallel with the NON.NOD mice, as controls, and they showed the expected resistance pattern (data not shown).



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  		Fig. 2. NON (H-2nb1) and NON.NOD (H-2g7) are susceptible to NI-induced tolerance using 50 µg of antigen. Female NON and NON.NOD mice had NI with HEL 7 days prior to s.c immunization with 100 µg HEL in CFA. Ten days later, the draining LNC were collected from individual mice and examined for proliferative recall responses to HEL (50 µg/ml). The results are expressed as {delta}c.p.m. and the mean of control wells (no antigen) ranged from 3935 to 5720 c.p.m. Using Student's t-test (unpaired, two-tailed) the mean c.p.m. from the HEL pretreated mice were compared to the saline-treated controls; *NON.NOD, P = 0.0196; **NON, P = 0.0178. P < 0.050 was considered significant. A repeated experiment gave similar results.

 
NOR (H-2g7) mice are not resistant to nasally induced tolerance with antigen
To evaluate the susceptibility of another I-Ag7-expressing mouse strain to nasally induced tolerance, 6-week-old female NOR mice (H-2g7) were NI with saline or 50 µg of OVA 7 days before immunization with OVA/CFA. The NOR (non-obese resistant) mouse is a recombinant congenic strain in which portions of the NOD/Lt genome have been replaced by genes from the C57BL/KsJ strain (23). Although NOR mice express the diabetogenic H-2g7 complex, they are free of insulitis and are resistant to diabetes. When the recall response was examined 10 days after parenteral immunization, OVA-pretreated NOR mice displayed a significant reduction in the proliferative recall response (60%), a response comparable to that of BALB/c mice (Table 2Go) [The minimum dose of OVA that was required to induce nasal suppression in BALB/c mice was 25 µg (data not shown).] On the other hand, there was no significant difference in the recall response between antigen-pretreated and saline-pretreated NOD mice (Table 2Go). Reductions in proliferative responses to homologous antigen were observed in NOR mice that were NI with 50 µg of HEL before immunization with HEL/CFA (data not shown). Thus, despite the homozygous expression of I-Ag7, NOR mice were discordant with NOD in their susceptibility to nasal tolerance.


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  		Table 2. NOD (H-2g7) mice are resistant to nasally induced tolerance with OVA
 
(NODxNOR)F1 (H-2g7) mice are resistant to nasally induced tolerance
It is possible that the resistance to nasally induced tolerance observed in (NODxBALB/c)F1 mice (Table 1Go and Fig. 1Go) merely reflects a dilution of the BALB/c-derived MHC alleles (I-Ad and I-Ed) on the surface of antigen-presenting cells. On the other hand, in the outcrossed strain, NOD mice may contribute a non-MHC gene that confers resistance to nasally induced tolerance in a dominant fashion. To determine if the resistance in F1 mice was due to heterozygosity at the MHC locus, we studied nasally induced tolerance in (NODxNOR)F1 mice. The parental strains in this case are discordant with regard to their susceptibility to nasally induced tolerance and autoimmune disease, but express identical MHC molecules. Interestingly, nasal pretreatment of (NODxNOR)F1 mice was unable to significantly reduce the proliferative recall response to homologous antigen (Table 3Go). Surprisingly, pretreatment with OVA did induce a significant increase in the number of antigen-responsive IL-5-secreting cells in (NODxNOR)F1 mice (Table 3Go); however, the number of IFN-{gamma} SFC was also slightly increased (Table 3Go). Since these mice are able to generate an IL-5 response after treatment it is unlikely that the resistance to nasally induced suppression in (NODxNOR)F1 mice stems from an inability to mount a Th2 response, but rather the failure appears to result from the inability to turn off the Th1 response to challenge. These data suggest that the elements that mediate suppression of the proliferative response and the preferential induction of Th2 responses upon NI are distinct and, most importantly, they show that the resistance to nasally induced tolerance in (NODxNOR)F1 is not solely mediated by I-Ag7. Given the distinction between NOR and (NODxNOR)F1 mice in susceptibility to IDDM and nasally induced tolerance, it seems likely that NOD-derived non-MHC gene product(s) are required for the resistance to nasally induced tolerance.


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  		Table 3. NI induces immune deviation but insignificant inhibition of proliferation in (NODxNOR)F1 mice
 
NI with very high doses of antigen reduces the recall response in NOD mice
To determine if the resistance to NI in NOD mice could be overcome by significantly higher or lower doses of antigen, female NOD mice had NI with 5, 50 or 500 µg of HEL or saline 7 days before being immunized with HEL/CFA. Only in mice pretreated with 500 µg of antigen did we observe a >50% reduction in the HEL-specific proliferation in draining lymph nodes as compared to saline-treated controls (Fig. 3Go). In additional experiments, we found that NI with 100 µg of HEL was also ineffective in significantly reducing the proliferative response in NOD mice (data not shown).



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  		Fig. 3. NI with a 10-fold higher dose of HEL (500 µg) reduces the proliferative recall response in NOD mice. Female NOD mice (6–8 weeks old) had NI with 5, 50 or 500 µg of HEL or saline (three mice per group) 7 days prior to s.c. immunization with 100 µg HEL in CFA. Ten days later, the draining LNC from individual mice were collected and tested for proliferative responses to HEL (50 µg/ml). The results are expressed as {delta}c.p.m. and the control wells (no antigen) ranged from 2781 to 9400 c.p.m. The results are representative of three independent experiments. The {delta}c.p.m. from each antigen-pretreated group was compared to that of saline-treated mice using Student's t-test (unpaired, two-tailed); *5 µg, P = 0.0951; **50 µg, P = 0.0622; ***500 µg, P = 0.0034. P < 0.050 was considered significant.

 
We then compared lymphocytic responses to OVA in NOD mice that were NI with 50 or 250 µg of OVA or saline prior to in vivo challenge with OVA/CFA. While the 50 µg dose was insufficient in reducing the recall response (13.4%) (Fig. 4AGo), the 250 µg dose reduced the proliferation by 50% (Fig. 4AGo). NOR mice pretreated in parallel with 50 µg OVA showed a 63% reduction in the proliferative response (data not shown). While a marginal difference was seen in the number of OVA-responsive IFN-{gamma}-producing cells in either group of antigen-pretreated NOD mice (Fig. 4BGo), only the NOD mice NI with the 250 µg dose of OVA showed a significant increase in the number of IL-5-producing cells (Fig. 4CGo). Two other experiments gave similar results. A nasal dose of 100 µg of OVA was not able to induce a similar reduction in proliferation in NOD mice, while NI with 25 µg of OVA was the minimal dose required in BALB/c mice (data not shown). Therefore, a substantially higher nasal dose of antigen was clearly necessary to reduce the proliferative response in NOD mice as compared to other mouse strains.





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  		Fig. 4. NI with 250 µg of OVA induces an increase in the number of IL-5 spot-forming cells without a concomitant reduction in IFN-{gamma} SFC in the draining lymph nodes of immunized NOD mice. Female NOD mice (three per group) had NI with 50 or 250 µg of OVA, or saline, 7 days prior to s.c. immunization with 50 µg OVA in CFA. Ten days after in vivo challenge, the draining LNC were collected, pooled and stimulated in vitro with OVA (50 µg/ml) or PPD. The proliferative response (A) was measured by [3H]thymidine incorporation, as described in Methods and the {delta}c.p.m. from antigen-pretreated mice was compared to that of saline-treated mice using Student's t-test (unpaired, two-tailed); *50 µg, P = 0.0375, **250 µg, P = 0.0011. P < 0.0250 was considered significant. The IFN-{gamma}-secreting and (B) IL-5-secreting (C) cells were enumerated using the ELISPOT assay as described in Methods. Values from the IL-5 and IFN-{gamma} media control wells ranged from 1 to 8 and 3 to 6 spots respectively. Similar results were obtained in two other experiments.

 
NOD T cell blasts are resistant to the inhibitory effects of TCR complex religation
The induction of peripheral T cell tolerance is predicated on the interaction between antigen-presenting cells and antigen-specific T cells, with the efficient delivery of certain signals through MHC molecules and the TCR complex. It is therefore possible that the resistance to nasally induced tolerance in NOD mice is due to a relative ineffectiveness of NOD T cells to receive tolerogenic signals rather than a deficiency in I-Ag7 to deliver them.

AICD has been reported to be involved in the loss of recall responses in the draining lymph nodes of mice pretreated by NI with antigen (24). Defects in mechanisms controlling peripheral T cell expansion could render antigen-responsive T cells resistant to the tolerogenic effects of nasal pretreatment with antigen. To determine if NOD T cells have an increased resistance to tolerogenic signals, newly activated Con A blasts from NOD (H-2g7), NOR (H-2g7) and BALB/c (H-2d) mice were re-stimulated using plate-bound anti-CD3 to induce cell death. Cells treated in this manner have been shown to be unresponsive and to undergo AICD (17–19). Since the anti-CD3-mediated ligation of the TCR is independent of MHC class II, the nature of the MHC class II molecule should not influence the outcome. T cell blasts from all the strains showed a dose-dependent reduction in proliferation when cultured with plate-bound anti-CD3 versus PBS coated wells (Table 4Go). However, unlike the inhibition seen in NOR and BALB/c T cell blasts (16.5 and 22.4% respectively), NOD Con A blasts were comparatively resistant to the inhibitory effects of anti-CD3 at 0.33 µg/ml (Table 4Go). The distinctive responses observed in NOD T cells were not caused by insufficient activation, since dose–response experiments with Con A showed that the initial proliferative responses in NOD spleen cells were indistinguishable from those of NOR mice (data not shown). The production of the cytokines IL-2, IL-5 and IFN-{gamma} was also similar among the strains (data not shown). Furthermore, FACS analysis revealed that CD44 and CD69 were expressed similarly on CD3+ blast cells from each of the strains (data not shown). The disparity between NOD mice and NOR or BALB/c mice in the religation experiments was similar when soluble anti-CD3 was used to induce T cell blast formation in spleen cells (data not shown). Together these data indicate that MHC-independent regulatory mechanisms can differentiate the sensitivity of peripheral T cells in NOD mice from that of other mice, including other I-Ag7 expressing strains.


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  		Table 4. The proliferative responses of NOD T cell blasts are resistant to the inhibitory effects of TCR complex religation
 
To demonstrate that the resistance to inhibition by TCR religation was applicable to NOD T cells displaying self-reactivity, recently activated T cells specific for the islet antigen GAD65 or OVA were cultured on anti-CD3-coated plates and tested for their proliferative responses (Table 5Go). While T cell clones produced from BALB/c mice were highly sensitive to the inhibitory effects of TCR religation, both the GAD65- and OVA-specific T cell clones produced from NOD mice were completely resistant (Table 5Go). The NOD- and BALB/c-derived clones were all characterized as being of the Th1 phenotype since they were potent producers of IFN-{gamma} after antigenic stimulation and secreted no IL-4 or IL-5 (data not shown).


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  		Table 5. NOD T cell clones specific for self or non-self antigens are resistant to the inhibitory effects of TCR complex religation
 
Defective AICD in NOD T cells
To evaluate the effect of TCR religation on AICD, NOD or BALB/c Con A blasts were added to anti-CD3-coated plates and then incubated with Annexin–V-FITC and propidium iodide (PI). Cells undergoing AICD or apoptosis would be expected to be Annexin V+ and PI–. While there was only a marginal increase in the number of Annexin V+ cells among the NOD blasts, twice as many became positive in the re-stimulated BALB/c blasts (Fig. 5Go). Since Annexin V binding is indicative of the early stages of AICD, NOD T cells may not effectively engage apoptotic pathways that normally control peripheral T cell expansion.



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  		Fig. 5. NOD mice have a reduced sensitivity to AICD. Viable 72-h Con A blasts from NOD or BALB/c spleen cells were plated onto 96-well plates pre-coated with anti-CD3, in rIL-2-containing medium, and incubated for 4 h at 37°C. The cells were then harvested from the wells and stained with Annexin V–FITC and –PI. The Annexin V+ PI– cells are undergoing apoptosis while Annexin V+ PI+ cells are dead cells.

 
Apoptosis can be induced through death receptors such as Fas (CD95), TRAIL (TNF-related apoptosis-inducing ligand) and TNFRI (TNF-receptor I)/p55. We examined the expression of these key components in NOD T cell blasts since the apparent resistance to AICD observed in NOD T cell cells could be due to differences in the apoptotic pathway. However, cell lysates collected 48 h after stimulation showed no differences between T cell blasts from NOD, NOR and BALB/c mice in the expression of CD95, TRAIL or TNFRIp55 (Fig. 6Go). While the RPA (Fig. 6Go) and FACS analysis (data not shown) indicated that surface expression of CD95 was similar among the strains studied, it was possible that signaling through CD95 was defective in NOD T cells. To compare the sensitivity to CD95-induced cell death in NOD T cells to that of mice sensitive to nasally induced tolerance, Con A blasts from NOD or NOR mice were incubated overnight (24 h) with anti-CD95 (clone Jo-2) and anti-guinea pig IgG to cross-link and stimulate CD95. The blasts were then fixed, stained with DAPI, and examined for apoptotic bodies (Fig. 7Go) or for Annexin V binding (data not shown). The results show that compared to activated NOR T cells, NOD-derived T cells have a reduced sensitivity to apoptosis induced by cross-linking CD95 (Fig. 7Go).



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  		Fig. 6. Activated T cells from NOD, NOR and BALB/c mice express similar levels of apoptosis-associated molecules. To determine if the resistance to activation-induced cell death observed in NOD T cells was due to differences in the expression of caspase-8, FasL, Fas (CD95), TRAIL or TNFRp55, we examined these key components in T cell blasts from NOD, NOR and BALB/c mice, using a commercial RPA, as described in Methods. `RNase-protected' probes were resolved on a polyacrylamide gel and imaged by autoradiography. The expression of specific mRNA species was analyzed 48 h after activation and detected probes have been labeled. GADPH and L32 were used as mRNA controls.

 


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  		Fig. 7. NOD splenocytes are less sensitive to death-inducing signals mediated through CD95. Con A blasts produced from NOD or NOR spleen cells were incubated overnight (24 h) with anti-CD95 (clone Jo-2) and anti-guinea pig IgG to cross-link and stimulate CD95 (Fas) death receptor. As a control, cells were also incubated with isotype control (guinea pig IgG) and the secondary antibody. The blasts were then fixed, stained with DAPI and examined for apoptotic bodies as described in Methods.

 
The engagement of CD95 and the subsequent activation of caspase-8 and caspase-3 may be important in the AICD of peripheral T cells (25). Triggering of the CD95 death receptor leads to the recruitment of pro-caspase-8 to the death-inducing signaling complex (26); as with all caspases, caspase-3 and -8 are present as inactive zymogens (procaspases). Once cleaved, activated and released into the cytosol, caspase-8 can then activate other caspases, resulting in the initiation of an apoptotic cascade. Although the level of caspase-8 may be crucial in the initiation of AICD, when activated T cells from NOD mice were investigated, we found the expression levels of caspase-8 were similar to those of control strains (Fig. 6Go), indicating that the defect (s) in NOD T cells are not due to altered expression of this step in the death pathway.

Caspase-3 is an executioner caspase, downstream of caspase-8, and is responsible for cleaving many of the substrates that mediate the biochemical and morphological characteristics of apoptosis. Upon cleavage by other previously activated caspases, such as caspase-8, 32-kDa procaspase 3 is cleaved into small (12 kDa) and large (19 kDa) subunits, the latter of which is subsequently cleaved into a 17-kDa form. To assess this step in apoptosis we assayed cell extracts from NOD or BALB/c Con A-stimulated splenocytes for immunoreactivity with an antibody specific for cleaved caspase-3. In both strains, activation of the cell death pathway increased with time, as indicated by cleaved caspase-3, and reached a maximum cleavage rate by 48 h (Fig. 8Go). Interestingly, slightly more of the 19-kDa cleavage product of caspase-3 is seen in NOD versus BALB/c T cells at 24 h. Whether this difference has an impact on the functional activity of the subsequent steps in the apoptotic machinery remains to be determined and awaits further study. Nonetheless, it appears that after stimulation both caspase-3 and -8 are cleaved and activated in NOD T cells.



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  		Fig. 8. c-FLIP expression increases in response to Con A stimulation. Activated T cell blasts were produced by stimulating spleen cells with Con A or anti-CD3 for 48 h. The cells were then pelleted, resuspended in lysis buffer and 20 µg of each sample was resolved on a gradient gel and blotted to Hybond-ECL overnight. Blots were probed with anti-FLIP, anti-caspase 3 or anti-Bcl-2.

 
Apoptosis is a complex phenomenon in which a number of regulatory mechanisms may control its effect on cell survival, including molecules that exert an antagonistic influence on the death pathway. Thus, in addition to members of the death program described above, we examined the expression of two anti-apoptotic molecules, c-FLIP and Bcl-2 in NOD T cells. c-Flip is a critical inhibitor of caspase-8 recruitment to the Fas death-inducing signaling complex and Bcl-2 blocks the mitochondrial-dependent activation of caspase-3. c-FLIP expression increased in Con A blasts from both BALB/c and NOD splenocytes within 24 h of stimulation, but only the p43 form was detected (Fig. 8Go)—the long form of c-FLIP is 55-kDa, but it is processed to produce the p43 form after CD95 stimulation (27). It has been reported that the p43 form of c-FLIP is the only one to interact with the Fas death-inducing signaling complex to prevent caspase-8 activation, in response to Fas stimulation. We were not able to detect any differences in c-FLIP expression between BALB/c and NOD mice (Fig. 8Go). Furthermore, the levels of Bcl-2 protein, an anti-apoptotic protein that protects cells from many apoptotic stimuli (28), were also comparable in BALB/c and NOD mice (Fig. 8Go). Therefore, the increased resistance to AICD does not appear to be associated with an increased expression of these anti-apoptotic molecules. While NOD T cells are relatively resistant to mechanisms that function in the maintenance or establishment of peripheral tolerance (immune deviation and apoptosis), several of the many components involved in the cell death pathway appear to function similarly to other strains.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
One of the primary strategies for revealing the causes of autoimmune disease lies in addressing the mechanisms by which pathogenic clones escape regulation and expand in the periphery of susceptible individuals. The goal of this study was to investigate the influence of the diabetes-associated H-2g7 locus on the pattern of resistance to nasally induced tolerance in NOD mice. We demonstrate that T cells from NOD mice have a heightened threshold for tolerogenic signals. Although previous studies performed in vitro have made similar assertions, using nasally induced tolerance as a model, we show here that NOD T cells are relatively resistant to tolerogenic signals delivered in vivo as well. While it has been suggested that I-Ag7may be ineffective in mediating tolerogenic signals to T cells, the expression of I-Ag7 was not sufficient to provide resistance to nasally induced tolerance in the recombinant congenic NOR (H-2g7) and the NON.NOD (H-2g7) mouse strains. Thus, in spite of their expression of the unique I-Ag7 molecule, both of these strains remain diabetes-free and therefore demonstrate the importance of non-MHC genes in providing protection from autoimmune disease (21,23). Moreover, there is a lack of consensus regarding the stability of I-Ag7 (12,29), its peptide binding characteristics (11,30) and its precise role in the orchestration of the disease. HEL and OVA were used as model proteins in this report to avoid the difficulty of factoring in the role of central tolerance in the regulation of peripheral immune responses to antigens, as would be the case for self proteins. However, the pattern of resistance to AICD was also shown to be present in NOD T cells specific for the self-protein GAD65, which has been shown to induce diabetogenic T cells (31). Whether such islet-reactive T cells can persist longer after activation in NOD mice compared to diabetes-resistant congenic NOR mice, owing to defective AICD, awaits further study. Nonetheless, the findings in this report suggest that genes other than I-Ag7 influence the resistance to nasally induced tolerance in NOD mice and that an increased resistance of NOD T cells to AICD contributes to their susceptibility to autoimmune disease.

The notion that the linkage between certain MHC alleles and autoimmune disease is a function of defective thymic deletion or central tolerance is widespread. Yet, the peripheral regulation of autoreactive T cells may be just as critical as central tolerance in controlling the unwanted expansion of such cells. Pathogenic lymphocytes may be specifically regulated in the periphery by mechanisms that actively suppress immune responses, and those that induce non-responsiveness through anergy or apoptosis (32,33). Recently it has been reported that NI with antigen sensitizes responsive T cells for the induction of apoptosis upon rechallenge with antigen by parenteral immunization (24). The data presented here show that T cells from NOD mice are resistant to the regulation mediated by nasal pretreatment with antigen as well as to the loss of proliferation induced by religation of the TCR with plate-bound anti-CD3. Although it is tempting to postulate that activated T lymphocytes from NOD mice are defective in their expression of a molecule(s) that is required for the initiation of programmed cell death (34,35), we were not able to demonstrate a difference between NOD, NOR and BALB/c T cells in the expression of several components that are involved in AICD, including CD95, Fas ligand (FasL), caspase-3 and caspase-8. However, we did observe that NOD T cell blasts had a reduced sensitivity to apoptosis induced via anti-CD95. Furthermore, there was a modest difference between NOD and BALB/c T cell blasts in the pattern of cleavage products from the proteolytic activation of caspase-3. Since caspase-8 contributes enzymatically to the activation of caspase 3, is possible that the functional activities of caspase-8 are altered in NOD mice, despite the fact that it is expressed at levels similar to BALB/c and NOR mice. Interestingly, the NOD Idd 5 locus, which has been implicated in the control of insulitis and diabetes in NOD mice, contains the Casp8 gene (36).

Our conclusion that NOD-derived factors other than I-Ag7 are responsible for the resistance of NOD mice to nasally induced tolerance is supported by the results from F1 mice produced by outcrosses of NOD mice with either of two tolerance-susceptible strains, BALB/c or NOR mice. F1 offspring from each cross are resistant to the suppressive effects of NI with 50 µg of antigen. The resistance to nasal-induced tolerance in F1 mice cannot be explained as a simple reduction in the expression of a susceptible MHC allele in the offspring since both parents of (NODxNOR)F1 offspring are of the same haplotype (H-2g7). Although (NODxBALB/c)F1 and (NODxNOR)F1 mice are NOD-like in their resistance to NI, it is likely that BALB/c-derived genes are able to compensate and provide protection from IDDM in (NODxBALB/c)F1 mice (37,38). In addition, since class I MHC molecules are necessary for the initiation of IDDM in NOD mice (39–42), it is clear that mechanisms independent of I-Ag7 can contribute to the loss of peripheral tolerance in NOD mice.

It should be noted that nasal treatment with islet antigens has been used successfully to alter the course of IDDM in NOD mice. Prediabetic mice nasally-treated with a high dose of four glutamic acid decarboxylase peptides (200 µg) (43) or multiple doses of an insulin ß chain peptide (44) were protected from diabetes and demonstrated a deviated response to ß cell antigens (a switch from a Th1 to a Th2 phenotype). However, it cannot be overemphasized that in contrast to this report, mice in the former studies were never challenged by immunization, a step that would definitively demonstrate that the mice were tolerant to specific antigen and thus unable to mount the immune response observed in control animals. In addition, the need for high doses of antigen to achieve protection in the former studies concurs with our results, which indicate that comparatively stronger immunological stimuli are required to engage regulation in NOD mice. It is not surprising then that some therapies that reduce the incidence of IDDM in NOD mice fail to eliminate the cognate autoreactive Th1 response (45–47) and vary in their long-term efficacy.

Like a number of other autoimmune-prone mouse strains, NOD mice also have an increased resistance to signals that are normally capable of inducing apoptosis (34,48–51). Interestingly, similar to the NOD situation, NOR mice have been reported to display a resistance to the apoptotic signals triggered by cyclophosphamide (52). Our findings that NOR mice are susceptible to nasally induced tolerance and their well-known complete resistance to insulitis and IDDM (23) suggest that NOR mice possess alternative regulatory mechanisms that can compensate for the putative defect (s) in their apoptotic machinery, which makes them insensitive to cyclophosphamide. These regulatory mechanisms may also provide some measure of protection to (NODxNOR)F1 mice which display a reduced incidence (0–30%) and a greatly delayed onset of diabetes compared to NOD mice (23,53).

Susceptibility to autoimmune disease is multi-factorial, with influences from MHC and non-MHC-encoded elements (54–58). A conclusion that NOD mice display a general defect in peripheral tolerance independent of I-Ag7 is consistent with our results and takes into account the findings of many others (34,48–51). Given the efforts to design immunotherapy for autoimmune diseases in humans, it is important to understand the mechanisms which contribute to susceptibility in a spontaneous autoimmune disease model such as the NOD mouse. While the expression of certain MHC alleles may increase the risk for developing autoimmune disease via forces determined by the positive/negative selection of a relevant T cell repertoire, other gene products are required in the chain of events leading to the onset of spontaneous autoimmune disease. The distinction between spontaneous and inducible autoimmune disease may be critical in terms of the approaches that would be useful for treatment. Thus, in contrast to inducible models of autoimmune disease, where the malady often innately resolves, those individuals who inadvertently develop autoimmune disease may be less effective in reinstating peripheral tolerance. Surprising as it sounds, the more intensely and early that one induces experimental autoimmune encephalomyelitis in susceptible B10.PL mice, the more readily the disease naturally resolves. In spontaneous disease, aggressive approaches (larger or more frequent doses of antigen) may be needed to effectively ameliorate the pathogenic response since the subjects have a proclivity towards heightened autoreactivity (3), coupled with a defective ability to regulate such responses.


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  		Table 6. Mouse strains genetically related to the NOD mouse
 

   Acknowledgments
 
The authors thank Sophia Tian for excellent technical assistance. This work was supported by AI28419 and a grant from The Juvenile Diabetes Foundation International. This is publication no. 283 from The La Jolla Institute for Allergy and Immunology.


   Abbreviations
 
AICD activation-induced cell death
Con A concanavalin A
CFA complete Freund's adjuvant
DAPI 4',6'-diamidino-2-phenylindole
FasL Fas ligand
HEL hen egg lysozyme
IDDM insulin-dependent diabetes mellitus
LNC lymph node cell
NI nasal installation
NOD non-obese diabetic
NON non-obese non-diabetic
NOR non-obese diabetes-resistant
OVA ovalbumin
PI propidium iodide
PPD purified protein derivative
RPA RNase protection assay
SFC spot-forming cell
SPC spleen cell
TNFRI tumor necrosis factor receptor I
TRAIL tumor necrosis factor-related apoptosis-inducing ligand

   Notes
 
3 Present address: Department of Immunology, Holland Laboratory, American Red Cross, Rockville, MD 208552, USA Back

Transmitting editor: G. Doria

Received 20 December 2000, accepted 13 July 2001.


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 Introduction
 Methods
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 Discussion
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