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*
Department of Immunology, Alliance Pharmaceutical, San Diego, CA 92121; and
Departments of Immunology and Neuropharmacology, Division of Virology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting T cells in pancreatic lymph nodes, as well as
intermolecular Th2 epitope spreading to glutamic acid
decarboxylase determinants. A critical role of IL-4 for the
Ag-specific protective effect triggered by plasmid administration was
revealed in female IL-4-/- NOD mice that developed
diabetes and higher Th1 responses. Coadministration of IL-4-expressing
plasmid or extension of the vaccination schedule corrected the
unfavorable response of male NOD mice to DNA vaccination with InsB
chain. Thus, plasmid-mediated expression of the InsB chain early in
diabetes-prone mice has the potential to prevent transition to
full-blown disease depending on the presence of
IL-4. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ag-based immune prophylaxis of IDDM has the potential to prevent progression to full-blown disease and avoid hormone replacement therapy. An ideal target for immune modulation would be an Ag common for humans and NOD mice, because the proof of concept can be addressed easier. Such an Ag is the insulin B (InsB) chain, recognized by T cells from DQ8+ humans predisposed to disease or with full-blown IDDM (6).
Ag-based therapeutic regimens may act via different mechanisms: 1)
deletion/anergy of pathogenic T cells; 2) induction of regulatory cells
that mediate bystander suppression; or 3) when initiated during the
prediabetic stage, redirection of T cell precursors toward
nonpathogenic cells. The significance of the Th2 shift noted in many
cases of protection from IDDM is still under debate. For example, in
the case of regimens inducing deletion/anergy of pathogenic cells, the
resulting Th2-biased response may reflect just a higher tendency for
Th1 cells for activation-induced cell death (7). Also,
based on observations that protective treatments are associated with a
Th (cytokine) switch toward Th2 (IL-4), Tr1 (IL-10), and/or Th3
(TGF-
) (8), it is not always clear whether such
modifications are causal or secondary. Indeed, some previous studies
documented protective effects of Th1 cells (9) and
detrimental effects of Th2 cells (10) in IDDM.
Intriguingly, the protective effect mediated by a plasmid expressing a
dominant myelin basic protein epitope in an experimental
autoimmune encephalomyelitis (EAE) model was dependent on the presence
of T1-driving CpG motifs (11). This was in apparent
discrepancy with a more recent report that suggested a critical role
for IL-4 in the protection against IDDM conferred by a glutamic acid
decarboxylase (GAD)-expressing plasmid (12). Thus,
there likely are multiple mechanisms that can restore the normal immune
homeostasis, depending on model, stage, as well as therapeutic
strategy.
Here, we investigated whether plasmid vaccination of NOD mice with an InsB-expressing vector would result in protection from IDDM. We reasoned that initiation of vaccination during the neonatal window may result in either anergy/deletion of specific autoreactive T cells in context of a poor expression of costimulatory molecules (13), or in induction of nonpathogenic/regulatory cells of Th2 profile (14). We found that plasmid vaccination of female NOD mice redirected the profile of autoreactive T cells and protected from IDDM. The Th2-associated cytokine IL-4 was critical for the protection, rather than being just a secondary outcome. Furthermore, coadministration of IL-4-expressing plasmid or extension of the vaccination schedule corrected the lack of protection of male NOD mice by plasmid vaccination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The plasmid expressing the InsB chain (pInsB) was described previously (15) and the pGAD plasmid was a generous gift of Dr. D. Kaufman (University of California, Los Angeles, CA). As control, we used pCMV plasmid devoid of Ag open reading frame. The plasmids were produced in Escherichia coli and purified using EndoFree purification kits (Qiagen, Valencia, CA).
We used the following custom-synthesized peptides: GAD-65 Ag7-restricted dominant epitopes 78–97 KPCNCPKGDVNYAFLHATDL, 217–236 EYVTLKKMREIIGWP GGSGD, 539–558 EYGTTMVSYQPLGDKVNFFR, and the InsB-derived peptides 9–23 SHLVEALYLVCGERG and 15–23 LYLVCGERG.
All Abs were purchased from BD PharMingen (San Diego, CA). For the
ELISPOT analysis, we used the mouse anti-cytokine mAbs recommended
by the manufacturer: capture rat anti-mouse IFN- clone R4-6A2
(catalog no. 18181D) and anti-mouse IL-4 clone BVD4-1D11 (catalog
no. 18031D); and detection biotinylated rat anti-mouse IFN-
clone XMG1.2 (catalog no. 18112D) and anti-mouse IL-4 clone
BVD6-24G2 (catalog no. 18042D), respectively. As stimulating Abs in
bioassays, we used anti-mouse CD3
and anti-mouse CD28 mAbs
purchased from the same manufacturer.
Mice and immunization
NOD/LtJ breeder mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in specific pathogen-free conditions. The rate of diabetes in the colony was 75–90% (females at 30 wk of age). The IL-4-deficient NOD mice were obtained using a speed-congenic approach described previously (16). The naive IL-4-/- x NOD female mice developed diabetes similar to IL-4-competent NOD female mice (16, 17).
The mice were immunized at the age of 1 wk into the gluteal muscle and later into the quadriceps muscle, bilaterally, with 100 µg of plasmid in 100 µl of normal saline. Mice were considered diabetic if they displayed at least two consecutively measured blood-glucose levels of 400 mg/dl.
T cell assay
The analysis of T cell responsiveness was conducted by ELISPOT using responder cells isolated from spleen and pancreatic lymph nodes (PLNs). The ELISPOT plates (Millipore, Molsheim, France) were coated with anti-cytokine Abs, blocked, and the responder cells were incubated (range from 2 x 104 to 5 x 105/well) in HL-1 medium, with or without stimulator cells (2 x 105/well) and 20 µg/ml of peptides (InsB 9-23, 15-23, or a mixture of GAD peptides). In the case of splenocytes, we used an initial 5-day stimulation with peptides, followed by a 3-day stimulation with rIL-2 (10 U/ml) and a 3-day stimulation with mitomycin-treated feeder cells plus peptides. In the case of PLN cells, we used a 5-day stimulation protocol in the presence of peptide-pulsed feeder cells and 10 U/ml of rIL-2 during the last 48 h. Alternatively, we used 72-h polyclonal stimulation with a mixture of anti-CD3 + anti-CD28 mAbs (5 + 1 µg/ml). The reaction was developed using detecting Abs (2 µg/ml) followed by streptavidin-HRP and 3-amino-9-ethylcarbazole substrate (Sigma, St. Louis, MO). The data were acquired using an automated system (Navitar, Rochester, NY) with ImagePro-Plus software (Media Cybernetics, Silver Spring, MD).
The cytokine production by islet-infiltrating cells was measured after overnight incubation of islets, using ELISA kits (BioSource International, Camarillo, CA and R&D Systems, Minneapolis, MN). For the assay, the islets were obtained by digestion with collagenase (Sigma) and Ficoll gradient centrifugation.
Statistical analysis
The comparison among disease profiles in the context of various treatment regimens was conducted by log-rank analysis. Values of p < 0.05 of the log-rank test were considered statistically significant, relative to the effect on kinetics of disease.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the InsB chain is a major potential target for Ag-based
immune prophylaxis of IDDM, we tested whether immunization with pInsB
initiated at day 7 after birth and repeated during the preclinical
stage (wk 4 and 8) affected the kinetics of disease in NOD mice. As
shown in Fig. 1
A, pInsB
delayed the development and suppressed the rate of IDDM in female NOD
mice (p of log-rank test
<0.005). The control plasmid and pGAD did not significantly alter the kinetics of disease (>p
> 0.05; Fig. 1A). The exclusion of the priming step on day
7 precluded suppression of IDDM by inoculation of pInsB (data not
shown).
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, B and
C). The total number of IFN-- and, particularly,
IL-4-producing SFC was higher in PLN from mice treated with pInsB, as
opposed to controls. In addition, polyclonal stimulation of PLN T cells
from pInsB-immunized mice had a limited additional effect on the number
of IL-4 relative to IFN- SFC (Fig. 1, B and
C), suggesting that most of the IL-4-producing T cells in
the PLN from protected mice are active. However, whereas the total
number of polyclonally stimulated IL-4 SFC was significantly higher (2
orders of magnitude) in pInsB-immunized mice, the number of IFN- SFC
was in the same range compared with that of naive controls. Treatment
with pGAD failed to similarly modify the number of SFC in the PLN (Fig. 1, B and C), resembling the administration of
mock plasmid (data not shown). Impact of vaccination with pInsB on the cytokine profile of Ag-specific T cells and islet-infiltrating lymphocytes
We next assessed whether plasmid vaccination persistently modified
the cytokine profile of InsB and GAD-specific T cells in PLN and
spleen. Short-term incubation of PLN T cells from 30-wk-old naive mice
with peptide-pulsed APC did not result in substantial generation of
IL-4 and IFN- SFC (Fig. 2, A and B). However, using a similar protocol of
stimulation, the number of both IFN-- and IL-4-producing
InsB-specific colonies was greatly enhanced in pInsB-vaccinated NOD
mice that were free of disease by wk 30. Immunization with pGAD failed
to reproduce a similar expansion in the pool of PLN SFC (Fig. 2, A and B), although it increased to a certain
extent the reactivity to both InsB and GAD.
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in
PLNs from pInsB-immunized NOD mice, there was a clear shift to T2
immunity in the spleen. We compared the cytokine profiles of SFC
generated ex vivo by stimulation with dominant InsB or major GAD
epitopes. As shown in Fig. 2D, the mice vaccinated with
pInsB and protected against IDDM displayed enhanced frequency of
IL-4-producing SFC generated with either GAD or InsB peptides. In
addition, the frequency of IFN--producing SFC generated with InsB
peptide was lower than that of naive NOD mice (Fig. 2C). In
contrast, pGAD vaccination did not result in the expansion of
IL-4-producing SFC (Fig. 2D). Conversely, we measured a
substantial increase in the frequency and total number of
IFN--producing SFC obtained with InsB and GAD peptides in
pGAD-immunized NOD mice (Fig. 2C). Collectively, the data
generated using splenocytes show opposite effects of plasmid
vaccination with InsB chain and GAD-65: Th2-biased profile in the first
case and a pattern dominated by T1 immunity in the latter.
Intermolecular epitope spreading was noted in both pInsB- and
pGAD-immunized mice (Fig. 2, C and D).
The effect of pInsB vaccination on the activity of islet-infiltrating T
cells was assessed by measuring in vitro cytokine production. Despite
its ability to prevent IDDM, plasmid vaccination did not preclude
peripheral infiltration of the islets (data not shown). Compared with
age-matched naive mice or mice injected with mock plasmid, the
islet-infiltrating cells from 12-wk-old pInsB-vaccinated mice produced
significantly less IL-1, but more IL-4 (Fig. 3). In addition, there was a trend for
decreased IFN- and increased TGF- production by cells from
pInsB-vaccinated mice. The cytokine profile of islet-infiltrating cells
from pInsB-treated mice was somewhat similar to that of naive NOD mice
that remained free of disease by the age of 30 wk (Fig. 3). No
detectable IL-10 production was measured (data not shown), arguing
against the induction of Tr1 cells by plasmid vaccination.
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IL-4 is required for protection from IDDM conferred by pInsB vaccination
Despite the fact that bystander suppression was documented in various protocols of peptide or protein-based immune therapy (18), use of plasmid expression vectors for a similar purpose (11, 12) is intriguing because unmethylated CpG motifs on bacterial DNA are strong Th1 inducers (19). Various mechanisms were previously proposed to explain the efficacy of plasmid vaccination in a transgenic model of autoimmune diabetes (15) and models of EAE (11, 20), ranging from bystander suppression, to anergy or Th1 cytokine-mediated protection.
Because the association of increased IL-4 production and protection from IDDM supports but does not demonstrate a causal role for IL-4, we sought to address this question by using recently described IL-4-/- x NOD mice (16). If expansion of IL-4-producing T cells is just an epiphenomenon, then pInsB should protect the IL-4-defective NOD mice against IDDM.
Culture of splenocytes from naive IL-4-/- NOD
mice, with or without polyclonal stimulation, confirmed the lack of
IL-4 production and showed an increased frequency of IFN--producing
SFC (Fig. 4, A and
B). This was expected due to the role of IL-4 as negative
regulator of T1 immunity (21). RT-PCR analysis showed that
sterile IL-4 transcripts were still produced in
IL-4-/- mice (data not shown), indicating that
the generation of Th2-like cells associated with specific chromatin
remodeling and opening of specific loci (22) was not
completely prevented.
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C;
p > 0.05). Furthermore, we showed that the number of
IFN- SFC obtained by stimulation of splenocytes with InsB and GAD
peptides was increased in the IL-4-/- mice, as
compared with IL-4-competent mice that developed IDDM (Fig. 4D) or were protected from disease (Fig. 2D). In
contrast to the pInsB-vaccinated NOD mice protected from IDDM, the
diabetic mice showed significantly decreased numbers of IL-4 but not
IFN--producing SFC in PLN (Figs. 2 and 4). Thus, vaccination with
pInsB of IL-4-deficient NOD mice failed to prevent IDDM and to
substantially change the T cell profile in spleen and PLN. Coadministration of IL-4 or expansion of the vaccination schedule ameliorates the response of male NOD mice to InsB-expressing plasmid
Because the male NOD mice develop disease with a lower rate and
delayed kinetics compared with females, we assessed whether pInsB
vaccination had an impact on disease in males. Interestingly, in
contrast to female NOD mice, the vaccination of males failed to
significantly influence the kinetics of IDDM (Fig. 5A; p of log-rank
test 0.05) or to shift the cytokine profile toward T2 immunity (data
not shown). We reasoned that this failure of pInsB vaccination in males
may be due to either one (or both) of the following factors: 1) an
ineffective exposure (timewise) of insulin-specific T cells to self-Ag
expressed by plasmid; or 2) a defect of male NOD mice to mount T2
immunity to InsB.
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A; p of
log-rank test
<0.05 in two independent experiments). Alternatively, coadministration of IL-4-expressing plasmid and pInsB resulted in significant suppression of disease (Fig. 5>A
similar to coadministration of IL-4). Interestingly, this is
reminiscent of a recently published study, that while confirming the
inability of GAD-expressing plasmid to suppress IDDM in female NOD
mice, showed that plasmid vaccination with an IgG-GAD chimeric
construct together with IL-4 resulted in significant disease
suppression (12). Profound effects of plasmid vaccination
were noted in the PLN of protected males (Fig. 5B). Although
administration of pInsB using an expanded schedule resulted in
substantial increase in the pool of IL-4-producing T cells (4-fold) and
IFN--producing T cells (2-fold), coadministration of IL-4- and
InsB-expressing plasmids resulted in diminution of the number of
cytokine-producing T cells in PLN. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We showed that protection against disease by plasmid vaccination with
the InsB chain of female NOD mice was associated with a substantial
increase in the total number of cytokine-producing T cells in PLN
(Figs. 1 and 2). The enhancement in the overall number of
IL-4-producing T cells was not paralleled by a reduction of the
IFN--producing T cell pool in the PLN from protected animals.
However, as revealed by the estimation of cytokine-producing cells in
various conditions of in vitro stimulation, a large percentage of
IL-4-producing cells were active in situ. Thus, the mere ex vivo
incubation of PLN T cells with APC was able to render substantial IL-4
production, with no additional effect of polyclonal activation (Fig. 1C). In contrast, a substantial percentage of
IFN--committed T cells in PLN from mice vaccinated with pInsB were
quiescent, because they required strong polyclonal activation to expand
in vitro (Fig. 1B). Together, our data support a T2-biased
profile in the PLN of pInsB-vaccinated female NOD mice protected
against disease. Vaccination with GAD-expressing plasmid failed to
trigger a similar expansion of T2 cells in PLN (Figs. 1C and
2B).
The effect of pInsB vaccination on the splenic T cell profile was more
clear-cut (Fig. 2, C and D). Collectively, the
data generated using splenocytes show opposite effects of plasmid
vaccination with InsB chain and GAD-65: Th2-biased profile in the first
case and a pattern dominated by T1 immunity in the latter.
Intermolecular epitope spreading was noted in both pInsB- and
pGAD-immunized mice. In addition, paralleling the pInsB-driven
modification of the T cell profile in secondary lymphoid organs, there
was a change in the cytokine pattern of islet-infiltrating cells,
consisting in down-regulation of proinflammatory and up-regulation of
suppressor cytokines (Fig. 3).
In our model, bacterial plasmid devoid of Ag-open reading frame lacked any effect on the kinetics of disease. In addition, underlining the Ag dependency, plasmids expressing different self-Ags displayed differential outcomes in terms of specific T cell responses.
Based on the T cell profile data, we tested whether IL-4 was an
essential cytokine participating in the protection conferred by
pInsB plasmid. We demonstrated that IL-4-defective NOD mice could not
be protected against diabetes by plasmid vaccination (Fig. 4). This
result points toward IL-4 as a central mediator of pInsB-triggered
protection from IDDM. This interpretation is also supported by the
intermolecular spreading of Th2 immunity toward GAD determinants in
pInsB-immunized mice (Fig. 2, C and D). Such a
phenomenon may be of paramount importance in deactivating
autoaggressive cells against multiple Ags (23). In
addition, a bystander/regulatory effect may explain the modification
noted in the profile of cytokines produced by islet-infiltrating
lymphocytes (Fig. 3). Conversely, pGAD-immunized mice displayed
Th1-biased immunity and epitope spreading toward the InsB determinant
(Fig. 2). This was associated with lack of protection against disease
and may be due to earlier recruitment of GAD determinants in the
autoimmune process (24) resulting in refractoriness of
GAD-specific T cells to modulation. The requirement for early priming
with pInsB for the protective effect to occur supports the model that
timing of Ag recruitment to the autoimmune process is an important
parameter. It is a likely possibility that IL-4 produced by Th2 cells
primed during neonatal window (25) may inhibit the
expansion of Th1 and CTL (26) or modulate the activity of
APC in the PLN and pancreas (27). This may explain the
limited induction of autoaggressive T cells recognizing a dominant, MHC
class I-restricted epitope on the InsB chain (28) in
pInsB-vaccinated IL-4-competent but not IL-4-deficient NOD mice (Fig. 4E).
Previous studies showed that in contrast to adult vaccination, neonatal
DNA immunization triggers both Th2 and Th1 cells (25)
despite the Th1 driving effect of CpG motifs. Similar to studies
addressing the mechanism of peptide-based immune modulation in
IL-4-deficient (29) or STAT-6-defective mice
(30), as well as to a recent study addressing the response
to a GAD peptide-expressing plasmid (12), our findings
underline a crucial role for the Th2-derived cytokine IL-4 in the
protection from IDDM. However, our model likely involves different
effector mechanisms than those described in a recent report
demonstrating a requirement for Th1-inducing factors (11)
and lack of bystander suppression (31) in the protection
against EAE after vaccination with an myelin basic protein-expressing
plasmid. Further emphasizing the pleiotropism of protective mechanisms,
various immunostimulatory protocols of preventing diabetes in NOD mice
depend to different extents on IL-4 (16, 32). Finally, the
dependency of protection on IL-4 in this model is not concordant
with a potential role for IL-10-producing CD8+
T
cells, usually triggered by mucosal vaccination
(33).
A similar vaccination schedule with pInsB was not effective in the case
of male NOD mice. However, extension of vaccination schedule or
addition of IL-4-expressing plasmid to the inoculum provided
significant protection against disease (Fig. 5). The outcome on the T
cell profile was different as compared with that measured in
pInsB-vaccinated female NOD mice. Thus, coadministration of IL-4 is not
merely shifting the profile from T1 to T2 but has a more profound
impact on the overall peripheral repertoire, consistent with an effect
on APC (27). Because neonatal inoculation of plasmid is
associated with substantially shorter persistence of transgene
(34), the prolongation of vaccination schedule may
optimize exposure of emerging self-reactive T cells to InsB chain in
circumstances when the intrinsic disease kinetics is slower, as in the
case of male NOD mice.
In summary, our study demonstrates the efficacy of InsB, an Ag common for DQ8+ humans and NOD mice (6, 35), in the prophylaxis of IDDM conducted by plasmid inoculation. The current data extend our previous results from a transgenic model (15) to a spontaneous disease model involving multiple epitopes (NOD mice) and underline the importance of the following factors in the success of plasmid-based immune modulation: 1) the nature of Ag, 2) timing of vaccination, 3) regulatory cytokines, and 3) gender (in a broader context, the heterogeneity regarding disease-kinetics and pathogenesis). Because the 9-23 peptide of the InsB chain is a dominant epitope in humans at risk to develop IDDM (6), plasmid vaccination with this Ag may be of clinical interest. The addition of IL-4 may improve the efficacy of Ag-based immunotherapy with plasmid vectors. Finally, in contrast to Ags recruited earlier in the autoimmune process (i.e., GAD), immune prophylaxis with InsB chain may be safer and more effective.
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Acknowledgments |
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Footnotes |
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2 Current address: Targeted Molecules Corp., San Diego, CA 92121.
3 Address correspondence and reprint requests to Dr. Matthias G. von Herrath, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: matthias{at}scripps.edu
4 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; InsB, insulin B; pInsB, plasmid expressing the InsB chain; NOD, nonobese diabetic; PLN, pancreatic lymph node; GAD, glutamic acid decarboxylase; EAE, experimental autoimmune encephalomyelitis; SFC, spot-forming colony.
Received for publication May 2, 2001. Accepted for publication June 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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islet-cell reactive T cell clones that suppress autoimmune diabetes in nonobese diabetic mice. J. Exp. Med. 182:187.
cell autoimmunity by plasmid DNA vaccination. J. Immunol. 166:2122.. Proc. Natl. Acad. Sci. USA 93:2879. cells. Science 284:1183. T cells as mediators of mucosal tolerance: the autoimmune diabetes model. Immunol. Rev. 173:109.[Medline]
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