By
From the * Department of Molecular and Medical Pharmacology, University of California, Los Angeles,
California 90095-1735; and the Department of Pathology, School of Medicine, Case Western
Reserve University, Cleveland, Ohio 44106
The nature (Th1 versus Th2) and dynamics of the autoimmune response during the development of insulin-dependent diabetes mellitus (IDDM) and after immunotherapy are unclear.
Here, we show in nonobese diabetic (NOD) mice that the autoreactive T cell response starts and spreads as a pure Th1 type autoimmunity, suggesting that a spontaneous Th1 cascade underlies disease progression. Surprisingly, induction of antiinflammatory Th2 responses to a single cell antigen (
CA) resulted in the spreading of Th2 cellular and humoral immunity to
unrelated
CAs in an infectious manner and protection from IDDM. The data suggest that
both Th1 and Th2 autoimmunity evolve in amplificatory cascades by generating site-specific, but not antigen-specific, positive feedback circuits. Determinant spreading of Th2 responses
may be a fundamental mechanism underlying antigen-based immunotherapeutics, explaining
observations of infectious tolerance and providing a new theoretical framework for therapeutic intervention.
We have been studying the development of insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse. Concurrent with the onset
of insulitis, a spontaneous proliferative T cell response arises
to a determinant of glutamic acid decarboxylase (GAD; references 1 and 2). Proliferative T cell responses subsequently
spread to other While autoantigen-induced Th2 responses are associated
with inhibition of Th1-mediated autoimmune disease (6-
11), the nature of the induced tolerance is a matter of intense debate. Although it is generally accepted that deletion/inactivation of autoreactive lymphocytes is a fundamental tolerance mechanism, infectious tolerance has often
been observed (12), which is inconsistent with deletional mechanisms. This regulatory tolerance has been attributed
to suppressor cells, and more recently to Th2 cells (6).
However, Th2 cell lines fail to mitigate Th1-mediated target tissue destruction in adoptive transfer experiments in
both NOD mice and in the experimental autoimmune encephalomyelitis model (13, 14), suggesting that a mechanism other than bystander suppression contributes to regulatory tolerance.
The involvement of Th1 and Th2 cells in both the spontaneous autoimmune process and in tolerance states has
been difficult to address in nontransgenic mice, primarily
because of the very low frequency of autoreactive T cells
within the T cell pool. Using an assay capable of characterizing T cells at the single cell level, we examined the natural development of Mice.
NOD (Taconic Farms, Germantown, NY), BALB/c,
and AKR mice (The Jackson Laboratory, Bar Harbor, ME) were
bred under specific pathogen-free conditions. Newborn mice
were treated intraperitoneally on days 1 and 3 with 200 µg of the
indicated antigen in 50% IFA (GIBCO BRL, Gaithersburg, MD).
Only female mice were used in these studies.
Antigens.
Mouse GAD (GAD65), myelin basic protein (MBP),
and control Escherichia coli ELISPOT Analysis.
Splenic T cells were isolated at 4 or 12 wk of age from individual antigen-treated mice as well as unmanipulated mice, and the frequency of antigen-specific T cells secreting IFN- Autoantibody Characterization.
At the time of sacrifice, sera
was collected and the isotype of GAD and insulin autoantibodies
were characterized using an ELISA assay as described in the legend to Fig. 1 and in reference 18. In brief, GAD (Synectics Biomedical, Stockholm) or insulin B chain at 10 µg/ml were bound
to 96-well plates (Nunc), in 0.1 M NaHCO3, pH 8.5 (GAD) or
pH 9.6 (insulin B chain), at 4°C overnight. The wells were rinsed
with PBS and then blocked with 3% BSA in PBS for 1 h. Mouse
sera was added (0.1 ml of a 1:500 dilution) and incubated for 1 h
at 37°C. After washing, bound Ig was characterized using affinity
purified HRP-coupled goat anti-mouse IgG+A+M (H+L) (Pierce Chemical Co., Rockford, IL), or HRP-coupled goat anti- mouse isotype-specific antibodies for IgG1 and IgG2a (Southern Biotechnology Associates, Birmingham, AL) and ABTS. Sera from untreated BALB/c and AKR mice were used as negative controls.
Table 1B.
Intermolecular Spreading of Th2 Responses to cell antigen (
CA) determinants. The
spontaneously primed GAD-reactive T cells secrete IFN-
(1), suggesting that a proinflammatory cascade might drive
disease progression. However, other studies suggest that Th2
cells could be involved in the disease process, e.g., (a) IFN-
-deficient mice still develop insulitis and IDDM (3), (b)
production of IL-10 in the islets accelerates diabetes in
transgenic NOD mice (4), and (c) pathogenic T cell populations express some Th2 cytokines (5). Thus, the characteristics of autoreactive T cell responses during the spontaneous
development of murine IDDM remains an open question.
cell autoimmunity in NOD mice and
the immunological impact of neonatal tolerization to a
CA. In the course of these studies, we observed a new
phenomenon, Th2 determinant spreading, which may be a
fundamental mechanism underlying the efficacy of antigen
based immunotherapies and may explain observations of infectious tolerance.
-galactosidase (
-gal) were purified as
previously described (1, 15). The GAD and heat shock protein
peptide 277 (HSP) peptides have been reported elsewhere (1, 16,
17). Control hen egg white lysozyme (HEL) peptide HEL11-25,
immunogenic in NOD mice, was provided by Eli Sercarz (La
Jolla Institute for Allergy and Immunology, La Jolla, CA). Insulin B
chain and HEL were purchased from Sigma Chemical Co. (St.
Louis, MO).
, IL-4, and IL-5 was determined using a modified
ELISA spot technique (15, 18). In brief, 106 splenic mononuclear
cells were added per well (in triplicate) of an ELISPOT plate
(Athersys, Cleveland, OH) that had been coated with cytokine
capture antibodies and incubated with peptide (20 µM) or whole
protein (100 µM) for 24 h for IFN-
, or for 40 h for IL-4 and
IL-5 detection. After washing, biotinylated detection antibodies
were added and the plates were incubated at 4°C overnight. Bound secondary antibodies were visualized using horseradish
peroxidase (HRP)-streptavidin (DAKO Corp., Carpinteria, CA)
and 3-amino-9-ethylcarbazole. Antibodies R4-6A2/XMG 1.2-biotin, 11B11/BVD6-24G2-biotin, and TRFK5/TRFK4-biotin
(all from PharMingen, San Diego, CA) were used for capture and
detection of IFN-
, IL-4, and IL-5, respectively.
Fig. 1.
Propagation of IgG1 responses to CAs. NOD mice were
treated with HEL, GAD, or insulin B chain, as described in Materials and
Methods. GAD (a) and insulin (b) antibodies were characterized at 12 wk
of age using antigen-specific ELISA assays (18). The background OD was
~0.05 ± 0.01 for all samples. Serial dilutions of sera showed a linear relationship with resulting OD. The data are represented as the mean absorbance values over background of triplicate samples from individual mice.
Experimental and control sera were tested simultaneously in two separate
assays (n = 5 for each group). The variance in absorbance values between
triplicate samples from the two sets of experiments was <8%. Humoral
responses to GAD and insulin in control NOD mice treated with HEL
were similar to those of unmanipulated NOD mice. BALB/c mice treated
with
CAs developed antibodies only against the injected antigen (data
not shown), consistent with the observed lack of Th2 spreading in these
mice (Table 1B). Antibodies to GAD and insulin in sera from untreated
BALB/c and AKR mice were at background levels (data not shown).
[View Larger Version of this Image (13K GIF file)]
CAs
Response to antigens
GAD
HSP
Insulin B-chain
-gal
Strain
Treatment
IL-4
IL-5
IFN-
IL-4
IL-5
IFN-
IL-4
IL-5
IFN-
IL-4
IL-5
IFN-
NOD
none (4 wk)
103
none
365
130
70
HEL
350
105
68
MBP
359
116
64
-gal
303
114
76
71
43
GAD
187
113
164
37
26
67
48
48
46
HSP
76
34
245
109
66
54
47
17
60
Insulin B
65
26
275
25
16
71
137
80
54
BALB/c
HEL
GAD
58
40
Insulin B
68
56
Spreading of Th2 responses to CAs. Mice were intraperitoneally injected neonatally with control HEL11-25 peptide, GADp35, and GADp6, which
constitute determinants (1); GADp11, which does not constitute a determinant; HSPp277; insulin B chain; or whole proteins GAD, MBP, HEL, or
-gal in IFA. T cells from individual spleens were isolated at 12 wk of age (except where indicated) and the frequency of antigen-specific T cells secreting IFN-
, IL-4, and IL-5 was determined by ELISA spot. The data are represented as the mean number of spot-forming colonies 106 splenic T cells
above background. The individual variation within each group was <15%. Most wells without antigen showed no responses, but a background of up
to five spots was observed in a few wells. Variation in the spot-forming cells among triplicate samples was <15%.
, no response over background. Experimental and control mice were tested simultaneously (in triplicate) in two separate experiments. n = 5 for each group.
When unmanipulated NOD mice were tested at the onset
of insulitis (4 wk of age), we detected vigorous IFN-, but
no IL-4 or IL-5 splenic T cell responses to a single determinant of GAD, GAD peptide 35, (hereafter called GADp35)
consistent with a unipolar Th1 response (Table 1A). By
12 wk of age, T cell autoimmunity had spread intramolecularly to additional GAD determinants (GADp6 and
GADp15) and intermolecularly to other
CAs (insulin B
chain and heat shock protein, supporting earlier observations [refs 1, 2]): all of these second wave reactivities were
also purely Th1 in nature (Table 1). Thus, the spontaneously developing autoimmune process is characterized by
the spreading of unipolar Th1 type anti-
CA reactivity.
Conceivably, the first wave of autoreactive Th1 cells, via
secretion of IFN-
and induction of IL-12, creates an environment that favors Th1 cell differentiation, generating a
positive feedback loop of Th1 reactivity and amplifying proinflammatory autoimmune responses to
CAs.
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We hypothesized that Th2 autoimmunity, like Th1 autoimmunity, might also spread, since the IL-4 produced by
Th2 cells is itself a Th2 differentiation factor (19). We
tested this hypothesis by inducing Th2 immunity to a single CA and characterizing the development of T and B
cell responses to unrelated
CAs.
NOD mice were neonatally treated with control antigens or CAs in IFA, a protocol which has recently been
shown to induce vigorous Th2 responses (15). NOD mice
injected with the control antigens mouse MBP, HEL, or
-gal displayed vigorous IL-4 and IL-5, but no IFN-
responses to the injected antigen, indicating the induction of
unipolar Th2 responses (Table 1). Thus, NOD mice are
not generally Th1 biased as has been thought, and can be
manipulated to mount Th2 responses. Notably, induction
of Th2 immunity to these control antigens did not affect
the spontaneous development of Th1-biased anti-
CA responses (Table 1B), nor disease incidence (see below).
Neonatal injection of NOD mice with a single CA peptide (GADp35, which contains the earliest known target determinant; reference 1), induced clear IL-4 and IL-5 responses
not only to the injected peptide, but also to other GAD
peptides (GADp6 and GADp15, which contain later target
determinants), indicating Th2 type intramolecular spreading (Table 1A). Similarly, neonatal treatment with GADp6 led to the spreading of Th2 immunity to GADp35 and
GADp15, indicating that primed Th2 responses can spread
to other autoantigen determinants, independent of the order in which spontaneous autoimmune responses arise to
these determinants (see Fig. 3 in reference 1). Indeed, after
treatment with a GAD peptide, Th2 responses became predominant to the injected peptide, as well as to uninjected
GAD peptides through intramolecular spreading. Furthermore, injection of
CAs individually (GAD, HSPp277, or
insulin B chain), led to the development of Th2 autoimmunity to noninjected
CAs (intermolecular spreading, Table 1B), creating an amplificatory cascade of this antiinflammatory limb.
CA-treated NOD mice failed to respond to nontarget tissue antigens (MBP, HEL or
-gal),
and in other strains of mice, primed Th2 responses to
CAs were restricted to the injected antigen (Table 1 and
data not shown). NOD mouse immune responses to control nontarget tissue antigens (MBP, HEL and
-gal) were
similar in magnitude to those induced by these antigens in
other strains of mice (data not shown) but failed to spread
to
CAs (Table 1). Thus, the spreading of Th2 responses
was limited to target tissue antigens and was dependent
upon a local inflammatory process.
Analysis of humoral responses showed that while unmanipulated and control antigen treated NOD mice had low levels of autoantibodies, GAD-treated animals had elevated autoantibodies to both GAD and insulin (Fig. 1), consistent with the intermolecular spreading of Th2 immunity. Similarly, neonatal treatment with insulin B chain raised the titer of GAD-specific antibodies in addition to the insulin-specific ones. The induced antibodies were of the IgG1 subclass, characteristic of Th2 responses (19). Thus, the spreading of Th2 immunity can lead to the diversification of humoral responses.
Furthermore, the GAD peptide-treated NOD mice had
significantly reduced long-term disease incidence (~30% of
GAD peptide-treated mice versus 96% of controls developed diabetes over 1 yr (P 0.02); Fig. 2). The lack of complete protection by GAD treatment may be due to the
diminution of Th2 responses later in life (11, 18). While
the induction of Th2 responses to some nontarget tissue
antigens has been associated with protection from autoimmune disease (23, 24), the control nontarget tissue antigens
used in this study did not induce Th2 spreading or protection from disease.
Although we induced Th2 immunity before the onset of
insulitis, CA-treated NOD mice still developed
CA-reactive Th1 cells, suggesting that there is an inherent islet
perturbation which promotes Th1 autoimmunity to
CAs.
However, the Th1 responses to
CAs were markedly reduced in
CA-treated animals relative to control groups
(Table 1). Despite the presence of significant Th1 responses,
CA (but not control) -treated mice displayed almost no
splenic T cell proliferative responses to the injected antigen and had greatly reduced proliferative responses to other
CAs (data not shown). As the autoantibody responses in
CA-treated mice were predominantly of the Th2 type
and the mice were protected from disease, it appears that
Th2 immunity can be functionally dominant and a potent
regulator of pathogenic Th1 activity.
In summary, we have shown in unmanipulated NOD
mice that the anti- cell response starts and spreads as a
pure Th1 type autoimmunity, suggesting that a Th1 cascade underlies disease progression. Induction of Th2 autoimmunity to a single
CA resulted in the spreading of
Th2 type T cell and antibody responses to other
CAs in
an infectious manner. These data suggest that both Th1 and
Th2 autoimmunity evolve in amplificatory cascades defined
by site-specific, but not antigen-specific, positive feedback
circuits, which may be generated by the cytokine milieu
(24) or induced changes in accessory molecule expression
(25). Th2 type determinant spreading introduces a
novel mechanism for Th2-mediated protection, providing an explanation for the infectious nature of tolerance and
why different autoantigens can be successfully used for immune therapy. Conversely, in Th2-mediated diseases, Th2
determinant spreading may contribute to the disease process, perhaps accounting for observations that in allergic
conditions, individuals gradually become sensitized to an increasing number of antigens. Thus, these findings may provide a new theoretical framework for understanding disease
progression in autoimmune and allergic conditions, as well
as for therapeutic intervention.
Address correspondence to Dr. Daniel L. Kaufman, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Rm 23-167 CHS, University of California, Los Angeles, Los Angeles, CA 90095-1735. Phone: 310-794-9664; FAX: 310-825-6267; E-mail: dkaufman{at}pharm.medsch.ucla.edu
Received for publication 25 July 1997 and in revised form 12 September 1997.
This work was supported by grants from the National Institutes of Health (Bethesda, MD), the Juvenile Diabetes Foundation International, and the Riva Foundation.
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