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Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting CD4+ and CD8+ T cells in
response to insulin B chain-specific peptides compared with untreated
mice. In contrast, treatment with pDNAs encoding insulin A chain-IgGFc
and IL-4 elicited a low frequency of IL-4-secreting Th cells and had no
effect on the progression of IDDM. Vaccination with pDNAs encoding
GAD65-IgGFc and IL-4, however, prevented IDDM. These results
demonstrate that insulin- and GAD65-specific T cell reactivity induced
by pDNA vaccination has distinct effects on the progression of
IDDM. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One general strategy to suppress ongoing autoimmunity is the induction
of Ag-specific regulatory Th cells. Under the appropriate conditions,
regulatory Th cells can be elicited in an Ag-specific manner that
secrete anti-inflammatory cytokines such as IL-4, IL-10, and/or TGF-
(6, 7). Once established, these regulatory Th cells
traffic to the appropriate tissue(s) and suppress the differentiation
and effector function of pathogenic T cells independent of autoantigen
specificity (8, 9, 10). A number of factors, including the
mode and route of immunization, dose of Ag, and use of adjuvant have an
impact on the immunotherapeutic efficacy of Ag-specific immune
deviation (11, 12, 13, 14). The most important and obvious factor,
however, is the autoantigen used to target the corresponding T cell
population. Studies in nonobese diabetic (NOD) mice, a model for IDDM,
have shown that not all autoantigens effectively mediate immune
deviation, especially after cell autoimmunity has been established
(15, 16). In part, this may reflect the relative size of
the pool of uncommitted Th cell precursors specific for an autoantigen,
which give rise to established Th1 or Th2 effector cells
(17). For example, if this frequency is low, the number of
regulatory Th cells induced may not be sufficient to modulate disease
progression. Furthermore, if the frequency of established T effector
cells is high, there is the possibility that immunization with
autoantigen may expand the pathogenic population and exacerbate
disease. Accordingly, the size of the pool of uncommitted self-specific
Th cells found in the periphery will be influenced by selection events
ongoing in the thymus, the relative immunogenicity and tissue
distribution of the autoantigen, and the stage of disease progression
(17, 18, 19, 20, 21).
We have been investigating the use of plasmid DNAs (pDNAs) as an
approach to mediate Ag-specific immune deviation for the prevention and
treatment of IDDM. pDNA vaccines have a number of properties which are
amenable for clinical use (22). Indeed, clinical trials
are ongoing to determine the immunotherapeutic efficacy of pDNA
vaccination to prevent various infectious diseases and cancers.
Furthermore, various studies have demonstrated that administration of
pDNAs encoding autoantigen (23, 24, 25, 26), antiinflammatory
cytokines (27, 28), or chemokines (29) can
effectively prevent disease in different models of autoimmunity.
Recently, we demonstrated that pDNA encoding IL-4 and a fragment of the
cell autoantigen glutamic acid decarboxylase 65 (GAD65) fused to an
IgFc can effectively induce regulatory Th2 cells and prevent the
differentiation of pathogenic Th1 effector cells in NOD mice
(30). Consequently, diabetes could be prevented at either
early or late preclinical stages of IDDM. To gain further insight into
the application of pDNA vaccination to mediate Ag-specific immune
deviation, we investigated the immunotherapeutic efficacy of pDNAs
encoding insulin. Insulin has been shown to be a critical cell
protein targeted by the diabetogenic response (31).
Furthermore, a number of reports have shown that IDDM can be prevented
by treating young NOD mice with insulin or insulin B chain administered
orally (32), intranasally (33, 34), or s.c.
when prepared in IFA (35). These studies have provided the
rational for ongoing clinical trials to test the efficacy of insulin
administration to prevent diabetes in high risk individuals. Here, we
demonstrate that despite using conditions that effectively induced
GAD65-specific Th2 cells, immunization with pDNA encoding an insulin B
chain-IgFc fusion protein elicited insulin B chain-specific
CD4+ Th1 and CD8+ Tc1 cells
and accelerated the progression of IDDM in NOD mice. In contrast,
immunization with pDNA encoding insulin A chain-IgGFc and IL-4 induced
Th2 cell reactivity but had no significant effect on the onset or
frequency IDDM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of pDNAs encoding GAD65-IgGFc (JwGAD65), hen egg lysozyme (HEL)-IgGFc (JwHEL), and IL-4 (JwIL4) have previously been described (30). To establish recombinants encoding insulin, cDNAs encoding either the full length murine insulin A or B chains were subcloned into the signal pIg vector (R&D Systems, Minneapolis, MN) which contains a human CD33 signal sequence, and genomic DNA consisting of the hinge, CH2, and CH3 exons derived from human IgG4. The recombinants were then subcloned into the Jw4303 vector, which contains a transcriptional unit composed of a CMV promoter/enhancer element, and polyadenylation and transcriptional termination sequences derived from the bovine growth hormone gene (36). To test expression and secretion of the insulin A chain-IgGFc and insulin B chain-IgGFc fusion proteins, COS7 cells were transfected with JwInsA and JwInsB, respectively, using Lipofectamine (Life Technologies, Gaithersburg, MD) as recommended by the manufacturer. Culture supernatants were harvested 2 days after transfection. The IgGFc fusion proteins were immunoprecipitated via protein G-Sepharose (Pharmacia, Piscataway, NJ), resolved on SDS-PAGE, and analyzed by Western blot using a mouse anti-human IgG-HRP conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA).
Mice
NOD/Lt mice were housed and bred under specific pathogen-free
conditions and fed NIH diet 31A (Purina, St. Louis, MO). Currently,
IDDM develops in 
80% of female NOD/Lt mice by 1 year of
age.
Assessment of diabetes and insulitis
Mice were monitored weekly for the development of glycosuria with Diastix (Ames, Elkhart, IN). Glycosuric values of >3 for two successive measurements was considered diagnostic of diabetes onset. Insulitis was assessed by histology. Pancreases were prepared for histology by fixing in neutral buffered formalin and then embedding in paraffin. The fixed blocks were sectioned and stained with hematoxylin and eosin. A minimum of five sections, each differing by 90 µm, were cut for each block, and slides viewed by light microscopy. A minimum of 30 individual islets was scored for each animal. The severity of insulitis was scored as either periinsulitis (islets surrounded by a few lymphocytes) or intrainsulitis (lymphocytic infiltration into the interior of the islets).
Immunizations
pDNA was prepared from DH5
Escherichia coli using
a Qiagen endotoxin-free kit (Qiagen, Chatsworth, CA) and resuspended at
1.0 mg/ml in PBS. Female NOD mice 4 wk of age received three i.m.
injections during 21 days of 50 µl (50 µg) pDNA in each
quadricep.
Antigens
The cloning and preparation of murine GAD65 have previously been described (37). Briefly, cDNA encoding murine GAD65 was engineered to encode six histidine residues at the C terminus of the protein. Recombinant GAD65 was expressed in a baculovirus expression system and purified using a Ni2+-conjugated resin (Qiagen). An additional purification step involved preparative SDS-PAGE, after which recombinant GAD65 was electroeluted and dialyzed extensively against PBS. Insulin A and B peptides were synthesized by using standard fluorenylmethoxycarbonyl chemistry on a Ranin Symphony at the Peptide Synthesis facility at the University of North Carolina (Chapel Hill, NC). The purity of the peptides was verified by reversed phase HPLC and mass spectroscopic analysis.
ELISPOT
ImmunoSpot M200 plates (Cellular Technology, Cleveland,
OH) were coated overnight at 4°C with either 2 µg/ml
anti-IFN- Ab (R4-6A2; BD PharMingen, San Diego, CA) or 4 µg/ml
anti-IL-4 Ab (11B11; PharMingen) prepared in PBS. Plates were
blocked with 1% BSA-PBS for a minimum of 2 h at room temperature
and then washed four times with PBS. Spleen cells were prepared as
described (30). Briefly, a spleen cell suspension was
prepared from individual mice in ice cold PBS. The spleen cell
suspension was immediately centrifuged at 400 x g for
5 min at 4°C and resuspended at 2.5 x 106
cells/ml in HL-1 medium (BioWhittaker, Walkersville, MD). Splenocytes
were then plated at 5 x 105/well (0.2
ml/well). Pancreatic lymph nodes were pooled within a given treatment
group, and the resulting suspension was prepared in HL-1 medium and
plated at 2.5 x 105/well with 5 x
105/well irradiated (3000 rad) splenocytes
harvested from NOD.IL4null mice.
CD4+ and CD8+ T cells were
purified from the spleen of individual mice via positive selection
using the OCTO-MACS system (Miltenyi Biotec, Auburn, CA). T cells were
resuspended in HL-1 medium and plated at 1 x
105/well with 5 x
105/well irradiated splenocytes from
NOD.IL4null mice. In all assays, Ag was added to
triplicate wells at a final concentration of 20 µg/ml. The plates
were incubated for either 24 (IFN-) or 48 (IL-4) h at 37°C in
5.5% CO2 and then washed three times with PBS
followed by an additional three washes with 0.025% Tween-PBS.
Biotinylated anti-IFN- (XMG1.2; BD PharMingen) or anti-IL-4
(BVD6-24G2; BD PharMingen) were added at 2 and 4 µg/ml, respectively,
in 1% BSA-PBS (0.1 ml/well). After overnight incubation at 4°C,
plates were washed three times with 0.025% Tween-PBS, and
streptavidin-HRP (BD PharMingen) was added at 1/2000 dilution for
2 h at room temperature. This was followed by three washes with
0.025% Tween-PBS and three washes with PBS only. Development solution
consisted of 0.8 ml 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO; 20
mg dissolved in 2.0 ml dimethylformamide) added to 24 ml 0.1 M sodium
acetate (pH 5.0) plus 0.12 ml 3.0% hydrogen peroxide; 0.2 ml was added
per well.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently demonstrated that overt diabetes could be prevented in
NOD mice treated with pDNAs encoding a secreted GAD65-IgGFc fusion
protein (JwGAD65) and IL-4 (JwIL4) before or after the establishment of
cell autoimmunity (30). Because studies have reported
that IDDM is prevented in NOD mice immunized at a young age with
insulin or insulin B chain peptide (32, 33, 34, 35), we
investigated whether pDNA encoding insulin could also mediate
protection. Specifically, pDNAs were engineered to encode secreted full
length murine insulin A (JwInsA) or B (JwInsB) chains fused to an
IgGFc. Expression of the recombinants was confirmed by detection of the
fusion proteins in culture supernatants harvested from COS7 cells
transfected with either JwInsA or JwInsB (Fig. 1
). Groups of 10 female NOD mice 4 wk of
age were left untreated or received three i.m. injections of JwIL4 with
either JwInsA, JwInsB, JwGAD65, or the control pDNA JwHEL which encodes
a HEL-IgGFc fusion protein. An additional group of NOD mice received
only JwInsB. As reported earlier, vaccination with JwGAD65 and JwIL4
significantly reduced the frequency of diabetes (1 of 10,
p < 10-3,
2) relative to untreated animals (10 of 10)
(Fig. 2). Furthermore, treatment with
JwInsA and JwIL4 or JwHEL and JwIL4 had no significant effect on
disease progression (Fig. 2). In contrast, administration of JwInsB
proved to be diabetogenic. The entire group of NOD mice receiving
JwInsB developed diabetes with an accelerated time of onset
(p = 0.006; Mann-Whitney rank sum test)
compared with untreated NOD mice (Fig. 2). Surprisingly, an accelerated
time of onset of diabetes (p = 0.005,
Mann-Whitney rank sum test) was also detected in NOD mice coimmunized
with JwInsB and JwIL4 relative to untreated animals (Fig. 2).
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). In contrast, NOD mice treated with
JwInsB alone or both JwInsB and JwIL4 exhibited a significant and
progressive increase in the frequency of intrainsulitis compared with
untreated animals (Fig. 3). In contrast, the majority of islets in NOD
mice receiving JwGAD65 and JwIL4 remained free of infiltration at both
8 and 12 wk of age (Fig. 3).
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T cell responses in NOD mice vaccinated with pDNA were
characterized via an ELISPOT assay (38) to gain insight
into the diabetogenic capacity of JwInsB and the apparent lack of an
immunotherapeutic effect after JwInsA administration. Spleen and
pancreatic lymph node cultures were prepared from 8- and 12-wk-old
female NOD mice immunized with the panel of pDNAs at 4 wk of age, and
the frequency of IFN- and IL-4 secreting T cells in response to
insulin A and B chains and GAD65 was measured. The pancreatic lymph
nodes are believed to be a key site for the initial activation of
cell-specific T cells (39, 40). A Th1-like cytokine
profile, characterized by IFN- and no IL-4 secretion in response to
the panel of cell autoantigens, was detected in spleen and
pancreatic lymph node cultures established from untreated NOD mice 8
(Fig. 4) and 12 (Fig. 5) wk of age.
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)- and 12 (Fig. 5)-wk-old NOD mice treated with JwGAD65 and JwIL4.
Similarly, spleen and pancreatic lymph node cultures established from
NOD mice treated with either JwInsA and JwIL4 or JwInsB and JwIL4
exhibited an increased frequency of IL-4 secreting T cells in response
to insulin A chain and insulin B chain, respectively, compared with
untreated animals (Figs. 4 and 5). In contrast, no IL-4-secreting T
cells in response to GAD65 and insulin were detected in spleen and
pancreatic lymph cultures prepared from either JwHEL and JwIL4 or
JwInsB-only immunized NOD mice (Figs. 4 and 5).
Although treatment with the GAD65 and insulin encoding pDNAs plus JwIL4
elicited Th2 cell reactivity specific for the respective cell
autoantigens, marked differences in the relative frequency of
IL-4-secreting T cells were evident between the treatment groups. In
general, a higher frequency of IL-4-secreting T cells was detected in
spleen and pancreatic lymph node cultures prepared from NOD mice
treated with JwGAD65 and JwIL4 vs JwInsA and JwIL4 or JwInsB and JwIL4
in response to the corresponding cell autoantigens (Figs. 4 and 5).
For example, the frequency of GAD65-specific IL-4-secreting T cells
detected in pancreatic lymph cultures prepared from 12-wk-old NOD mice
treated with JwGAD65 and JwIL4 was 5.2-fold greater than the frequency
of insulin A chain-specific Th2 cells observed in JwInsA and JwIL4
immunized animals (Fig. 5). The difference was even greater (7.4-fold)
when compared with the frequency of insulin B chain-specific IL-4
secreting T cells detected in cultures prepared from NOD mice receiving
JwInsB and JwIL4 (Fig. 5).
The general cytokine profile in response to the panel of cell
autoantigens also differed significantly between the treatment groups.
In addition to an increased frequency of GAD65-specific Th2 cell
reactivity, JwGAD65 and JwIL4 treatment elicited IL-4-secreting T cells
in response to insulin A and B chains in both spleen and pancreatic
lymph node cultures (Figs. 4 and 5). Furthermore, the frequency of
IFN--secreting T cells in response to GAD65 and insulin A and B
chains was also markedly reduced in these cultures relative to
untreated NOD mice (Figs. 4 and 5). In contrast, spleen and pancreatic
lymph node cultures prepared from JwInsA- and JwIL4- immunized NOD mice
lacked IL-4-secreting T cells in response to insulin B chain or GAD65,
and Th1 cell reactivity specific for the panel of cell autoantigens
was not significantly altered compared with untreated animals (Figs. 4 and 5). A third type of cytokine profile was detected in spleen and
pancreatic lymph node cultures prepared from JwInsB- and
JwIL4-immunized NOD mice. Here, no difference in the frequency of
IL-4-secreting T cells in response to insulin A chain and GAD65 was
detected compared with responses observed in untreated NOD mice (Figs. 4 and 5). A significant increase in the frequency of IFN--secreting
T cells in response to insulin B chain was detected. In fact, this
frequency was similar to that detected in cultures prepared from NOD
mice treated with JwInsB only (Figs. 4 and 5).
Immunization with JwInsB stimulates insulin B chain-specific CD4+ and CD8+ T cells
To further define the T cell response induced by JwInsB
immunization, CD4+ and CD8+
T cells were purified from the spleens of individual NOD mice
vaccinated with JwInsB, JwInsB, and JwIL4 or left untreated. Included
in the analysis were T cells purified from NOD mice immunized with
JwGAD65 and JwIL4. The frequencies of IFN-- and IL-4-secreting T
cells in response to the I-Ag7- and
H-2Kd-restricted insulin-specific epitopes
B9–23 (34) and
B15–23 (41), respectively, and
GAD65 were then examined via ELISPOT. Consistent with the above
results, a significant increase in IL-4 and a marked decrease in
IFN--secreting T cells in response to GAD65 were detected in
cultures containing CD4+ T cells isolated from
JwGAD65- and JwIL4-immunized NOD mice relative to untreated animals
(Fig. 6). CD8+ T
cell reactivity to intact GAD65 was not observed. A significant
frequency of both CD4+ and
CD8+ T cells secreting IFN- in response to the
respective insulin B chain-specific peptides was detected in cultures
established from NOD mice immunized with JwInsB alone or with JwInsB
and JwIL4 (Fig. 6). As seen above, a low frequency of IL-4-secreting
CD4+ T cells was also detected in cultures
prepared from animals immunized with JwInsB and JwIL4, but not with
JwInsB alone. Earlier work has demonstrated that some insulin B
chain-specific CD4+ T cell clones recognized a
minimal epitope consisting of B13–23
(42). The observed CD4+ T cell
reactivity to the H-Kd-restricted
B15–23 peptide likely reflects recognition of
this minimal epitope (Fig. 6). Conversely, CD8+ T
cell reactivity to B9–23 was likely due to
degradation of the peptide in culture resulting in some
B15–23 that was bound by
H-2Kd (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), which correlated with the inhibition
of insulitis (Fig. 3) and the development of overt diabetes (Fig. 2).
In marked contrast, administration of JwInsB with or without JwIL4
significantly enhanced insulin B chain-specific
CD4+ Th1 and CD8+ Tc1 cell
reactivity (
Figs. 4–6), which coincided with an accelerated
progression of insulitis (Fig. 3) and an early onset of overt diabetes
(Fig. 2). In contrast, immunization with JwInsA and JwIL4 had no
significant effect on the progression of IDDM (Figs. 2 and 3).
The frequency of Th2 cells elicited by pDNA vaccination appears to be a
key factor in determining protection. For example, coimmunization with
JwInsA and JwIL4 induced an insulin A chain-specific Th2 response
(Figs. 4 and 5), yet had no significant effect on either insulitis or
diabetes (Figs. 2 and 3). Accordingly, the frequency of IL-4-secreting
T cells specific for insulin A-chain detected in the spleen and
pancreatic lymph nodes was markedly lower than that for GAD65-specific
Th2 cells induced by JwGAD65 and JwIL4 treatment (Figs. 4 and 5).
Importantly, immunization with JwGAD65 and JwIL4 effectively mediated
bystander suppression of cell-specific Th1 reactivity (
Figs. 4–6),
in addition to intermolecular determinant spread of Th2 reactivity
(
Figs. 4–6). The latter was evident by detection of IL-4 secreting Th2
cells in response to insulin A and B chain in the pancreatic lymph
nodes (Figs. 4 and 5). In contrast, JwInsA and JwIL4 treatment failed
to mediate either set of events (Figs. 4 and 5). The relatively low
frequency of insulin A chain-specific Th2 cells induced by JwInsA and
JwIL4 treatment may in part reflect a low frequency of uncommitted T
cell precursors. Our finding that the frequency of established Th1
effector cells specific for insulin A-chain is significantly lower than
for insulin B chain- and GAD65-specific Th1 cells in untreated NOD mice
(Fig. 4) is consistent with this notion. The relative immunogenicity of
insulin A chain-IgFc would also be expected to influence the magnitude
of the Th2 cell response.
The failure of JwInsB and JwIL4 coimmunization to prevent IDDM was
unexpected. In this study and in previous work, JwIL4 effectively
promoted Th2 cell reactivity when coadministered with JwGAD65 (
Figs. 4–6). IL-4-secreting T cells in response to insulin B chain were
detected in spleen and pancreatic lymph node cultures prepared from
JwInsB and JwIL4 immunized animals (Figs. 4 and 5). However, this
frequency was reduced compared with the frequency of insulin A-chain-
and GAD65-specific IL-4-secreting Th cells elicited by JwInsA and JwIL4
and by JwGAD65 and JwIL4 treatment, respectively (Figs. 4 and 5). One
explanation for the skewed expansion of Th1 and Tc1 effector cells by
JwInsB despite the presence of JwIL4 is that the two pDNAs were
differentially taken up in vivo. The majority of pDNA prepared in
saline is released systemically after i.m. injection and as a result is
taken up by a variety of cell types and tissues (43).
Furthermore, pDNA prepared in saline generally mediates Th1 cell
reactivity in vivo in the absence of appropriate cytokines
(44). Therefore, preferential uptake of JwInsB would
result in localized expression of insulin B chain-IgFc under conditions
that promote Th1 cell development, especially if IL-4 expression was
limiting. However, we believe that this explanation is unlikely in view
of our observation that significant Th1 cell reactivity was detected in
NOD mice immunized with a pDNA encoding both insulin B chain-IgGFc and
IL-4 (R. Tisch, unpublished data). An alternative possibility is that
immunization with JwInsB and JwIL4 led primarily to the expansion of
established insulin B chain-specific Th1 and Tc1 effector cells.
Indeed, a recent study demonstrated that a high frequency of
B15–23-specific CD8+ T
cells can be detected in the pancreas of young NOD mice
(41). IL-4 expressed by JwIL4 would have a minimal effect
on the differentiation status of committed Th1 and Tc1 cells in vivo
(45, 46). In fact, IL-4 and IL-12 together has been
reported to enhance Th1 cell proliferation in vitro (47).
The systemic release and subsequent uptake of JwInsB and JwIL4 by
different cells and tissues may result in broad expression of insulin B
chain-IgGFc. This in turn would increase the likelihood of Ag encounter
with pools of established insulin B chain-specific Th1cells, found for
instance in the pancreatic lymph nodes. Interestingly, the frequency of
insulin B chain-specific Th1 cells was 2-fold greater than
GAD65-specific Th1 cells in the pancreatic lymph nodes of untreated NOD
mice (Figs. 4 and 5). Consistent with the above hypothesis is the
observation that s.c. but not systemic administration via i.p.
injection of insulin B chain or B9–23 peptide
prepared in IFA prevented diabetes in NOD mice (48).
Recently, it was demonstrated that immunization with pDNA encoding
lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) failed to
prevent diabetes in BALB/c mice expressing LCMV-NP in the pancreas
after LCMV infection (25). However, administration of pDNA
encoding porcine insulin B chain was protective. This group concluded
that the lack of protection by LCMV-NP pDNA immunization was also due
to a high frequency of established NP-specific Th1/Tc1effector cells,
whereas a sufficient frequency of uncommitted insulin B chain T cells
must have been present (25). The IgGFc fragment may also
impact on the immunogenicity of insulin B chain. For example, flanking
sequences found in the IgGFc molecule may influence the efficiency of
processing and presentation events and/or the fine specificity of
insulin B chain epitopes leading to enhanced Th1 and/or Tc1 cell
stimulation (49). In addition, secreted insulin B
chain-IgGFc may preferentially be taken up by specific types of APCs,
such as B cells which are known to have a critical role in initiating
cell-specific T cell reactivity (50, 51).
The observation that JwInsB alone had no protective effect (Figs. 2 and 3) is in agreement with our earlier findings that immunization with
JwGAD65 alone also failed to prevent diabetes in NOD mice
(30). In both instances, significant Th1 cell reactivity
specific for the respective autoantigens was induced (Figs. 4 and 5)
(30). However, immunization with JwInsB accelerated the
progression of insulitis and the development of overt diabetes (Figs. 2 and 3). This contrasts with our earlier observation that IDDM was not
significantly enhanced after treatment with JwGAD65 alone
(30). The intrinsic adjuvant effect associated with the
two pDNAs may differ, which could influence the diabetogenicity of the
respective recombinants. For example, the capacity of pDNA to mediate
Th1 cell reactivity has been correlated with the frequency of CpG
motifs found in the vector (52, 53). In this instance,
however, both JwInsB and JwGAD65 contain two CpG motifs. The difference
in diabetogenicity seen between JwInsB and JwGAD65 may reflect the
respective role(s) of these two cell autoantigens in IDDM. For
example, the diabetogenicity of JwInsB is consistent with previous work
demonstrating that insulin B chain-specific Th1 cell clones exacerbate
IDDM in NOD recipient mice (31). In contrast, adoptive
transfer of GAD65-specific Th1 cell clones resulted in an increased
frequency of intrainsulitis in young NOD recipients, but did not
accelerate the onset of diabetes (R. Tisch, unpublished results).
Nevertheless, our results indicate that modulating T cell
reactivity specific for insulin B chain and GAD65 can significantly
impact disease progression, further supporting the notion that these
two autoantigens are key targets early in the autoimmune response.
Currently, we are examining whether the route of immunization and/or
the structure of the recombinant protein influence the
diabetogenicity associated with JwInsB (and JwIL4).
In conclusion, i.m. pDNA vaccination has revealed that significant differences exist between insulin and GAD65 in terms of mediating and immunoregulating the progression of autoimmune diabetes in NOD mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Roland Tisch, Department of Microbiology and Immunology, Mary Ellen Jones Building, Room 804, Campus Box 7290, University of North Carolina, Chapel Hill NC 27599-7290. E-mail address: rmtisch{at}med.unc.edu
3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; GAD65, glutamic acid decarboxylase 65; HEL, hen egg lysozyme; pDNA, plasmid DNA; JwGAD65, pDNA encoding GAD65-IgGFc; JwHEL, pDNA encoding HEL-IgGFc; JwIL4, pDNA encoding IL-4; NOD, nonobese diabetic; LCMV-NP, lymphocytic choriomeningitis virus nucleoprotein.
Received for publication March 6, 2001. Accepted for publication April 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/-transgenic model. J. Exp. Med. 182:1579.1 somatic gene therapy prevents autoimmune disease in nonobese diabetic mice. J. Immunol. 161:3950. cell autoimmunity by plasmid DNA vaccination. J. Immunol. 166:2122.
T cells that prevent murine insulin dependent diabetes. J. Exp. Med. 184:2167. transcription. J. Clin. Invest. 95:628.
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G. Han, Y. Li, J. Wang, R. Wang, G. Chen, L. Song, R. Xu, M. Yu, X. Wu, J. Qian, et al. Active Tolerance Induction and Prevention of Autoimmune Diabetes by Immunogene Therapy Using Recombinant Adenoassociated Virus Expressing Glutamic Acid Decarboxylase 65 Peptide GAD500-585 J. Immunol., April 15, 2005; 174(8): 4516 - 4524. [Abstract] [Full Text] [PDF]
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P. Y. Arnold, A. R. Burton, and D. A. A. Vignali Diabetes Incidence Is Unaltered in Glutamate Decarboxylase 65-Specific TCR Retrogenic Nonobese Diabetic Mice: Generation by Retroviral-Mediated Stem Cell Gene Transfer J. Immunol., September 1, 2004; 173(5): 3103 - 3111. [Abstract] [Full Text] [PDF]
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C. Seifarth, S. Pop, B. Liu, C. P. Wong, and R. Tisch More Stringent Conditions of Plasmid DNA Vaccination Are Required to Protect Grafted Versus Endogenous Islets in Nonobese Diabetic Mice J. Immunol., July 1, 2003; 171(1): 469 - 476. [Abstract] [Full Text] [PDF]
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W. Karges, K. Pechhold, S. Al Dahouk, I. Riegger, M. Rief, A. Wissmann, R. Schirmbeck, C. Barth, and B. O. Boehm Induction of Autoimmune Diabetes Through Insulin (but Not GAD65) DNA Vaccination in Nonobese Diabetic and in RIP-B7.1 Mice Diabetes, November 1, 2002; 51(11): 3237 - 3244. [Abstract] [Full Text] [PDF]
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P. Halbout, J.-P. Briand, C. Becourt, S. Muller, and C. Boitard T Cell Response to Preproinsulin I and II in the Nonobese Diabetic Mouse J. Immunol., September 1, 2002; 169(5): 2436 - 2443. [Abstract] [Full Text] [PDF]
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 K. V. Tarbell, M. Lee, E. Ranheim, C. C. Chao, M. Sanna, S.-K. Kim, P. Dickie, L. Teyton, M. Davis, and H. McDevitt CD4+ T Cells from Glutamic Acid Decarboxylase (GAD)65-specific T Cell Receptor Transgenic Mice Are Not Diabetogenic and Can Delay Diabetes Transfer J. Exp. Med., August 19, 2002; 196(4): 481 - 492. [Abstract] [Full Text] [PDF]
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 F. J. Baker, M. Lee, Y.-h. Chien, and M. M. Davis Restricted islet-cell reactive T cell repertoire of early pancreatic islet infiltrates in NOD mice PNAS, July 9, 2002; 99(14): 9374 - 9379. [Abstract] [Full Text] [PDF]
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), periinsulitis (
), and intrainsulitis
(
). The number of islets counted for each treatment group were:
untreated (8 wk, 255; 12 wk, 159); JwHEL plus JwIL4 (8 wk, 240; 12
wk,189); JwInsA plus JwIL4 (8 wk, 216; 12 wk, 155); JwInsB (8 wk, 195;
12 wk, 177); JwInsB plus JwIL4 (8 wk,188; 12 wk,162); and JwGAD65 plus
JwIL4 (8 wk, 271; 12 wk, 178). The results are representative of two
independent experiments. *, p <
10-3 vs untreated NOD mice as determined by
