Download Makes the Difference One-Amino Acid Change in the B Chain

Insulin in Oral Immune ''Tolerance'': A
One-Amino Acid Change in the B Chain
Makes the Difference
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of June 16, 2017.
Dirk Homann, Thomas Dyrberg, Jacob Petersen, Michael B.
A. Oldstone and Matthias G. von Herrath
J Immunol 1999; 163:1833-1838; ;
http://www.jimmunol.org/content/163/4/1833
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Copyright © 1999 by The American Association of
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References
Insulin in Oral Immune “Tolerance”: A One-Amino Acid
Change in the B Chain Makes the Difference1
Dirk Homann, 2* Thomas Dyrberg,2† Jacob Petersen,† Michael B. A. Oldstone,*
and Matthias G. von Herrath3*
T
ype 1 diabetes is considered a T cell-mediated autoimmune disease that results from specific destruction of insulin-producing b cells residing in the pancreatic islets of
Langerhans. Pathogenetically, MHC genes, aberrant immune responses, and possibly viruses have been implicated (1); however,
the precise nature of self-Ags involved in the initiation of the autoimmune process remains largely unknown. Currently, no cure or
effective prevention is available, and despite treatment with insulin, long term complications are frequent. Oral administration of
self (islet) Ags has been shown to reduce diabetes in various animal models (2, 3), and trials using human insulin orally or intranasally have been initiated. However, the precise mechanism(s)
and antigenic requirements are not completely understood, and no
certain predictions about the efficacy of an orally administered Ag
are available. These issues need further exploration, especially
since human trials using oral self-Ags are ongoing or planned and
are the focus of this article.
For this study we used two different models for type 1 diabetes.
Diabetes in the nonobese diabetic (NOD)4 mouse is genetically
*Department of Neuropharmacology, Division of Virology, The Scripps Research
Institute, La Jolla, CA 92037; and †Novo Nordisk, Bagsvaerd, Denmark
Received for publication October 29, 1998. Accepted for publication June 3, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by joint grants from the National Institutes of Health
(JDF/DK 995005 to M.B.A.O. and M.G.v.H.). M.G.v.H. was also supported by a
Career Development Award from the Juvenile Diabetes Foundation International
(Grant 296120) and National Institutes of Health Grants DK51091 and AI44451. D.H.
was supported by National Institutes of Health Training Grant AG-00080 and a grant
by the Studienstiftung des Deutschen Volkes. This is publication No. 11341-NP from
the Department of Neuropharmacology, Division of Virology, The Scripps Research
Institute.
2
D.H. and T.D. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Matthias G. von Herrath, Department of Neuropharmacology, Division of Virology, The Scripps Research Institute, La Jolla, CA 92037. E-mail address: [email protected]
4
Abbreviations used in this paper: NOD, nonobese diabetic; RIP, rat insulin promoter; LCMV, lymphocytic choriomeningitis virus; NP, nucleoprotein; ARM, LCMV
Armstrong strain; pCTL, precursor CTL; GP, guinea pig.
Copyright © 1999 by The American Association of Immunologists
predetermined, is linked to the MHC class II complex (IAg7), and
develops within 3–9 mo in most females (4). RIP-NP transgenic
mice develop autoimmune diabetes upon infection with lymphocytic choriomeningitis virus (LCMV) (5, 6). In this model the immune response against the virus also targets b cells expressing the
nucleoprotein (NP) of LCMV as a transgene. In both models
mononuclear cell infiltration of islets (requiring islet Ag or viral
NP-specific CD81 CTL as well as CD41 T cells) eventually leads
to destruction of b cells, resulting in cessation of insulin production and hyperglycemia.
Successful prevention of autoimmune diabetes has been reported by oral insulin administration in NOD and RIP-NP mice (7,
8). A likely mechanism for oral tolerance has been termed bystander suppression (2, 3) and relies on the induction of regulatory
cells in the gut specific for the orally administered self Ag. During
the prediabetic phase (preceding spontaneous onset in NOD mice
and following virus infection in RIP-NP mice), initial b cell destruction leads to release of various self Ags, including insulin,
which can be presented by resident APCs. After migration to the
diseased organ, regulatory cells could be activated, and by secretion of immunosuppressive cytokines (IL-4, IL-10, and TGF-b) (2,
3) lead to suppression of the ongoing autoimmune response to an
unrelated self Ag, e.g., the viral NP expressed as a transgene in the
islets of the RIP-NP mice. Evidence suggests that immune regulation and bystander suppression occur with intermediate oral Ag
doses via the induction of insulin B chain-specific T cells, whereas
deletion of Ag-reactive lymphocytes is detected at high dosages (2,
9 –10). Therefore, the term “oral tolerance” may, but not necessarily does, signify the deletion of specific lymphocytes (2, 3).
In this report we have delineated several crucial aspects of oral
Ag therapy for the prevention of type 1 diabetes. Protection occurs
via bystander suppression, thus circumventing the need for identification of the initiating autoantigen(s). However, the fed Ag has
to be specific for the target cell under destruction (pancreatic b
cells). Hormonal activity of insulin, potentially causing a b cell
rest, is not required. Most importantly, minute differences in Ag
composition, such as a 1-aa change in the immunogenic insulin B
chain, can abrogate the protective effect.
0022-1767/99/$02.00
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Oral administration of self-Ags can dampen or prevent autoimmune processes by induction of bystander suppression. Based on
encouraging results from experiments in nonobese diabetic (NOD) mice, clinical trials have been initiated in type 1 diabetes using
human insulin as an oral Ag. However, neither the precise antigenic requirements nor the mechanism of bystander suppression
are currently understood in detail. Here we report that 1) a 1-aa difference in position 30 of the insulin B chain abrogated the
ability of insulin to confer protection in both NOD as well as a virus-induced transgenic mouse model for type 1 diabetes. In the
latter model transgenic mice express the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) under the control of
the rat insulin promotor (RIP) in the pancreatic b cells and develop diabetes only following LCMV infection; and 2) protection
could be transferred with insulin B chain-restimulated but not LCMV-restimulated splenocytes from RIP-NP transgenic mice,
demonstrating that the mechanism of diabetes prevention in the RIP-NP model is mediated by insulin B chain-specific, IL-4producing regulatory cells acting as bystander suppressors. The Journal of Immunology, 1999, 163: 1833–1838.
1834
INSULIN IN ORAL IMMUNE TOLERANCE
Table I. Insulin sequence alignmenta
Insulin
Sequence
A-Chains
1
Porcine
Human
Human Analog
Mouse I
Mouse II
G
z
z
z
z
B-Chains
1
Porcine
B30 A3T
B25 F3N, B30 A3T
Mouse I
Mouse II
F
z
z
z
z
5
I
z
z
z
z
V
z
z
z
z
E
z
z
D
D
Q
z
z
z
z
10
C
z
z
z
z
C
z
z
z
z
T
z
z
z
z
S
z
z
z
z
5
V
z
z
z
z
N
z
z
K
K
Q
z
z
z
z
H
z
z
z
z
I C
z z
z z
z z
z z
15
S
z
z
z
z
L
z
z
z
z
Y
z
z
z
z
10
L
z
z
z
z
C
z
z
z
z
G
z
z
z
z
S
z
z
P
z
H L
z z
z z
z z
z z
Q L
z z
z z
z z
z z
20
E
z
z
z
z
N
z
z
z
z
Y
z
z
z
z
15
V
z
z
z
z
E
z
z
z
z
A
z
z
z
z
L Y
z z
z z
z z
z z
C N
z z
z z
z z
z z
20
L
z
z
z
z
V
z
z
z
z
C
z
z
z
z
G E
z z
z z
z z
z z
25
R
z
z
z
z
G
z
z
z
z
F F Y
z z z
z N z
z z z
z z z
30
T P
z z
z z
z z
z z
K
z
z
z
z
A
T
T
S
S
Materials and Methods
Animals
Female NOD mice were obtained from Bommice (Ry, Denmark) at 4 wk
of age and were housed in a specific pathogen-free environment. The average incidence of diabetes in untreated mice housed under identical conditions during the study period was 60 –75% at 40 wk of age. The transgenic RIP-NP 25-3 H-2d line used in this study expressed the NP of LCMV
under control of the rat insulin promoter (RIP) in the pancreatic b cells as
well as in the thymus, but not in any other tissues (5, 6). BALB/c nontransgenic H-2d mice were used for the evaluation of metabolic activity
after feeding of oral Ags and for assessment of CTL precursor frequencies.
The virus used was LCMV Armstrong (ARM) strain (clone 53b). Four to
twenty-one-week-old RIP-NP 25–3 mice were inoculated i.p. with 1 3 105
PFU LCMV ARM in a volume of 0.2 ml to initiate diabetes.
Analysis of blood glucose
NOD mice were screened for diabetes twice a week from 10 wk of age by
testing for glucosuria. Subsequently, diabetes was defined by two consecutive blood glucose analyses (Accucheck III, Boehringer Mannheim, Indianapolis, IN) with values .300 mg/dl. Blood samples from RIP-NP mice
were analyzed biweekly accordingly.
Oral Ags
The human and analogue insulin as well as glucagon were recombinant
proteins; porcine insulin was purified from pancreatic glands, all from
Novo Nordisk (Bagsvaerd, Denmark). All proteins were obtained as dry
crystals from the very last stage of the purification process, immediately
before formulation into the injectable product used for patients. Porcine,
human, and murine (I1II) insulin B chains were also provided by Novo
Nordisk. All insulins were solubilized in acid buffer, the pH was adjusted,
and the solution was stored at 220°C until used. Oral Ag was administered
via a blunt-ended curved feeding tube inserted into the esophagus/stomach.
In a total of three sequential studies NOD mice were fed buffer and human
and porcine insulins from 5 wk of age at doses of 1 mg twice a week. From
10 wk of age the mice were given insulin only once a week. In two of the
studies the animals were followed until 45 wk of age, and in the third they
were followed until 30 wk of age. There was no difference in the diabetes
incidence relative to treatment in the three studies, so the combined data are
presented. RIP-NP mice were fed biweekly with 0.5 ml of an aqueous
solution containing 2 mg/ml Ag. Feeding was started 1 wk before infection
with LCMV and was discontinued after 8 wk. Control groups received
saline or BSA at a concentration of 2 mg/ml.
Cytotoxicity assays
LCMV-specific CTL activity in spleens harvested 7 days after inoculation
with 105 LCMV ARM i.p. was assessed in a standard 4- to 5-h 51Cr release
assay in LCMV-infected and uninfected, MHC-matched (BALB/c17
[H-2d]) and mismatched (MC57 [H-2b]) target cells (11). For determination
of LCMV-specific CTL precursor frequencies (pCTL) 7 days after infection, spleen cells from immunized mice were serial diluted and cultured in
96-well flat-bottom plates (12 wells/dilution; highest dilution, 16,000 cells/
well) with LCMV-infected and irradiated (2,000 rad) macrophages as well
as irradiated spleen feeder cells. After 8 days, cells from each well were
split and tested on LCMV infected and uninfected BALB/c17 targets in a
4- to 5-h 51Cr release assay. The pCTL frequencies were assessed by plotting the fraction of negative cultures on a semilogarithmic scale against the
number of splenocytes per culture; pCTL frequencies are defined by the
slope of the linear regression among at least three separate data points.
Adoptive transfers
Splenocytes from diabetic or protected porcine insulin-fed RIP-NP mice
were cultured for 3 days in the presence of 100 mg/ml porcine insulin B
chain or 1025 M of the immunodominant, MHC class I-restricted, LCMV
NP (aa 118 –126). Supernatants were analyzed for IFN-g and IL-4 as described previously (11), and 5 3 106 cells were transferred i.p. into nonirradiated, prediabetic (day 5 after LCMV) RIP-NP recipients.
Results
A single amino acid difference in the insulin B chain abrogates
its protective effect to induce “oral tolerance”
To define the precise sequence (structural) requirements for oral
Ags, we investigated several different pancreatic hormones (Table
I and Figs. 1 and 2). Oral administration of porcine insulin significantly reduced the development of diabetes in both NOD (Fig. 1)
and RIP-NP (Fig. 2) mice. In marked contrast to an earlier report
using NOD mice (12), oral treatment with human insulin, which
differs by a single amino acid in position 30 of the B chain from
porcine insulin (Table I), did not result in protection from diabetes
in either NOD or RIP-NP mice. Protection was associated with
peri-islitis in the absence of MHC class I up-regulation, while diabetic mice showed profound islet infiltration by CD8 and CD4 T
cells as well as up-regulation of MHC class I and II (data not
shown) (7).
Hormonal activity of insulin is not required for oral tolerance
induction
We further explored whether hormonal activity was required for
“oral tolerance” induction. Prophylactic insulin treatment of individuals at risk for type 1 diabetes has been explored as a means to
prevent or delay the onset of disease (13, 14), and the protective
effect in this situation has been hypothesized to be mediated
through induction of a “b cell rest”, making b cells less sensitive
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a
Sequence alignment of porcine, human, analog, and mouse insulins. Differences between experimental insulins affect the carboxy-terminal end of the B-chain. Human insulin
differs from porcine insulin by one amino acid (Ala B303 Thr). Metabolically inactive analog insulin has been engineered by substitution of one amino acid in human insulin
(Phe B253 Asn). In addition, analog insulin preparation did not contain zinc required for insulin homodimer and homo hexamer formation. All three insulins differ by several
amino acids (3–5) from mouse insulin I or II.
The Journal of Immunology
1835
FIGURE 1. Cumulative incidences
of diabetes in oral Ag-treated NOD
mice. Biweekly administration of 1 mg
of oral Ag was started at 5 wk of age
(NOD). Protection from diabetes was
observed only in porcine insulin-treated
mice. PBS treatment, n 5 90; porcine
insulin treatment, n 5 79; human insulin (B30A3 T) treatment, n 5 52 (combined results from three different experiments, data corrected for death).
Target cell specificity is required for effective oral tolerogens
It has been hypothesized that organ specificity of the oral Ag may
be sufficient for successful tolerance induction. More specifically,
if “oral tolerance” is to occur via bystander suppression as outlined
above, target cell specificity should be a necessary condition. In
human type 1 diabetes, as in our two animal models, b cell destruction is specific, and glucagon-producing a cells in the immediate vicinity remain largely untouched. To study whether other
islet-derived Ags derived from non-b islet cells can induce oral
tolerance we fed RIP-NP mice recombinant glucagon. This treatment did not influence the development of diabetes, indicating that
bystander suppression does not occur if the orally administered Ag
is not derived from the target cell (Fig. 2). However, the lack of
immunogenicity or the absence of a glucagon-specific T cell repertoire could also account for the failure to induce protection.
Oral Ag treatment does not affect the systemic immune response
We have previously shown that protection from diabetes after oral
administration of porcine insulin in RIP-NP mice is associated
with abrogation of virus (transgene)-specific CTL activity in the
pancreas, but not in the spleen. As expected, none of the orally
administered Ags used in the present study affected the systemic
generation of LCMV-specific CTL as determined by CTL activity,
lytic units, and precursor frequencies (Table II). Thus, protection
does not occur via systemic deletion of NP (self)-specific CTL. As
expected (7), pancreatic NP-specific CTL activity was only observed in diabetic RIP-NP, not protected mice (data not shown).
Protection from type 1 diabetes occurs via bystander
suppression
We have previously demonstrated, by comparison of cytokine profiles in protected and diabetic RIP-NP mice, that bystander suppression is the likely mechanism for protection after oral insulin
administration (7). We now show that protection can be adoptively
transferred. Although ex vivo transfers of splenocytes from protected mice did not abrogate the development of diabetes (data not
shown), short term in vitro stimulation with insulin B chain, but
not the MHC class I-restricted immunodominant LCMV NP (aa
FIGURE 2. Cumulative incidences
of diabetes in oral Ag-treated RIP-NP
mice. Biweekly administration of 1 mg
oral Ag was started 1 wk before i.p. immunization with 1 3 105 PFU LCMV
ARM and was continued until 8 wk after immunization. Sixty-five percent of
porcine insulin-treated mice were protected from diabetes, while no protection was observed in buffer/BSAtreated mice (diabetes incidence, 94%),
human insulin-treated mice (92%), analogue insulin (B30A3 T, B25F3 N)treated mice (91%), or glucagon-treated
mice (89%). No new cases of diabetes
were recorded 6 wk after infection.
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to immune destruction. We find that oral administration of porcine
and human insulin to fasted mice resulted in acute, but transient,
blood glucose reduction (Fig. 3). Statistically significant alterations in blood glucose only occurred 10 min after feeding, and
blood glucose returned to normal levels in all mice after they were
left to feed freely. Although these observations could suggest a
metabolic component in oral tolerance and thus lend support to the
b cell rest hypothesis, both porcine and human insulin had similar
metabolic effects, but only porcine insulin was protective. Therefore, b cell rest is not the mechanism by which oral insulin can
prevent diabetes.
1836
118 –126), led to significant production of IL-4 and diabetes protection after subsequent adoptive transfer into prediabetic RIP-NP
mice (Table III). Transfer of cells cultured in the absence of Ag
also failed to prevent diabetes (data not shown). It is of interest to
note that splenic CTL activity was not affected by the presence of
regulatory cells (Table II), presumably due to low precursor frequencies of B chain-specific cells in the spleen, which also may
account for the failure of unstimulated cells to confer protection
after transfer. Nevertheless, a 3-day stimulation of splenocytes derived from protected, but not diabetic, donors proved sufficient to
activate and probably expand the regulatory cell population.
Discussion
“Oral tolerance” induction provides an attractive experimental
concept for the prevention of autoimmune diseases. Although bystander suppression as well as deletion and anergy of autoreactive
cells have been proposed as potential mechanisms, a better understanding of the characteristics of “oral tolerogens” is also needed.
The emphasis of the present study lies on an analysis of antigenic
requirements for oral Ag treatment of autoimmune diabetes as well
as bystander suppression as the potential mechanism for diabetes
protection. By using oral Ags that differ from the initiating autoantigens in two models for type 1 diabetes (unknown in NOD
mice, viral transgene in RIP-NP mice), we assessed the efficacy of
various islet cell proteins to confer protection from spontaneous
(NOD) and virus-induced (RIP-NP) diabetes. Our data clearly
demonstrate that a single amino acid change (B30A3 T) can abrogate the protective effect after oral administration. Previous studies using porcine, equine, bovine, and ovine insulin, which differ
from porcine insulin by one, two, and three amino acids, respectively, in the A, but not the B, chain, have also been shown to
confer diabetes protection in NOD mice after oral (porcine,
equine) (8, 12, 15) or i.v. (bovine, ovine) (16) administration. Further, s.c. administration of the metabolically inactive B chain (17)
or B9-23 (18), but not of the A chain (17), in IFA was also protective in NOD mice. Although tolerance induction after parenteral
insulin administration is likely to have a mechanism different from
that of mucosal tolerance induction, the oral B chain administration has also been associated with a shift from Th1 to Th2 cytokines and diabetes protection in the NOD cotransfer model (10).
Furthermore, porcine, but neither human nor mouse, B chains were
protective in RIP-NP mice (M. von Herrath, unpublished observations). The single amino acid difference in the present study did
not affect the insulin epitope B9-23 (18), indicating that flanking
sequences might affect processing of the B chain before presentation by the NOD MHC (DbKdIAg7) as discussed below.
The idea that small structural differences in the primary sequence can produce dramatic differences in the clinical outcome
are supported by data from a different autoimmune model. Studies
in experimental autoimmune encephalitis demonstrated a preventive effect after a single amino acid change in the immunogenic
myelin basic protein peptide (19). Furthermore, experimental autoimmune encephalitis protection induced in Lewis rats with
guinea pig (GP) MBP68 – 88 or rat MBP68 – 88 was observed after
feeding of GP68 – 88, but not rat 68 – 88, which differs from GP68 – 88
by one amino acid (Ser683 Thr) (20). It should be noted, however,
that these studies used related peptides for both tolerance and autoimmune disease induction, while the present study used Ags (insulin) different from the initiating autoantigens. Our present findings stand in contrast to those from studies in NOD mice
demonstrating a protective effect of human insulin after oral (12)
or aerosol (21) administration in NOD mice. Other studies using
oral human insulin treatment of NOD (22, 23) mice, while not
having investigated the prevention of spontaneous onset diabetes,
have reported the generation of regulatory cells that, when cotransferred with diabetogenic cells, protected against autoimmune diabetes. In our NOD colony, cotransfers were much more effective
using porcine than human insulin-induced regulatory cells (T. Dyrberg, unpublished observations). These discrepancies may be related to differences in colonies, feeding protocols, or preparation of
the Ag, but a clear explanation remains elusive, since a direct
comparison based on experimental evidence is and will be difficult
to achieve. However, given these considerations, probably very
subtle differences can greatly influence the outcome of oral Ag
therapy, a fact that should be taken into account for human
applications.
Our observation of a temporary decrease in blood glucose was
observed only in mice fasted before insulin administration. Furthermore, significant blood glucose reductions were found only 10
min after feeding. Later measurements were not significantly different from initial blood glucose values and are in agreement with
other reports that have not found such effects on blood glucose (8,
12). More importantly, however, standard techniques for oral Ag
administration in mice may result in uptake of unprocessed Ag.
Because both porcine and human insulin had an intrinsic effect
after feeding, but only porcine insulin protected against diabetes,
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FIGURE 3. Effect of orally administered Ags on blood glucose levels in
fasted mice. Orally administered, intrinsically active Ag partially reaches
the bloodstream in its intact form, as determined by the effect on blood
glucose levels compatible with respective hormonal activity. Five or six
mice per group were fasted for 4 h before administration of 1 mg of Ag and
during the time of the study. Blood glucose levels were determined 10, 20,
40, 60, and 120 min after feeding. Statistically significant alteration of
blood glucose levels with p , 0.035 (by paired t test) were observed 10
min after feeding in all groups except the BSA-treated control group.
INSULIN IN ORAL IMMUNE TOLERANCE
The Journal of Immunology
1837
Table II. Oral Ag treatment does not affect generation of anti-LCMV (“self”) CTLa
Effectors
Splenocytes after
LCMV 7 days
BSA
Porcine insulin
Human insulin
Analog insulin
51
Cr Release from Targets
E:T
H-2d uninfected
H-2d LCMV
H-2b LCMV
Lytic Units
Virus-Specific pCTL
50:1
25:1
12.5:1
6.25:1
50:1
25:1
12.5:1
6.25:1
50:1
25:1
12.5:1
6.25:1
50:1
25:1
12.5:1
6.25:1
50:1
25:1
12.5:1
6.25:1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
67 6 2
40 6 10
25 6 9
20 6 4
65 6 5
43 6 6
27 6 6
17 6 3
54 6 14
31 6 15
22 6 10
10 6 8
46 6 4
33 6 7
24 6 10
866
67 6 4
53 6 8
37 6 11
13 6 9
463
1.30
1/1425 6 53
261
1.28
1/1217 6 174
463
1.00
1/1300 6 495
463
1.00
ND
864
1.35
1/1425 6 194
a
BALB/c (H-2d) mice were treated orally with porcine, human, and analog insulin or glucagon as described. All mice were infected with 1 3 105 PFU LCMV, and 7 days
later spleens were removed to test for CTL activity and determine LCMV-specific precursor frequencies (pCTL). Target cells were syngeneic (H-2d) or allogeneic (H-2b)
fibroblasts infected with LCMV (MOI 5 1) or uninfected. All samples were run in triplicate, displayed are mean 6 1 SE. Lytic units were defined as the reciprocal of the number
of 3104 effector cells required for 25% lysis of 104 targets determined by correlating four different E:T ratios with the respective 51Cr release values. Precursor frequencies are
shown as CTL precursors per number of splenocytes.
we conclude that hormonal activity is not necessary for oral tolerance induction. Therefore, our observation that oral administration of an insulin analogue with 100-fold lower activity receptor
affinity (B25F3 N) had no protective effect (Fig. 1) is probably not
related to its reduced hormonal activity, but, rather, to its similarity
to human insulin, in particular the threonin in position B30. This
finding does not necessarily stand in contrast to a recent study that
has demonstrated a protective effect of this analogue in NOD mice
after s.c. administration (24), because different mechanisms might
apply depending on the administration route.
Further analysis of antigenic requirements indicates that tolerizing Ags need to be target cell specific. Oral administration of
glucagon, produced by pancreatic a cells that are not subject to
autoimmune destruction in type 1 diabetes, is not protective. Here,
lack of local glucagon peptide presentation may have precluded
activation of glucagon-specific regulatory cells. Furthermore, it
cannot be excluded that such regulatory cells were not generated
by glucagon feeding. Nonetheless, studies using other b cell Ags,
such as glutamate decarboxylase have support the notion that the
orally (25) or nasally (26) administered Ag has to be derived from
the target cell under immunological attack.
Transfer of protection by B chain-stimulated, but not NP-stimulated, splenocytes derived from protected mice establishes bystander suppression as the mechanism for oral tolerance induced
by feeding of intermediate dosages of Ag. The clear distinction
between the specificity of autoaggressive CTL (viral NP expressed
as transgene in b cells) and IL-4-producing regulatory cells induced by oral insulin (insulin B chain) makes the RIP-NP model
the only diabetes model to date in which bystander suppression
could be unequivocally demonstrated. The reasons for incomplete
protection after oral porcine insulin are not clear, but may lie in the
generation of regulatory cells at different precursor frequencies.
We have recently generated insulin-specific cell lines that exhibit
a Th2-like cytokine profile and prevent type 1 diabetes when infused into prediabetic RIP-NP mice and thus allow this hypothesis
to be tested (D. Homann, unpublished observations).
In summary, our study points to the importance of oral Ag selection for treatment of human autoimmune disease. Protection via
Table III. Adoptive transfer of regulatory lymphocytes prevents type 1 diabetes in RIP-NP transgenic micea
Donor Splenocytes
Stimulation with
Protected RIP-NP mouse
Porcine B-chain
Protected RIP-NP mouse
LCMV NP118–126
Diabetic RIP-NP mouse
Porcine B-chain
Diabetic RIP-NP mouse
LCMV NP118–126
Cytokine Production (ng/100 ml)
IL-4: 0.25 6 0.1
IFN-g: 0.21 6 0.1
IL-4: ,0.05
IFN-g: 1.1 6 0.32
IL-4: ,0.05
IFN-g: 0.3 6 0.11
IL-4: ,0.05
IFN-g: 0.9 6 0.20
Adoptive Transfer Recipient
RIP-NP mouse
LCMV
RIP-NP mouse
LCMV
RIP-NP mouse
LCMV
RIP-NP mouse
LCMV
Type 1 Diabetes Incidence
After Transfer
day 5 after
50% (3/6)
day 5 after
100% (4/4)
day 5 after
83% (1/6)
day 5 after
100% (4/4)
a
Donor splenocytes were derived from protected and diabetic mice treated with porcine insulin. After a 3-day stimulation with porcine B-chain or the immunodominant MHC
class I-restricted LCMV NP-peptide (aa 118 –126), cytokine production (IL-4 and IFN-g) was determined in culture supernatants as described in Materials and Methods and 5 3
6
10 cells were transferred intraperitoneally into nonirradiated, prediabetic (day 5 after LCMV) RIP-NP recipients. Recipients were monitored biweekly for determination of blood
glucose.
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Glucagon
%
1838
bystander suppression obviates the need for autoantigen identification. However, while hormonal function is not related to protection, the Ag chosen probably has to be specific for the target cell
under attack. Minute differences in Ag structure/sequence can have
significant biological implications for the induction of “oral tolerance”. Thus, an appreciation of the full therapeutic potential of
orally administered Ags for treating or preventing human autoimmune diseases depends on a better understanding of the underlying
structural requirements as well as a defined testing system that can
predict their efficacy. In vitro testing of MHC class II-restricted
binding of insulin B chains does not appear to be a suitable criterion to predict protective capacity of oral Ags (unpublished observations). Parameters critical for induction of regulatory cells thus
probably include Ag processing and specific T cell repertoires.
INSULIN IN ORAL IMMUNE TOLERANCE
12.
13.
14.
15.
16.
17.
Acknowledgments
We thank Diana Frye for assistance with the manuscript preparation.
18.
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26.
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