By
From The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville 3050, Australia
Cellular immune hyporesponsiveness can be induced by the presentation of soluble protein antigens to mucosal surfaces. Most studies of mucosa-mediated tolerance have used the oral route of antigen delivery and few have examined autoantigens in natural models of autoimmune disease. Insulin is an autoantigen in humans and nonobese diabetic (NOD) mice with insulindependent diabetes mellitus (IDDM). When we administered insulin aerosol to NOD mice after the onset of subclinical disease, pancreatic islet pathology and diabetes incidence were both
significantly reduced. Insulin-treated mice had increased circulating antibodies to insulin, absent splenocyte proliferation to the major epitope, insulin B chain amino acids 9-23, which
was associated with increased IL-4 and particularly IL-10 secretion, and reduced proliferation
to glutamic acid decarboxylase, another islet autoantigen. The ability of splenocytes from insulin-treated mice to suppress the adoptive transfer of diabetes to nondiabetic mice by T cells of
diabetic mice was shown to be caused by small numbers of CD8 T cells. These findings reveal a novel mechanism for suppressing cell-mediated autoimmune disease. Induction of regulatory CD8
T cells by aerosol insulin is a therapeutic strategy with implications for the prevention of human IDDM.
Insulin-dependent diabetes mellitus (IDDM)1 results from
the selective destruction of insulin-producing An autoantigen can be assumed to be pathogenic if its
administration modifies the natural history of autoimmune
disease. Autoantigen-specific strategies of immune tolerance induction have been shown to favorably modify the
natural history of experimental autoimmune disease in rodents (5, 16). The presentation of soluble protein antigen to mucosal surfaces, classically via the oral route, results in selective suppression of antigen-specific, T cell-mediated,
delayed-type hypersensitivity (DTH) and IgE responses
(16). "Oral tolerance" has been associated with the deviation of immunity away from T cell (Th1) to antibody
(Th2) responses, with the induction of regulatory T cells
and, at higher antigen doses, with both T cell anergy and T
cell deletion (16, 19). Despite widespread interest in the
possibility of preventing IDDM, relatively few studies have
evaluated mucosa-mediated tolerance in the natural NOD
mouse model. Zhang et al. (20) found that oral porcine insulin (1 mg twice weekly) delayed the onset and reduced
the incidence of diabetes, and was associated with splenic
T cells that partially blocked the transfer of diabetes to
young NOD mice by spleen cells from diabetic mice. Subsequently, Bergerot et al. (21) reported that the regulatory
cells induced by oral insulin were CD4+ T cells. In studies
of oral tolerance to guinea pig myelin basic protein in the
Lewis rat model of experimental autoimmune encephalomyelitis (reviewed in reference 16), however, both CD4+
and CD8+ regulatory T cells that secrete IL-4, IL-10,
TGF- Holt and co-workers described the suppression of T cell
and IgE responses to OVA after its inhalation in rats or
mice, which was transferable by CD8+ T cells (18) and was
subsequently found to be effected by small numbers of
OVA-specific CD8 Aerosol Treatment and Diabetes Assessment.
Semisealed boxes of
eight female NOD mice were each aerosolized by connection to
a standard patient electric pump (Maymed Aerosol MKV; Anaesthetic Supplies, Sydney, Australia) and an Aeroflo nebulizer
(Waite & Co., Sydney, Australia). Recombinant human insulin
(Humulin R; Aza Research, West Ryde, Australia) or control OVA protein, at 4 mg/ml, was delivered over 10 min at an air
flow rate of 6 liters/min in a rated droplet size of <5.8 µm, to
groups of 24-32 mice. All treatments were given between 0900 and 1100 h. Protocols and mouse care were approved and supervised by the institutional Animal Ethics Committee. Retroorbital
venous blood was sampled at least every 28 d from 100 d of age,
and mice were considered diabetic if their blood glucose, confirmed by a repeat test, was >11 mM. Glucose was measured
with BM-Test Glycemie® strips and a Reflolux® II meter (Boehringer Mannheim GmbH, Mannheim, Germany) on a drop of
blood aspirated via a glass capillary tube from the retroorbital venous plexus of unanesthetised mice.
Histology.
Mice were killed by CO2 inhalation, and the pancreas and salivary glands were immediately removed into Bouin's
fixative and embedded in paraffin. The insulitis score, a measure
of the severity of islet infiltration, was determined blindly by two
independent investigators by grading and then averaging a minimum of 15 separate islets in serial 6-µm pancreas sections stained
with hematoxylin and eosin. The grading scale was as follows: 0, no infiltration, islet intact; 1, <10 periislet lymphoid cells, islet
intact; 2, 10-20 peri- and intraislet lymphoid cells, islet intact; 3, >20 peri- and intraislet lymphoid cells, <50% of islet replaced or destroyed; 4, massive lymphoid infiltrate with >50% of islet replaced or destroyed. Infiltration of the salivary glands was graded
by the number of lymphoid cells in clusters: 0, no cells; 1, <10 cells; 2, 10-50 cells; 3, >50 cells.
Immune Responses.
Spleen cells from individual normoglycemic mice were treated with a red cell lysis buffer, resuspended,
and incubated in quadruplicate at 2 × 105/200 µl of serum-free
HL-1 medium (Hycor, Portland, ME) containing 50 µm 2-MER
in round-bottom wells with the indicated concentrations of antigen. After 3 d at 37°C in 5% CO2/air, 100-µl aliquots from each
replicate supernatant were collected and stored at cells in
the islets of the pancreas, within an autoimmune inflammatory "insulitis" lesion (1, 2). The primary role of autoreactive T cells in mediating
cell destruction has been shown
directly in two spontaneous animal models of IDDM, the
Bio-Breeding (3) rat and the nonobese diabetic (NOD) mouse
(4). Target autoantigens that trigger or drive immune reactivity to
cells not only have diagnostic applications, but
are potential agents for specific immunotherapy (5). Several potentially pathogenic islet/
cell autoantigens have
been identified by their reactivity with circulating antibodies or T cells in rodents and humans with subclinical or clinical IDDM, particularly insulin, glutamic acid decarboxylase (GAD), and a tyrosine phosphatase, IA-2 (9). Insulin and its precursor, proinsulin, however, are the only
IDDM autoantigens that are
cell specific. Insulin autoantibodies are a risk marker for the development of clinical
IDDM (10) and have been detected before autoantibodies
to other islet antigens in the offspring of diabetic mothers
(11). Increased proliferation of peripheral blood T cells to
human insulin can be demonstrated in up to half of subclinical and recently diagnosed IDDM subjects (12), but responses are relatively low. This is possibly because the
dominant human T cell epitope is in proinsulin; a peptide
that spans the natural cleavage site between the B chain of
insulin and the connecting (C) peptide in proinsulin elicits
T cell proliferation in a majority of subclinical subjects (13).
In the NOD mouse, insulin autoantibodies are reported to
be a risk marker for the development of diabetes (14) and
the majority of T cell clones generated from the insulitis lesion react to the insulin B chain, amino acids 9-23 (15).
, or TGF-
, respectively, have been described. Daniel
and Wegmann (15) recently reported that intranasal administration of insulin B chain amino acids 9-23 protected
NOD mice from diabetes, but did not define a mechanism;
Tian et al. (22) reported that a single intranasal dose of
GAD T cell epitope peptides given to NOD mice before
the onset of insulitis induced the deviation of antiislet immunity towards Th2 responses and reduced diabetes incidence,
in association with regulatory CD4, not CD8, T cells.
T cells (23). There is increasing evidence for the immunoregulatory role of
T cells in general (24). We therefore decided to investigate the effect
of aerosol insulin inhalation in the NOD mouse. This
mode of delivery of autoantigen to the nasopharyngeal and
upper bronchial mucosa could have advantages over oral
insulin in humans. It is convenient and direct, might require a smaller dose, and be less likely to be associated with
insulin degradation and variability of absorption. In particular, we wished to determine if aerosol insulin would be
therapeutic if given after the onset of insulitis and to define the nature of the regulatory cells that it might potentially
induce.
70°C for
cytokine assays; the cells were then pulsed with [3H]thymidine,
harvested 16 h later, and counted on a microscintillation counter
(Topcount; Packard Instruments, Meriden, CT). Insulin was recombinant human (Humulin R; Eli Lilly), as used for aerosol treatments. Insulin B chain peptide corresponding to amino acids 9-23 of mouse insulin II (Peptide Express, Fort Collins, CO) was >90% pure, as determined by HPLC analysis. GAD65 was the
recombinant human form expressed with a COOH-terminal hexahistidine in a baculovirus system and purified by Ni2+ chelation
affinity chromatography. It was resolved as a single band in SDSPAGE, and was endotoxin-free by the quantitative limulus lysate
assay (BioWhittaker, Walkersville, MD).
were measured by ELISAs with mAb
pairs (PharMingen, San Diego, CA); the lower limits of detection were 62, 16, 16, and 55 pg/ml, respectively. TGF-
1 was measured with an ELISA kit (Promega, Madison, WI) with a lower
limit of detection of 16 pg/ml.
Adoptive Transfer of Diabetes. Male NOD mice, 6-9 wk old (16 per group), were irradiated (800 rad) from a cobalt source, and 3-6 h later, received 2 × 107 pooled splenocytes from recently diabetic 14-19-wk-old female NOD mice, together with 2 × 107 splenocytes (or cells fractionated from this number) from either aerosol insulin- or OVA-treated mice, in 200 µl via the tail vein. The onset of diabetes was then monitored by measuring blood glucose starting 2 wk after transfer.
Fractionation of Spleen Cell Populations. Spleen cells were treated with a red cell lysis buffer and resuspended in mouse tonicity PBS. Total T cells were purified by non-adherence to nylon wool. CD4 and CD8 cells were positively selected/depleted magnetically with mAbs directly bound to MACS MicroBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany), according to the manufacturer's protocols, and were counted as viable cells (trypan blue stain negative). Flow cytometry revealed >95% depletion of CD4 or CD8 cells, with recoveries of ~80 and ~50%, respectively.
Aerosol human insulin or OVA was administered in different schedules to female NOD mice from 28 d of age, the earliest time at which insulitis is detectable in our colony, and their incidence of diabetes and severity of insulitis were subsequently measured.
The incidence of diabetes was only marginally affected by a single aerosol insulin treatment at 28 d of age, being 72% by 240 d of age, compared to 88% after aerosol OVA. However, treatment for 3 or 10 consecutive days and then weekly significantly delayed the onset and reduced the incidence of diabetes. In five separate experiments, diabetes incidence at 156 d of age was reduced from a median of 47% in OVA-treated mice to 23% in insulin-treated mice; at 240 d of age, when the cumulative incidence of diabetes approaches a maximum, the values were 79 and 49%, respectively (P = 0.005, Kaplan-Meier survival statistic). There was no difference if the initial treatment was for 3 or 10 d. In another experiment, in which treatment was given for 10 consecutive days and then weekly, but not started until 49 d of age, when insulitis was well established, aerosol insulin still significantly reduced diabetes incidence at 156 d from 58 to 25% (P = 0.001). Insulin treatment was associated with a significant reduction in the severity of the islet lesion, as judged by the "insulitis score," which paralleled the decrease in diabetes incidence (Table 1). Infiltration of the salivary glands by lymphoid cells (sialitis), which also occurs in NOD mice, was unaffected by aerosol insulin.
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In the absence of absorption-promoting agents, systemic uptake of insulin from the nasopharyngeal mucosa in humans is insignificant (30). In NOD mice, blood glucose was not altered in the short-term by aerosol insulin (data not shown). Insulin solutions labeled with 10% Evan's blue dye were observed to be deposited in the nasopharynx, trachea, and main bronchial divisions, as well as in the esophagus. While it may be difficult, if not impossible, to avoid some gastrointestinal exposure after aerosol or intranasal delivery of soluble protein, delivery into the nasopharynx alone is sufficient to induce tolerance (18, 31, 32).
Immune Responses.We investigated if aerosol insulin
treatment had altered immune responses to insulin. Unprimed T cell proliferative responses to islet antigens, including insulin, have been reported in NOD mice (33), but
have not always been reproducible (34). Proliferative responses of spleen cells (0.5-2.5 × 106/ml) from either insulin- or OVA-treated mice, 56-105 d old, to human insulin
or OVA (0.2, 2.0, 20, and 40 µg/ml), in different serumsupplemented or serum-free media varied by less than twofold above basal and were usually depressed below basal at
the highest concentration of insulin. Insulin at high concentrations has been reported to inhibit T cell responses
(35). In contrast, in the OVA-treated control mice, but not
the insulin-treated mice, responses to insulin B chain peptide amino acids 9-23, a dominant epitope for NOD
mouse islet-derived T cell clones (15), were significant (Table 2). Furthermore, OVA mice had significantly higher
responses than insulin mice to human GAD65, which was
previously reported to stimulate splenic T cells in NOD
mice (33). In mice from both treatment groups, proliferative responses to non-antigen-specific stimulation by Con
A or the T cell receptor CD3 mAb, 145-2C11 were similar
(Table 2) and no different to untreated mice (not shown),
indicating that aerosol treatment did not cause general immunosuppression. IL-2, IFN-, and TGF-
1 secretion in
response to insulin B chain amino acids 9-23 were not significantly different between insulin- and OVA-treated mice;
however, the levels of IL-4 and particularly IL-10 were
higher in the wells from insulin-treated mice (Table 3).
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Insulin antibodies were measured by a standard immunoprecipitation assay with sera (n = 12 per group) from insulin- and OVA-treated mice aged 70-105 d. Precipitation
of 125I-insulin radioactivity by antibodies in sera from insulin-treated mice (12.7 ± 3.6%; mean precipitated cpm ± SD) was significantly higher (P <0.01, Mann-Whitney U test)
than in OVA-treated mice (6.9 ± 2.6%). This increase in
the "level" of insulin antibodies after aerosol insulin, together with the suppression of T cell proliferation and the
increase in IL-4 and IL-10 responses to the insulin B chain
peptide, is consistent with the phenomenon of immune deviation, as described after oral myelin basic protein in Lewis
rats (16) and intranasal GAD peptides in NOD mice (22). cell destruction within the DTH lesion of IDDM is an
example of a Th1-mediated process (1, 2), whose inhibition by aerosol insulin might be expected to shift the Th1/
Th2 balance towards Th2 in response to key islet antigens.
Defective suppressor T cell function has been postulated to
shift the balance towards Th1 in IDDM (2). It seems unlikely that the reduced T cell proliferative response to GAD
could reflect "bystander" suppression caused by the secretion of the Th2 cytokines IL-4 and IL-10 (16) by insulin
aerosol-induced regulatory cells because, apart from an absence of added insulin in the cultures with GAD, responses to Con A and anti-CD3 were not impaired. A direct explanation is that the reduced response to GAD reflects the
protective effect of aerosol insulin on insulitis and
cell
destruction. This implies that at least some GAD immunity
is secondary, and that immunity to (pro)insulin may have a
more proximal role in
cell destruction. Although NOD
mouse T cell responses to human GAD65 have been reported to be stronger and to appear earlier than those to
native human insulin (33), we have recently shown (36)
that the transgenic expression of mouse proinsulin II in
NOD mouse APCs completely prevents insulitis and diabetes.
As mucosal tolerance has
been associated with the appearance of "regulatory" cells in
the spleen (16, 18, 20), we asked if aerosol insulin-
induced regulatory cells that could inhibit the adoptive
transfer of diabetes by pathogenic effector T cells. In the
classic adoptive transfer model (37; see Fig. 3), spleen cells
from diabetic NOD female mice transferred intravenously to young, irradiated, nondiabetic, syngenic male or female
recipients cause clinical diabetes in the majority within 4 wk. When we coinjected 2 × 107 spleen cells from older,
diabetic mice with an equal number of spleen cells from
aerosol OVA mice, the majority of young recipients developed diabetes within 4-5 wk; in contrast, after coinjection with spleen cells from aerosol insulin mice, only a minority
developed diabetes (Fig. 1 A). Diabetes incidence was suppressed by 75% in six separate experiments with either
splenocytes or nylon wool-nonadherent splenocytes (enriched for T cells) from aerosol insulin mice.
Spleen cells were then fractionated to identify the regulatory cells responsible for the suppression of diabetes transfer. Depletion and positive selection of CD4 and CD8 cells clearly showed that CD8 cells were wholly responsible for the suppression of transfer (Fig. 1, B-E). Depletion of CD4 cells did not alter the ability of residual spleen cells from aerosol insulin mice to suppress transfer (Fig. 1 B), and positively selected CD4 cells did not suppress transfer (Fig. 1 C). On the other hand, there was no suppression by CD8depleted spleen cells from aerosol insulin mice (Fig. 1 D), whereas positively selected CD8 cells suppressed transfer (Fig. 1 E). The partial suppression by positively selected CD8 cells, in contrast to the rapid development of diabetes after their depletion, is probably caused by inefficient recovery of CD8 cells; in this experiment, 7 × 105 purified CD8 cells were coinjected into each recipient with 2 × 107 spleen cells from diabetic mice.
T cells bearing receptors have been shown to have an
immunoregulatory role (24). Interestingly, it has been
reported that total peripheral blood
cells decrease concomitantly with the loss of
cell function in humans with
subclinical IDDM (38). McMenamin et al. (23) found that
small numbers of
T cells could adoptively transfer the
suppression of OVA-specific IgE responses induced in mice
by repeated inhalation of OVA. To determine if the suppression of diabetes transfer that we observed was caused by
T cells, we fractionated spleen cells with the anti-
T cell mAb GL3-1A (39). Depletion of
T cells, like that of CD8 cells, completely abrogated the ability of nylon wool-
nonadherent spleen cells from insulin aerosol-treated mice
to suppress the adoptive transfer of diabetes (Fig. 2 A).
Conversely, relatively small numbers of
T cells from insulin aerosol-treated mice could suppress transfer. Diabetes
incidence after transfer was decreased by 50% for at least 70 d
when 1.4 × 105
T cells were coinjected with 2 × 107
spleen cells from diabetic mice (Fig. 2 A). The splenic CD8
and
T cells that suppressed diabetes transfer were one
and the same, and not two interdependent populations.
Thus, the ability of CD8 cells from insulin aerosol-treated
mice to suppress transfer was abolished if they were first depleted of
T cells, whereas small numbers of
cells purified from the CD8 cells prevented transfer (Fig. 2 B). A
summary of the results from 11 different cotransfer experiments is presented in Fig 3.
FACS® analysis revealed that cells reactive with GL3
antibody constitute 1.6-2.4% of total and ~1% of CD8+
T cells in the spleens of 12-16-wk-old female NOD mice.
These values were no different between groups of mice
treated with insulin or OVA aerosol. However, because of
their low abundance distinct subpopulations of antigenspecific CD8
T cells would be difficult to distinguish
this way. The higher protection with fractionated cells,
e.g., sequentially purified CD8
cells (Fig. 3), is quantitative and reflects their higher absolute number relative to
that in unfractionated cells.
Aerosol inhalation as a mode of insulin delivery to the
mucosa was as effective as oral insulin (20, 21) in reducing
diabetes incidence in the NOD mouse. The fact that it was
therapeutic after the onset of insulitis is especially relevant to
the prevention of IDDM in at-risk humans with subclinical
disease in whom the presence of circulating islet antigen-
reactive antibodies and T cells is taken to reflect underlying
insulitis. Indeed, compared to humans with recently diagnosed IDDM, NOD mice have more intense insulitis and
the majority of females progress to diabetes (1, 2, 4). Aerosol insulin had no obvious metabolic effect, but it induced a
population of regulatory CD8 T cells, small numbers of
which suppressed the ability of pathogenic effector T cells
to adoptively transfer diabetes. These antigen-induced "suppressor" T cells that are protective against cell-mediated autoimmune pathology have not been described previously.
The striking potency of these cells, unparalleled in "infectious suppression," was noted by McMenamin et al. (23) in
their experimental model of OVA aerosol-induced suppression of OVA-specific IgE and T cell responses, and implies they have the capacity to expand in vivo after transfer.
The ability of CD8
T cells to suppress, on the one hand,
cell-mediated autoimmune disease in NOD mice and, on
the other, an "allergic" response to exogenous OVA in
C57Bl mice (23) or Brown Norway rats (40) seems paradoxical within the framework of the Th1-Th2 paradigm.
However, the overriding consideration is that mucosa-mediated tolerance is manifest in genetically susceptible strains
by suppression of both DTH and IgE responses (18); either
the strict dichotomy of the Th1-Th2 paradigm may not
apply and/or there may be rodent strain variations in mucosa-mediated tolerance responses that reflect differences in
Th1-Th2 balance. It would be interesting to determine if
OVA aerosol induces CD8
T cells that suppress OVAspecific IgE responses in NOD mice.
Oral tolerance has been associated with a decrease in cellular and sometimes an increase in humoral antigen-specific
immunity, and with either CD8 or CD4 T cells that secrete, respectively, TGF- or IL-4, IL-10, and TGF-
1
(16). However, these regulatory cells have not been identified as bearing
receptors. In NOD mice, oral tolerance
to insulin was attributed to regulatory CD4 T cells (21).
We have shown that CD8
T cells account entirely for
the regulatory cells induced by aerosol insulin. This finding raises the possibility that the mechanisms and regulatory
cells that mediate mucosal tolerance in the upper airways
and gut are different. Experiments are in progress to identify the origin, fate, antigen specificity, and exact mode of
action of insulin aerosol-induced regulatory CD8
T cells.
Total T cells in the spleens of NOD female mice decrease with age similarly to BALB/c mice (41), but the role
of specific
T cells in the natural history of diabetes in the
NOD mouse remains to be defined. In subclinical IDDM
subjects, a decrease in the number of peripheral blood
V
9V
2 T cells signaled loss of
cell function and progression to clinical diabetes (38). We suggest, therefore, that
peripheral regulatory CD8
T cells could have a critical
role in determining the natural history of islet autoimmunity. Moreover, the induction of these regulatory cells by
aerosol delivery of insulin (or other islet antigens) has implications for the prevention of clinical diabetes in at-risk humans.
Received for publication 23 April 1996
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