The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Winer, S.
Right arrow Articles by Dosch, H.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Winer, S.
Right arrow Articles by Dosch, H.-M.
The Journal of Immunology, 2000, 165: 4086-4094.
Copyright © 2000 by The American Association of Immunologists

Peptide Dose, MHC Affinity, and Target Self-Antigen Expression Are Critical for Effective Immunotherapy of Nonobese Diabetic Mouse Prediabetes1

Shawn Winer2,*, Lakshman Gunaratnam2,*, Igor Astsatourov*, Roy K. Cheung*, Violetta Kubiak*, Wolfram Karges*, Denise Hammond-McKibben*, Roger Gaedigk*, Daniel Graziano{dagger}, Massimo Trucco{dagger}, Dorothy J. Becker{dagger} and H.-Michael Dosch3,*

* Department of Immunology, University of Toronto, and Immunity, Infection, Injury and Repair Program, The Hospital for Sick Children, Research Institute, Toronto, Ontario, Canada; and {dagger} Pittsburgh Children’s Hospital, University of Pittsburgh, Pittsburgh, PA 15219


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cross-reactive T cells that recognize both Tep69 (dominant nonobese diabetic (NOD) T cell epitope in ICA69 (islet cell autoantigen of 69 kDa)) and ABBOS (dominant NOD T cell epitope in BSA) are routinely generated during human and NOD mouse prediabetes. Here we analyzed how systemic administration of these mimicry peptides affects progressive autoimmunity in adoptively transferred and cyclophosphamide-accelerated NOD mouse diabetes. These models were chosen to approximate mid to late stage prediabetes, the typical status of probands in human intervention trials. Unexpectedly, high dose (100 µg) i.v. ABBOS prevented, while Tep69 exacerbated, disease in both study models. Peptide effects required cognate recognition of endogenous self-Ag, because both treatments were ineffective in ICA69null NOD congenic mice adoptively transferred with wild-type, diabetic splenocytes. The affinity of ABBOS for NOD I-Ag7 was orders of magnitude higher than that of Tep69. This explained 1) the expansion of the mimicry T cell pool following i.v. Tep69, 2) the long-term unresponsiveness of these cells after i.v. ABBOS, and 3) precipitation of the disease after low dose i.v. ABBOS. Disease precipitation and prevention in mid to late stage prediabetes are thus governed by affinity profiles and doses of therapeutic peptides. ABBOS or ABBOS analogues with even higher MHC affinity may be candidates for experimental intervention strategies in human prediabetes, but the dose translation from NOD mice to humans requires caution.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In autoimmune (insulin-dependent or type I) diabetes, autoreactive CD8+ as well as CD4+ T cells mediate progressive destruction of insulin-producing ß-cells in the islets of Langerhans (1, 2, 3). Genetic and environmental factors are required for disease development in both patients and diabetes-prone nonobese diabetic (NOD)3 mice (4, 5).

Prediabetes proceeds in discrete stages (6). Autoreactive T cells are detectable in NOD mice within 2 wk of weaning (3, 7), and peri-insulitis is slowly established over the next several weeks (8). Progression to invasive insulitis is prominent by 2–3 mo, with the beginning of significant islet destruction and consequent autoantibody production (9). The pace of ß-cell destruction peaks close to overt disease, at 5–7 mo of age (10). Within this progression program, many immunotherapies prevent NOD mouse diabetes when applied before the invasive insulitis phase of prediabetes; few such strategies are effective after the islet is breached (5). Because study subjects for clinical intervention trials are identified through measurements of islet autoantibodies, i.e., relatively late in prediabetes (11), immune intervention strategies for the human disease must be effective in mid to late prediabetes. To understand the effects of immunotherapy in late prediabetes is therefore essential.

While nearly 20 genes contribute to disease susceptibility (12), much of the genetic risk is focused on class II MHC, mainly DQ and its murine equivalent, I-A (13). There are striking similarities between prototypic, diabetes-associated human and NOD mouse class II molecules: both have risk-associated (14, 15) aspartate substitutions at position 57 of the DQ/I-A ß-chain and may bind peptides poorly (16, 17, 18). In contrast, the diabetes-protective DR2 and DQB1*0602 alleles encode high affinity peptide binders (18, 19), and addition of peptide-competent I-E or I-A transgenes halts disease development in NOD mice (20, 21). However, it remains unclear exactly how these risk-associated class II molecules promote autoimmunity or autoimmune disease (22) and how the diabetes-protective DR2/DQ6 alleles allow development of autoimmunity in multiple sclerosis (23). The properties of risk-associated class II heterodimers probably not only play a role in disease development, but will ultimately be important for the design and success of immune interventions to halt the progression of prediabetes (11).

With twin concordance of only 20–30%, environmental factors are critical elements of diabetogenesis (4). Diabetes risk has been associated with viral vectors, but definitive links remain elusive (24, 25). One candidate nutrition-related diabetes risk factor is abnormal immunity of susceptible infants and diabetes-prone NOD mice to cow milk protein (26). This is the basis for the first primary diabetes prevention trial (TRIGR) (27, 28). A role for the cow milk protein BSA has been suggested by several groups, based on abnormal Ab and T cell responses in newly diagnosed patients (29, 30, 31, 32, 33) as well as T cell mimicry between the major BSA epitope, ABBOS (dominant NOD T cell epitope in BSA), and the dominant self-epitope, Tep69 (dominant NOD T cell epitope in ICA69 (islet cell autoantigen of 69 kDa)), in the islet cell autoantigen, ICA69 (34, 35). The fully conserved Tep69 epitope is routinely targeted in diabetic patients and NOD mice. Immunization of young NOD, but not other strains of mice, with BSA or ABBOS generates cross-reactive ICA69/Tep69 responses and vice versa (34). NOD mice weaned to a diet free of intact proteins including cow milk do not develop Tep69/ABBOS cross-reactive T cell pools, suggesting that diet-derived ABBOS plays a role in the development or maintenance of these T cells (26).

Here we investigated the therapeutic potential and mechanisms of Tep69 and ABBOS peptides in accelerated models of NOD mouse diabetes. High dose i.v. Tep69 treatment exacerbated while high dose ABBOS halted progressive autoimmunity. Small amounts of ABBOS imitated high dose Tep69 and precipitated diabetes. The opposite effects of Tep69 and ABBOS mimicry peptides were related to opposite MHC class II affinities. The high MHC affinity ABBOS mimicry peptide may be attractive for immunotherapies, but peptide doses require caution in translation to human experimental therapies of established prediabetes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

NOD/Lt (H-2g7) mice were bred in our rodent facility according to facility guidelines in our conventional vivarium, where the diabetes incidence is 83% in female mice 7 mo of age. ICA69null mice were generated by homologous recombination in 129-line embryonal stem cells, disrupting the Tep69 coding sequence in exon 2 (36). A speed congenic approach with published microsatellite markers (37) was employed to develop the animals used here in their sixth backcross generation to NOD, with all idd loci being of NOD origin (I. Astsatourov et al., manuscript in preparation).

Peptides

Peptides were purchased HPLC purified (>98%), and purity was confirmed by mass spectroscopy (numbers indicate the N-terminal amino acid position): OVA152, EYQDNRVSFLGHFI; glutamic acid decarboxylase (GAD) 65–524, SRLSKVAPVIKARMMEYGTT; ICA69–36 (Tep69), AFIKATGKKEDE; and BSA150 (ABBOS), FKADEKKFWGKYLYE. Grade V BSA and OVA were purchased from Sigma (St. Louis, MO), and the human ICA69-ß isoform was purified as previously described (38).

E12.3 T cell hybridoma

NOD mice were immunized s.c. with 100 µg of BSA emulsified in CFA. Ten days later, draining nodes were removed and cultured (for 3 days) in the presence of 5 µg/ml of ABBOS peptide. Cultured cells were fused to the BW5147 TCR-deficient thymoma cell line (American Type Culture Collection, Manassas, VA) by the standard polyethelene glycol fusion protocol. Hybridomas were screened by a cell death assay, which measures the hydrolytic degradation of 2,7-bis-2-carboxyethyl-5–6-carboxyfluorescein (BCECF) dye (Molecular Probes, Eugene, OR) into fluorescent intermediates by viable, but not dying, cells (39, 40). Briefly, replicate cultures of 10 x 103 E12.3 cells were plated in the presence of 1 µg/well of Ag and 10 x 103 irradiated (2500 rad), stably I-Ag7-transfected C3G7 B lymphoma cells (a gift from Dr. E. Unanue, St. Louis, MO) (17) or 2 x 105 irradiated (2500 rad) spleen cells from NOD or other strains of mice. After 72 h of culture, 8 µg/ml BCECF was added for 45 min, and cells were moved to a 96-well filtration plate where free dye was removed through filtration before measurement of fluorescent BCECF derivatives in the cytosol of viable cells (IDDEX Screen Machine, Mundelein, ME). Data were expressed as relative fluorescence units compared with an internal standard (39). This assay provided a rapid, sensitive, and reproducible alternative to cytokine release assays during the screening for T cell hybridomas described here.

Adoptive transfer

Splenocytes from at least four diabetic females were pooled and transferred i.v. in 100 µl of PBS to 7- to 9-wk-old sublethally irradiated (650 rad) NOD males. Cells (10 x 106) were transferred unless noted otherwise. One day following transfer, recipients were given a single i.v. injection of either 100 or 400 µg of peptide in PBS. In all experiments, glucosuria (TesTape, Lily, Toronto, Canada) was used to screen for diabetes until 35 days post-transfer. Diabetes was confirmed by blood glucose measurements (SureStep, Life Technologies, Burnaby, Canada) (34).

Cyclophosphamide-accelerated diabetes

Diabetes was induced in 8- to 12-wk-old NOD females by a single i.p. injection of cyclophosphamide (Sigma; 250 mg/kg if not indicated otherwise). Peptide (100 µg) was given i.v. 5 days before cyclophosphamide injection.

Histology

Thirty-five days after cyclophosphamide treatment, nondiabetic mice were sacrificed, and their pancreata were preserved in 10% buffered formalin. Histologic sections were stained with hematoxylin and eosin. Two blinded observers scored the degree of insulitis with the following scale: 0, normal islet; 1, periinsulitis or <25% infiltration; 2, infiltration of 25–50% of islet surface area; 3, infiltration of >50% of islet surface area; and 4, complete (100%) infiltration of islet or a small retracted islet.

Tolerance induction and proliferative recall assay

Female NOD mice, aged 8–12 wk, were given a single i.v. injection of usually 100 µg of peptide dissolved in PBS. Four or 20 days later, mice were immunized s.c. with 100 µg of peptide emulsified in CFA. Lymph nodes were removed 9–10 days after immunization and were cultured (4 x 105 cells/well) in serum-free AIM V medium (Life Technologies, Mississauga, Canada) in the presence of 0.1–10 µg of peptide. On day 3, cultures were pulsed with 1 µCi of [3H]thymidine overnight and subjected to liquid scintillation counting. When analyzing T cell responses in animals not formally immunized, cells were cultured in the presence of Ag or medium and 10 U of human rIL-2, which enhanced the amplitudes of positive responses.

Affinity studies

Biotinylated peptides at different concentrations were incubated (16 h, 106 cells/ml) with I-Ag7-transfected C3G7 cells (17) or DR4/DQB1*0201-homozygous PRIESS cells (41) in 100 µl of complete RPMI 1640 supplemented with 10% horse serum. Cells were then washed and incubated (45 min) with FITC-labeled streptavidin. Cells incubated with FITC-streptavidin alone served as negative controls. After two additional washes, fluorescence was measured by flow cytometry. Relative fluorescence intensity was estimated as mean equivalents of fluorescein with Rainbow Calibration Particles according to the manufacturer’s instructions (Spherotech, Libertyville, IL). For measurements of dissociation kinetics, C3G7 cells were labeled with peptide as described above, washed, and incubated in complete medium for variable periods before labeling with FITC-streptavidin and flow cytometry. For in vitro affinity studies, murine I-Ag7 molecules were purified from lysates of C3G7 cells (42). Binding of biotinylated peptides to purified I-Ag7 (500 ng) was performed in 100 µl of PBS (pH 7.4) and overnight incubation at room temperature. The incubations were transferred to 96-well plates coated with 100 µl of purified capture Abs (10-2.16) overnight and blocked with 10% horse serum-PBS. The peptide-protein mixtures were incubated for 1 h at room temperature. After four washes (10 mM Tris/140 mM NaCl/0.05% Tween-20, pH 8), Streptavidin-conjugated HRP or alkaline phosphatase was used to detect bound biotinylated peptides in an automated plate reader.

Statistics

Numeric data were compared by Mann-Whitney tests. Incidence tables were analyzed by Fisher’s exact test. All p values were two-tailed, and significance was set at 5%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The bulk of BSA- and ICA69-specific T cells are mimicry cells

We have previously reported that T cell pools responsive to Tep69 and ABBOS peptides are routinely detectable in NOD mice and patients with recent-onset diabetes (35, 38). Immunization of young NOD mice with either peptide (or with either of the native proteins) raises T cell responses to both peptides (34). To analyze T cell mimicry at a clonal level and to determine whether cross-reactive T cells constituted a minor or major proportion of ICA69- and BSA-specific T cell pools, we have now cloned a panel of Tep69/ABBOS-specific T cell hybridomas from BSA-immunized NOD mice. Four clones, E12.2, -3, -4, and -5 were obtained. Each clone behaved similarly in vitro; data are shown for E12.3.

E12.3 cells were exposed to Ags/peptides in the presence I-Ag7-transfected C3G7 lymphoma cells as APC (17). Cognate recognition of BSA, recombinant ICA69-ß, ABBOS, and Tep69 (Fig. 1GoA), but not OVA or OVA152 peptide (Fig. 1GoB) produced rapid activation-induced cell death as measured in an automated, fluorescence-based viability assay (see Materials and Methods; Fig. 1Go, A–C). Dose kinetics of the ABBOS and Tep69 mimicry peptides were strikingly similar (Fig. 1GoA). In six independent experiments no difference in dose or time kinetics could be established between Tep69 and ABBOS. On a molar basis, the native proteins, BSA and ICA69, had a 30- to 50-fold lower activation threshold than the two peptides (p < 0.0001, by Mann-Whitney test), but the two proteins also had nearly identical dose responses. C3G7 cells and irradiated NOD splenocytes provided necessary and effective APC function, while irradiated C57BL/6 (H-2b) or BALB/c (H-2d) APC failed to do so (Fig. 1GoB). These data extend evidence for I-Ag7-restricted mimicry between ABBOS and Tep69 (34) to the level of a single T cell clone, and they implied, erroneously (see below), that the two peptides would have similar functional properties.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 1. Antigenic mimicry between Tep69 and ABBOS in the E12.3 hybridoma and NOD splenocytes. A, Ag dose-response curves of E12.3. The T cell hybridoma E12.3 undergoes activation-induced cell death in the presence of C3G7-presented, mimicry peptides or the respective proteins. One of seven experiments with similar results is shown. See Materials and Methods for details of the fluorescence-based cell survival assay. The assay was calibrated with untreated, dye-labeled E12.3 cells. B, Mimicry T cells are I-Ag7 restricted. I-Ag7-transfected C3G7 lymphoma cells and irradiated spleen cells from NOD, C57BL/6 or BALB/c mice were used as APCs. C, ALA scan of the ABBOS peptide. Peptides with single ALA replacement or replacement of the native ALA residue by aspartate (*) were used to stimulate E12.3 hybridoma cells as in A. Shaded bars, residues conserved in ABBOS and Tep69. D, Proliferative responses of lymph node cells from BSA-immunized NOD mice to the ABBOS-derived ALA replacement peptides were measured by [3H]thymidine incorporation (mean counts per minute + SD). A limited number of Tep69-derived ALA replacement peptides were tested in parallel. Response profiles resemble that of E12.3 cells (C).

 
Alanine scanning of the ABBOS peptide demonstrated that the central residues are critical for T cell responses, including the KAxyKK motif conserved between ABBOS and Tep69 (38) (Fig. 1GoC). We used positive/negative response profiles to alanine (ALA) replacement peptides to estimate the in vivo prevalence of mimicry T cells in NOD mice (Fig. 1GoD). The bulk proliferative responses of BSA-immunized NOD mice resembled the E12.3 profile, because ALA replacement of the identical residues eliminated these responses. The amplitudes of recall responses to BSA, ABBOS, ICA69, and Tep69 were similar; OVA152 peptide served as a negative control in BSA-immunized mice. Collectively, these data suggest that the bulk of BSA/ICA69-specific T cells in NOD mice are Tep69/ABBOS-specific mimicry T cells.

Immunotherapies with Tep69 and ABBOS have opposite outcomes

To analyze peptide-based immunotherapy of the Tep69/ABBOS-specific T cell pool in NOD mice, we employed the adoptive transfer model of NOD diabetes, because it has characteristics of progressive diabetic autoimmunity and provides a stringent read-out for immunotherapy. This is important when considering translation to humans with progressive, autoantibody-positive prediabetes (11), where the appearance of Tep69/ABBOS-specific T cells has been associated with a high risk to develop overt disease (35).

Engraftment of 0.5, 5, 10, 15, or 20 x 106 diabetic spleen cells in sublethally irradiated (650 rad) recipients produced 0, 20, 50–60, 75, and 100% overt diabetes, respectively, by 35 days post-transfer (data not shown). We aimed for an ~50% incidence to observe both positive and negative peptide effects on diabetes development. Unless indicated otherwise, we transferred 107 diabetic spleen cells in subsequent experiments.

Systemic (i.v.) administration of a single dose of 400 µg of Tep69 peptide 1 day following spleen cell transfer exacerbated disease development (87 vs 60%; p = 0.02; Fig. 2GoA). Mice injected with OVA152 peptide or PBS developed diabetes at the same rate. In contrast, a single dose of 400 µg of ABBOS peptide 1 day following transfer resulted in protection from disease (37%; p = 0.05). The difference between the effects of Tep69 and ABBOS was highly significant (p < 0.0001). A dose of 100 µg of Tep69 or ABBOS peptide had the same effect as a dose of 400 µg (p > 0.5; data not shown).



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 2. Tep69 and ABBOS peptides have opposing effects on adoptively transferred IDD. A, Irradiated NOD males received 107 pooled spleen cells from diabetic donors. The four groups of mice indicated were injected i.v. with a single dose of 400 µg of Tep69, OVA152, or ABBOS or with PBS only. The graph shows the development of overt IDD in each group. B, A single peptide injection protocol as described in A is compared with dual i.v. injection of Tep69 into diabetic donors 1 day before transfer, and into recipients 1 day after transfer. C, Single and dual i.v. ABBOS (100 µg) injection protocols are compared in mice adoptively transferred with a fully diabetogenic spleen cell dose (2 x 107 cells, i.v.).

 
Peptide effects could be boosted by repeated administration. Spleen cell donors received 100 µg Tep69 or ABBOS i.v., one day before transfer and the recipient received the same treatment one day after transfer (Fig. 2BGo, C). In three such experiments, 92–100% of mice treated twice with Tep69 developed diabetes. In contrast, dual ABBOS treatment significantly protected from overt disease, even following transfer of larger numbers of diabetic spleen cells (20 x 106) which caused diabetes in all untreated control mice (Fig. 2CGo, p = 0.0003). Systemic treatment with Tep69 thus exacerbates, while ABBOS prevents disease.

It was unexpected that mimicry peptides had opposing in vivo effects, not predicted by their near identical behavior in vitro (e.g., Fig. 1GoA). We therefore decided to seek confirmation in the other accelerated diabetes model, cyclophosphamide (CY)-induced NOD diabetes (43). Pilot experiments established optimal peptide administration kinetics and CY dose/diabetes relationships, where we chose a relatively low dose that generated overt disease in ~50% of treated mice (250 mg/kg; data not shown). Intravenous injection of 100 µg Tep69 5 days before administration of CY significantly increased the diabetes incidence 35 days after CY injection (90 vs 40%; p = 0.05; Fig. 3GoA). Animals receiving a low, subdiabetogenic CY dose (150 mg/kg) developed disease rarely (0–10%), but i.v. Tep69 still precipitated disease (56%; p = 0.008; Fig. 3GoB).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3. Intravenous Tep69 exacerbates and i.v. ABBOS protects from cyclophosphamide-induced IDD. A, NOD mice received 250 mg/kg cyclophosphamide 5 days after an i.v. injection of 100 µg of Tep69 or OVA152 peptide. Animals were monitored for the development of overt IDD until 35 days after cyclophosphamide treatment. B, The same experiment as that in A, except that animals received a subdiabetogenic cyclophosphamide dose of 150 mg/kg. C, ABBOS (100 µg) was injected i.v. instead of Tep69 (CY dose, 250 mg/kg).

 
As in the adoptive transfer experiments, i.v. injection of 100 µg ABBOS peptide provided significant protection from CY-induced disease (28 vs 54%; p = 0.03; Fig. 3GoC), with a concordant reduction of insulitis severity (Fig. 4Go), either counting islets with no or minor islet infiltration (insulitis score, <=1; p = 0.0001 vs PBS-treated mice, by Fisher’s exact test) or the number of breached islets (insulitis score, >=2; p = 0.0017). This observation mapped peptide effects to islets. Treatments with OVA152 peptide (Fig. 3Go, A and B), with an unrelated control peptide (BSA193; not shown) or with a Tep69-derived K(43)->ALA replacement peptide (see Fig. 9Go, below), all resulted in the same diabetes incidences as that in PBS-injected mice.



View larger version (69K):
[in this window]
[in a new window]
  		FIGURE 4. High dose i.v. ABBOS reduces insulitis. A, Islets (100/group) of hematoxylin/eosin-stained pancreata from i.v. PBS-treated (n = 5) or ABBOS-treated mice (n = 5). Pancreata were obtained 35 days after cyclophosphamide treatment from mice in either group that had not developed overt IDD. Islets were scored blindly for the presence and severity of islet infiltration using the standard scale (0, no infiltration; 1, peri-insulitis or <25% islet area infiltrate; 2, 25–50% islet area infiltrate; 3, >50% islet area infiltrate; 4, 100% islet area infiltrate or small retracted islet. B and C, Typical hematoxylin/eosin-stained islets from two ABBOS-treated, nondiabetic mice at x100 magnification. There is very little insulitis. D and E, Typical hematoxylin/eosin-stained islets from PBS-treated, nondiabetic mice at x50 and x100 magnification, respectively. Severe insulitis occurred in many islets.

 


View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 9. Low dose i.v. ABBOS exacerbates prediabetes. NOD mice were examined for the incidence of cyclophosphamide-induced insulin-dependent diabetes after receiving a single injection of 3 or 100 µg of ABBOS i.v., PBS, or 100 µg of the K(43)->ALA replacement peptide.

 
These data demonstrate that high dose i.v. administration of Tep69 or ABBOS peptide can exacerbate or protect from disease in two models of progressive NOD prediabetes. The peptide effects were Ag specific and thus targeted T cells with relevant (mimicry) TCRs. The mimicry T cell pool therefore is an intrinsic element of the prediabetes process, able to affect disease outcome via unknown mechanisms.

Peptide effects require expression of cognate self-Ag

A number of studies associated the outcome of NOD mouse immunotherapies with a shift in overall cytokine bias among autoreactive T cells (44, 45, 46, 47), however, the issue is not fully resolved (48). To determine whether Tep69 or ABBOS peptides acted through such systemic bystander effects on other autoreactive T cells, we adoptively transferred wild type diabetic NOD splenocytes into NOD congenic ICA69null mice, and treated with i.v. ABBOS or Tep69 as before. Injection of Tep69 or ABBOS in adoptively transferred ICA69null mice had no effect on diabetes development (Fig. 5Go). Thus, the manifestation of i.v. Tep69/ABBOS effects requires the expression and cognate recognition of the endogenous self-Ag, rather than a prominent, systemic bystander effect. Tep69/ABBOS effects thus differ from other immunotherapies, where diabetes development was suggested to be modulated through peptide-induced systemic cytokine release (46) and/or the development of regulatory cells (44). The observation of equal diabetes transfer in PBS-treated ICA69-/- homozygotes and ICA69-/+ heterozygotes indicated that, like other diabetes autoantigens such as GAD65 (49), ICA69 is a facultative, but not obligate, autoimmune target in diabetogenesis.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 5. Peptide-mediated modification of adoptively transferred IDD requires expression of the cognate self-Ag. ICA69-deficient and heterozygote male NOD congenics were adoptively transferred with 107 pooled spleen cells from diabetic wild-type NOD mice. Groups of 9–11 mice each were i.v. injected as described in Fig. 2Go with 100 µg of Tep69, ABBOS, or PBS. The graph shows the incidence of IDD up to 35 days post-transfer.

 
ABBOS and Tep69 differ in MHC affinity

The molecular basis for opposite in vivo peptide function was unclear, and we asked whether differential MHC class II binding could explain the opposite peptide effects. The relative I-Ag7 binding affinities of the two peptides were compared by flow cytometry as well as in vitro binding to purified I-Ag7. Binding of biotinylated ABBOS to C3G7 cells (17) was easily detected following labeling with FITC-conjugated streptavidin, while Tep69 binding was weak (Fig. 6GoA). Tep69 was easily displaced by unconjugated ABBOS, while unconjugated Tep69 displaced very little ABBOS in the dose range tested (not shown). When Tep69- or ABBOS-loaded C3G7 cells were washed and incubated at 37°C, cell-bound Tep69 was lost rapidly (Fig. 6GoC). In contrast, ABBOS binding was higher even at 1/10th the initial peptide loading dose, and binding was still detectable as late as 24 h postlabeling (Fig. 6GoB), suggesting an uncommonly high avidity of ABBOS to I-Ag7. NOD class II may be a poor binder for only some peptides (17). Major differences in I-Ag7 binding were confirmed with insolubilized, purified I-Ag7 (Fig. 6GoD), where only ABBOS binding was sufficiently strong to allow consistent measurement of its binding constant (k = 0.344 µM; Fig. 6GoD, inset); binding of Tep69 was erratic over the dose range tested. Peptide binding to the human PRIESS B cell line homozygous for diabetes-associated class II (41) followed a similar pattern as the C3G7 line, with much lower relative affinity for Tep69 compared with that for ABBOS (Fig. 6GoE).



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 6. Tep69 and ABBOS have grossly different affinity for MHC. A, Weak binding of biotinylated Tep69 (thin line) and strong binding of biotinylated ABBOS (solid line) to C3G7 cells. The dashed line represents control cells exposed to FITC-streptavidin only. B, C3G7 cells were loaded at two loading concentrations and washed. The graph shows the retention of ABBOS peptide over time. C, An experiment similar to that in B, except that 200 µM Tep69 was loaded. Tep69 is rapidly released from I-Ag7. D, Binding of biotinylated ABBOS and Tep69 peptides to purified I-Ag7. Inset, Determination of the ABBOS binding affinity by Scatchard plot. E, Weak binding of biotinylated Tep69 (thin line) and strong binding of biotinylated ABBOS (solid line) to human PRIESS cells. The dashed line represents control cells exposed to FITC-streptavidin only.

 
We concluded that ABBOS has a much higher binding affinity to relevant mouse and human class II heterodimers than Tep69 and that, in addition, ABBOS is released from I-Ag7 at a much slower rate. In functional terms, high on-rates and slow off-rates of ABBOS and the opposing profile of Tep69 would predict dramatic differences in their in vivo function. On face value, these data clash with the near identity of in vitro peptide dose responses (Fig. 1GoA). The two datasets might be reconciled if opposite MHC affinities were balanced by high average TCR affinities for Tep69 (TCRhigh) and low averages for ABBOS (TCRlow) (50).

Tolerance induction following i.v. peptide treatment

While effector mechanisms of high dose i.v. peptides are generally complex, peptide persistence and sustained TCR stimulation probably contribute to induction of unresponsiveness (51, 52). The assumption of opposite peptide affinity profiles for MHC and TCRs allowed us to derive some testable predictions, with MHC binding affinity the most important variable in vivo, as it governs the rate of irretrievable peptide loss. In vivo TCR-MHC occupancy rates should 1) only transiently be sufficient for induction of unresponsiveness to Tep69, while 2) the opposite would be expected for ABBOS, unless 3) ABBOS doses were sufficiently small to mimic the low TCR/MHC occupancy rates of high dose Tep69 (50).

To test the first two predictions, 100 µg of Tep69, ABBOS, or OVA152 peptide were injected i.v. as before, followed by immunization with the same peptide in CFA 4 days (Fig. 7Go, A–C) or 20 days (Fig. 7Go, D–F) later. In vitro proliferative recall responses were measured in lymph node cells obtained 9–10 days after the immunization.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 7. Short- and long-term unresponsiveness following i.v. peptide treatments. A–C, NOD mice received a single i.v. injection of 100 µg of Tep69, ABBOS, or OVA152. Four days later mice were immunized with the cognate peptide in CFA. Lymph node cells were obtained 9 days later and in vitro recalled with Tep69 (A), OVA152 (B), or ABBOS (C). Proliferative responses to purified protein derivative were measured in parallel. All three peptides induce short-term unresponsiveness. D and E, Similar experiment as that in A–C, except that i.v. peptide-treated mice were rested for 20 days before immunization and subsequent recall responses to Tep69 (D), OVA152 (E), and ABBOS (F). Intravenous ABBOS and OVA152, but not Tep69, induce lasting unresponsiveness.

 
High dose i.v. injection with any of the peptides generated short-term unresponsiveness to the cognate peptide (Fig. 7Go, A–C). Purified protein derivative (PPD) control responses were unaffected. As predicted, the unresponsiveness induced in i.v. Tep69-treated NOD mice was transient, and mice immunized 20 days after i.v. Tep69 administration no longer showed unresponsiveness, but had elevated responses to immunization and in vitro recall (Fig. 7Go, D and F). In contrast, unresponsiveness following i.v. ABBOS (or OVA152) administration (Fig. 7Go, B and C) was lasting, and immunization 3 wk after i.v. treatment failed to generate T cell responses as measured by in vitro proliferative recall responses (Fig. 7Go, D–F). ABBOS-treated mice also lacked detectable Tep69 responses (Fig. 7GoD), while i.v. Tep69-treated mice also had high ABBOS responses to immunization 3 wk after i.v. injection (Fig. 7GoF). These data associated long-term unresponsiveness to mimicry peptide with disease prevention, and its failure with disease progression.

The effects of mimicry peptides were further analyzed by measuring in vitro responses in i.v. peptide-treated, adoptively transferred, otherwise unimmunized diabetic mice (Fig. 8Go). Tep69-treated animals not only retained Tep69/ABBOS-specific T cells, but in vitro responses were much higher than those of PBS-injected control mice (Fig. 8GoA; p < 0.0001, by Mann-Whitney test). Tep69-induced disease exacerbation is most likely due to an expansion of the mimicry T cell pool size, but a change in activation status and cognate responsiveness could also play a role. When we analyzed the few (n = 3) nondiabetic mice that escaped disease following i.v. Tep69 treatment (Fig. 8GoB), they were found to lack detectable mimicry T cells; only diabetic mice had these cells. In contrast, the same mimicry T cells were undetectable in ABBOS-treated mice, including six animals that developed disease despite treatment (Fig. 8GoA). While ABBOS-induced unresponsiveness of the mimicry T cell pool is associated with disease protection, this protection appears less absolute than Tep69-mediated disease exacerbation following expansion of this T cell pool.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 8. High dose i.v. Tep69 expands mimicry T cells in adoptive transfer recipients. A, NOD mice received adoptive transfer grafts and i.v. Tep69, ABBOS, or PBS as before. Proliferative in vitro T cell responses were measured when transfer recipients became diabetic. Mimicry T cell pools are large in i.v. Tep69-treated mice and absent in ABBOS-treated animals. One of three similar experiments is shown. B, Spleen cells from nondiabetic and diabetic, i.v. Tep69-treated mice are compared for their proliferative responses. In mice escaping IDD, i.v. Tep69 treatment failed to expand the mimicry T cell pool. One of three similar experiments is shown.

 
Our third prediction was that a low i.v. ABBOS dose might mimic the Tep69 effects. This was indeed the case. As shown in Fig. 9Go, a 50-fold decrease in the i.v. ABBOS dose accelerated cyclophosphamide-induced diabetes compared with controls injected with PBS or the Tep69-derived K(43)->ALA replacement peptide. Low dose ABBOS was nearly as effective as high dose i.v. Tep69, while a low dose of Tep69 was, as expected, without effect (not shown).

Collectively, these data demonstrate that mimicry Ags/peptides can have a place in the immunotherapy of established diabetic autoimmunity. Mimicry peptides do not necessarily have equivalent function, and their in vivo action may require cognate recognition of the endogenous self-Ag. We also note that disease precipitation is a realistic concern in the immunotherapy of progressive diabetic autoimmunity. We discuss below how opposing functional profiles of endogenous self- and exogenous mimicry peptides may synergize in the maintenance and progression of diabetic autoimmunity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Prediabetic autoimmunity targets a fairly constant, similar set of autoantigens in humans and NOD mice (5, 35). Although immunotherapy with any one of these target Ags/peptides can prevent NOD diabetes, effective immunotherapy almost always requires treatment early in prediabetes (5). We consciously focused on models for progressive prediabetes, because human subjects recruited into clinical intervention trials are identified through markers of progressive autoimmunity (11).

The Tep69 effects described here were both unexpected and unusual, although there is precedence for adverse effects of immunotherapy (48, 53, 54, 55, 56). Disease precipitation by Tep69 does not assign a unique, primary role to ICA69 as diabetes autoantigen. The data presented here demonstrate that diabetes develops in ICA69-deficient mice adoptively transferred with wild-type diabetic splenocytes. Like GAD65-deficient animals (49), ICA69null NOD congenics develop IDD spontaneously,5 and T cell autoreactivities to GAD or ICA69 appear to be facultative rather than obligate or exclusive elements of diabetogenesis.

However, the present data also indicate that in the prediabetic islet locale, infiltrated by different pools of autoreactive T cells, the selective expansion of Tep69-specific T cells as well as their unresponsiveness following ABBOS immunotherapy can, respectively, enhance or halt diabetogenesis. The failure of Tep69/ABBOS treatments in ICA69 knockout mice indicates that systemic bystander effects such as those demonstrated for disease prevention with Calmette-Guérin bacillus (46, 57) are not of major relevance here. However, our data do not rule out a possible, peptide-induced cytokine bias within the islet. The failure to observe Tep69 and ABBOS effects in ICA69null mice could then reflect a failure to migrate to and encounter presented Tep69 in the pancreas. To clarify mechanisms of immunotherapy at cellular and molecular levels we are in the process of generating a mimicry TCR transgene. At this point the most plausible hypothesis is that Tep69 expands the mimicry T cell pool, while high dose ABBOS generates anergic T cells that can migrate to the islet and produce protective cytokines such as IL-4 (58) in response to endogenous ICA69 processed and presented by infiltrating APC. It will be interesting to determine whether the same holds true for GAD-specific autoimmunity, which has been associated with the induction of cytokine bias in several, but not all, studies (44, 48).

Dramatically different MHC affinities for Tep69 and ABBOS peptides applied to diabetes-relevant human and NOD mouse class II heterodimers. Low MHC affinity of Tep69-like peptides to diabetes-associated DR has been reported by the Roep laboratory (18). To seek and establish similarities between mouse and human autoimmunity is important when considering translation of murine data to prediabetic humans, and it will be necessary to expand these present studies to the natural disease in NOD females to determine how late in this process peptide therapy is effective, and if repeated administrations are necessary and of benefit.

We proposed that the best explanation that reconciles MHC binding data and the nearly identical peptide dose responses in the E12 hybridoma is opposite affinity profiles for MHC and TCR. Thus, the high MHC affinity of ABBOS would be balanced by low E12 TCR affinity, and the low MHC affinity of Tep69 would be balanced by high affinity of this TCR for that self-peptide. Thymic ICA69 expression levels are low, but not negative (59). Positive selection would favor high affinity TCRs, as few I-Ag7 molecules would be expected to carry sufficient numbers of Tep69 peptide for periods of time sufficient to stimulate any but high affinity TCRs. Functionally reminiscent of unopposed positive selection (60), there may rarely be enough peptide presentation for negative selection. By themselves, these thymic emigrants are rather unlikely candidates for pathogenic functions, unless these cells are "helped along" by ABBOS in the periphery.

The alternative MHC/TCR affinity profiles would fit with a disease-promoting effect of ABBOS, as the small amounts of peptide that may pass enteric censure (61) would be efficiently captured and retained by APCs. We observed that low level ABBOS exposure can enhance disease development, and low level natural ABBOS exposure may prevent the decline of the mimicry T cell pool due to neglect. However, once the islet is breached and invasive insulitis established, more endogenous Tep69 becomes available to lesional APCs, sufficient for activation of the mimicry pool by its cognate self-Ag.

Antigenic mimicry is a prominent concept in the pathoetiology of autoimmune diseases, but examples of naturally occurring mimicry are infrequent (62, 63), and mechanisms that lead to disease are not always clear (24). The implicit assumption of equivalent functions is common and probably at times correct, but is rarely tested.

ABBOS-containing BSA fragments are naturally generated following ingestion of cow milk (61). Complex, multiantigenic weaning diets are a prerequisite for diabetes development in NOD mice and BB rats, and these rodents are solidly protected from disease when weaned to nonantigenic diets such as casein hydrolysates (4). Hydrolyzed diets prevent the development of Tep69/ABBOS mimicry T cells, and, consistent with a role of diet in the shaping of diabetogenic T cell repertoires, such mice do not respond to immunization with ICA69 even in complete adjuvant (26). However, the role of cow milk proteins is controversial (64), and addition of BSA in drinking water of hydrolysate-fed BB rats did not lead to diabetes, but, instead, afforded a small, but significant, protective effect (65): in this report consumption of several milligrams of daily BSA in mothers and offspring may have provided sufficient systemic ABBOS exposure for protective ABBOS effects early in the life of some animals.

Collectively, our observations have uncovered several unexpected facts, and they suggest that immunotherapeutic targeting of a common mimicry T cell pool with high affinity ABBOS and possibly homologues with even higher MHC affinity can halt the progression of diabetic autoimmunity. Opposing effects of mimicry peptides provide a plausible scenario of how this natural pair of mimics may act in synergistic fashion. Environmentally acquired ABBOS would sustain thymic emigrant mimicry cells, while Tep69-mediated effector function in the islet would be enhanced when the self-Ag supply from dying ß-cells reaches a significant level in progressive prediabetes. Our data suggest caution in the design of immunotherapies applied during mid to late stage prediabetes, where T cell access to the target organ has already appeared. The similarities of mimicry T cell pools and ABBOS and Tep69 affinities in humans and NOD mice are striking, making this an attractive system for the development of a tailored immunotherapeutic strategy. The peptide effects were clearly boostable, and repeated peptide treatments can be considered when the reappearance of target T cells is detected. Peptides that have homologues in relevant animal models, parallel study of humans and rodents, peptide dose, and high MHC affinity should be factors in the design of immunotherapeutic peptides considered for human use.


   Acknowledgments
 
We thank Dr. D. Mathis (Harvard Medical School, Boston, MA) and Drs. T. Watts, J. Danska, P. Poussier, P. Ohashi, D. Winer, and E. Iakhnina (University of Toronto, Toronto, Canada) for helpful discussions of this work, and J. C. Ortiz for technical support. We gratefully acknowledge the efforts of A. Darnley, S. Pietropaolo, K. Riley, and the Pittsburgh General Clinical Research Center nurses.


   Footnotes
 
1 This work was supported by grants from the Canadian Medical Research Council, the National Institutes of Health (General Clinical Research Center Grants MO1RR00084 and RO1DK24021), and the Renziehausen Fund. Back

2 S.W. and L.G. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. H.-Michael Dosch, The Hospital For Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Back

4 Abbreviations used in this paper: NOD, nonobese diabetic; ABBOS, dominant NOD T cell epitope in BSA; ICA69, islet cell autoantigen of 69 kDa; BCECF, 2,7-bis-2-carboxyethyl-5–6-carboxyfluorescein; Tep69, dominant NOD T cell epitope in ICA69; CY, cyclophosphamide; GAD, glutamic acid decarboxylase. Back

5 I. Astsatourov, S. Winer, R. Gaedigk, M. Pilon, D. Hammond-McKibben, R. K. Cheung, V. Kubiak, W. Karges, and H.-M. Dosch. 2000. ICA69-deficient NOD congenic mice develop diabetes but resist disease acceleration by cyclophosphamide. Submitted for publication. Back

Received for publication April 12, 2000. Accepted for publication July 13, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Katz, J. D., C. Benoist, D. Mathis. 1995. T helper cell subsets in insulin-dependent diabetes. Science 268:1185.[Abstract/Free Full Text]
  2. Serreze, D. V., H. D. Chapman, D. S. Varnum, I. Gerling, E. H. Leiter, L. D. Shultz. 1997. Initiation of autoimmune diabetes in NOD/Lt mice is MHC class I-dependent. J. Immunol. 158:3978.[Abstract]
  3. Wong, F. S., J. Karttunen, C. Dumont, L. Wen, I. Visintin, I. M. Pilip, N. Shastri, E. G. Pamer, Jr C. A. Janeway. 1999. Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library. Nat. Med. 5:1026.[Medline]
  4. Karges, W., J. Ilonen, B. H. Robinson, H.-M. Dosch. 1995. Self and non-self antigen in diabetic autoimmunity: molecules and mechanisms. Mol. Aspects Med. 16:79.[Medline]
  5. Atkinson, M. A., E. H. Leiter. 1999. The NOD mouse model of type 1 diabetes: as good as it gets?. Nat. Med. 5:601.[Medline]
  6. Gonzales, A., J. D. Katz, M.-G. Mattei, H. Kikutani, C. Benoist, D. Mathis. 1997. Genetic control of diabetes progression. Immunity 7:873.[Medline]
  7. Tisch, R., X. D. Yang, S. M. Singer, R. S. Liblau, L. Fugger, H. O. McDevitt. 1993. Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 366:72.[Medline]
  8. Garchon, H. J., P. Bedossa, L. Eloy, J. F. Bach. 1991. Identification and mapping to chromosome 1 of a susceptibility locus for periinsulitis in non-obese diabetic mice. Nature 353:260.[Medline]
  9. Reddy, S., N. J. Bibby, R. B. Elliott. 1988. Ontogeny of islet cell antibodies, insulin autoantibodies and insulitis in the non-obese diabetic mouse. Diabetologia 31:322.[Medline]
  10. Shimada, A., B. Charlton, E. C. Taylor, C. G. Fathman. 1996. ß-cell destruction may be a late consequence of the autoimmune process in nonobese diabetic mice. Diabetes 45:1063.[Abstract]
  11. Becker, D. J., R. E. LaPorte, I. Libman, M. Pietropaolo, H.-M. Dosch. 2000. Prevention of type I diabetes: is now the time?. J. Clin. Endocrinol. Metab. 85:494.[Free Full Text]
  12. Becker, K. G.. 1999. Comparative genetics of type 1 diabetes and autoimmune disease: common loci, common pathways?. Diabetes 48:1353.[Abstract]
  13. Wicker, L. S., J. A. Todd, L. B. Peterson. 1995. Genetic control of autoimmune diabetes in the NOD mouse. Annu. Rev. Immunol. 13:179.[Medline]
  14. Morel, P. A., J. S. Dorman, J. A. Todd, H. O. McDevitt, M. Trucco. 1988. Aspartic acid at position 57 of the HLA-DQ beta chain protects against type I diabetes: a family study. [Published erratum appears in 1989 Proc. Natl. Acad. Sci. USA 86:1317.]. Proc. Natl. Acad. Sci. USA 85:8111.[Abstract/Free Full Text]
  15. Quartey-Papafio, R., T. Lund, P. Chandler, J. Picard, P. Ozegbe, S. Day, P. R. Hutchings, L. O’Reilly, D. Kioussis, E. Simpson, et al 1995. Aspartate at position 57 of nonobese diabetic I-Ag7 ß-chain diminishes the spontaneous incidence of insulin-dependent diabetes mellitus. J. Immunol. 154:5567.[Abstract]
  16. Dorman, J. S., R. E. LaPorte, R. A. Stone, M. Trucco. 1990. Worldwide differences in the incidence of type I diabetes are associated with amino acid variation at position 57 of the HLA-DQ ß chain. Proc. Natl. Acad. Sci. USA 87:7370.[Abstract/Free Full Text]
  17. Carrasco-Marin, E., J. Shimizu, O. Kanagawa, E. R. Unanue. 1996. The class II MHC I-Ag7 molecules from non-obese diabetic mice are poor peptide binders. J. Immunol. 156:450.[Abstract]
  18. Geluk, A., K. E. van Meijgaarden, N. C. Schloot, J. W. Drijfhout, T. H. Ottenhoff, B. O. Roep. 1998. HLA-DR binding analysis of peptides from islet antigens in IDDM. Diabetes 47:1594.[Abstract]
  19. Ettinger, R. A., A. W. Liu, G. T. Nepom, W. W. Kwok. 1998. Exceptional stability of the HLA-DQA1*0102/DQB1*0602 {alpha}ß protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus. J. Immunol. 161:6439.[Abstract/Free Full Text]
  20. Hanson, M. S., M. Cetkovic-Cvrlje, V. K. Ramiya, M. A. Atkinson, N. K. Maclaren, B. Singh, J. F. Elliott, D. V. Serreze, E. H. Leiter. 1996. Quantitative thresholds of MHC class II I-E expressed on hemopoietically derived antigen-presenting cells in transgenic NOD/Lt mice determine level of diabetes resistance and indicate mechanism of protection. J. Immunol. 157:1279.[Abstract]
  21. Singer, S. M., R. Tisch, X. D. Yang, H. O. McDevitt. 1993. An Abd transgene prevents diabetes in nonobese diabetic mice by inducing regulatory T cells. Proc. Natl. Acad. Sci. USA 90:9566.[Abstract/Free Full Text]
  22. Antoniou, A. N., J. Elliott, E. Rosmarakis, P. J. Dyson. 1998. MHC class II Ab diabetogenic residue 57 Asp/non-Asp dimorphism influences T-cell recognition and selection. Immunogenetics 47:218.[Medline]
  23. Wucherpfennig, K. W., I. Catz, S. Hausmann, J. L. Strominger, L. Steinman, K. G. Warren. 1997. Recognition of the immunodominant myelin basic protein peptide by autoantibodies and HLA-DR2-restricted T cell clones from multiple sclerosis patients: identity of key contact residues in the B-cell and T-cell epitopes. J. Clin. Invest. 100:1114.[Medline]
  24. Horwitz, M. S., L. M. Bradley, J. Harbertson, T. Krahl, J. Lee, N. Sarvetnick. 1998. Diabetes induced by coxsackie virus: initiation by bystander damage and not mimicry. Nat. Med. 4:781.[Medline]
  25. Juhela, S., H. Hyoty, A. Hinkkanen, J. Elliott, M. Roivainen, P. Kulmala, J. Rahko, M. Knip, J. Ilonen. 1999. T cell responses to enterovirus antigens and to ß-cell autoantigens in unaffected children positive for IDDM-associated autoantibodies. J. Autoimmun. 12:269.[Medline]
  26. Karges, W., D. Hammond-McKibben, R. K. Cheung, M. Visconti, N. Shibuya, D. Kemp, H. M. Dosch. 1997. Immunological aspects of nutritional diabetes prevention in NOD mice: a pilot study for the cow’s milk-based IDDM prevention trial. Diabetes 46:557.[Abstract]
  27. Akerblom, H. K., M. Knip. 1998. Putative environmental factors in type 1 diabetes. Diabetes Metab. Rev. 14:31.[Medline]
  28. Knip, M., H. K. Å kerblom. 1998. IDDM prevention trials in progress: a critical assessment. J. Pediatr. Endocrinol. Metab. 11:371.
  29. Karjalainen, J., J. M. Martin, M. Knip, J. Ilonen, B. H. Robinson, E. Savilahti, H. K. Å kerblom, H.-M. Dosch. 1992. A bovine albumin peptide as a possible trigger of insulin dependent diabetes mellitus. N. Engl. J. Med. 327:302.[Abstract]
  30. Pigny, P., G. Mortreux, A. Racadot, C. Stuckens, A. Boersma. 1995. Humoral immune response to bovine serum albumin in new onset and established insulin-dependent diabetes mellitus. Acta Diabetol. 32:135.[Medline]
  31. Pardini, V. C., J. G. Vieira, W. Miranda, S. R. Ferreira, G. Velho, E. M. Russo. 1996. Antibodies to bovine serum albumin in Brazilian children and young adults with IDDM. Diabetes Care 19:126.[Abstract]
  32. Miyazaki, I., R. K. Cheung, R. Gaedigk, M. F. Hui, J. Van der Meulen, R. V. Rajotte, H.-M. Dosch. 1995. T cell activation and anergy to islet cell antigen in type 1 diabetes. J. Immunol. 154:1461.[Abstract]
  33. Saukkonen, T., S. M. Virtanen, M. Karppinen, H. Reijonen, J. Ilonen, L. Räsänen, H. K. Å kerblom, E. Savilahti, C.D.I.F.S. Group. 1998. Significance of cow’s milk protein antibodies as risk factor for childhood IDDM: interactions with dietary cow’s milk intake and HLA-DQB1 genotype. Diabetologia 41:72.[Medline]
  34. Karges, W., D. Hammond-McKibben, R. Gaedigk, N. Shibuya, R. Cheung, H.-M. Dosch. 1997. Loss of self-tolerance to ICA69 in non-obese diabetic mice. Diabetes 46:1548.[Abstract]
  35. Dosch, H.-M., R. K. Cheung, W. Karges, M. Pietropaolo, D. J. Becker. 1999. Persistent T cell anergy in human type 1 diabetes. J. Immunol. 163:6933.[Abstract/Free Full Text]
  36. Gaedigk, R., W. Karges, M. F. Hui, H.-M. Dosch. 1996. Genomic organization of human and mouse ICAp69, a target antigen in diabetic autoimmunity. Genomics 38:382.[Medline]
  37. Serreze, D. V., H. D. Chapman, D. S. Varnum, M. S. Hanson, P. C. Reifsnyder, S. D. Richard, S. A. Fleming, E. H. Leiter, L. D. Shultz. 1996. B lymphocytes are essential for the initiation of T cell-mediated autoimmune diabetes: analysis of a new "speed congenic" stock of NOD.Ig µ null mice. J. Exp. Med. 184:2049.[Abstract/Free Full Text]
  38. Karges, W., R. Gaedigk, M. F. Hui, R. K. Cheung, H.-M. Dosch. 1997. Molecular cloning of murine ICA69: diabetes-prone mice recognize the human autoimmune-epitope, Tep69, conserved in splice variants from both species. Biochim. Biophys. Acta 1360:97.[Medline]
  39. Leeder, J. S., A. Nakhooda, S. P. Spielberg, H. M. Dosch. 1991. Cellular toxicity of sulfamethoxazole reactive metabolites. II. Inhibition of natural killer activity in human peripheral blood mononuclear cells. Biochem. Pharmacol. 41:567.[Medline]
  40. Leeder, J. S., H. M. Dosch, P. A. Harper, P. Lam, S. P. Spielberg. 1989. Fluorescence-based viability assay for studies of reactive drug intermediates. Anal. Biochem. 177:364.[Medline]
  41. Compagnone-Post, P., E. Turco, C. Robinson, M. Trucco. 1988. The ß-chains of DP4 molecules from different haplotypes are encoded by the same gene. Genomics 2:8.[Medline]
  42. Harrison, L. C., M. C. Honeyman, S. Trembleau, S. Gregori, F. Gallazzi, P. Augstein, V. Brusic, J. Hammer, L. Adorini. 1997. A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice. J. Exp. Med. 185:1013.[Abstract/Free Full Text]
  43. Harada, M., S. Makino. 1984. Promotion of spontaneous diabetes in non-obese diabetic-prone mice by cyclophosphamide. Diabetologia 27:604.[Medline]
  44. Tisch, R., B. Wang, D. V. Serreze. 1999. Induction of glutamic acid decarboxylase 65-specific Th2 cells and suppression of autoimmune diabetes at late stages of disease is epitope dependent. J. Immunol. 163:1178.[Abstract/Free Full Text]
  45. Sai, P., A. S. Rivereau, C. Granier, T. Haertle, L. Martignat. 1996. Immunization of non-obese diabetic (NOD) mice with glutamic acid decarboxylase-derived peptide 524–543 reduces cyclophosphamide-accelerated diabetes. Clin. Exp. Immunol. 105:330.[Medline]
  46. Qin, H. Y., J. F. Elliott, J. R. T. Lakey, R. V. Rajotte, B. Singh. 1998. Endogenous immune response to glutamic acid decarboxylase (GAD67) in NOD mice is modulated by adjuvant immunotherapy. J. Autoimmun. 11:591.[Medline]
  47. Homann, D., A. Holz, A. Bot, B. Coon, T. Wolfe, J. Petersen, T. P. Dyrberg, M. J. Grusby, M. G. von Herrath. 1999. Autoreactive CD4+ T cells protect from autoimmune diabetes via bystander suppression using the IL-4/Stat6 pathway. Immunity 11:463.[Medline]
  48. Cetkovic-Cvrlje, M., I. C. Gerling, A. Muir, M. A. Atkinson, J. F. Elliot, E. H. Leiter. 1997. Retardation or acceleration of diabetes in NOD/Lt mice mediated by intrathymic administration of candidate ß-cell antigens. Diabetes 46:1975.[Abstract]
  49. Kash, S. F., B. G. Condie, S. Baekkeskov. 1999. Glutamate decarboxylase and GABA in pancreatic islets: lessons from knock-out mice. Horm. Metab. Res. 31:340.[Medline]
  50. Madrenas, J., L. A. Chau, J. Smith, J. A. Bluestone, R. N. Germain. 1997. The efficiency of CD4 recruitment to ligand-engaged TCR controls the agonist/partial agonist properties of peptide-MHC molecule ligands. J. Exp. Med. 185:219.[Abstract/Free Full Text]
  51. Kearny, E. R., K. A. Pape, D. Y. Loh, M. K. Jenkins. 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1:327.[Medline]
  52. Bercovici, N., J. Delon, C. Cambouris, N. Escriou, P. Debre, R. S. Liblau. 1999. Chronic intravenous injections of antigen induce and maintain tolerance in T cell receptor-transgenic mice. Eur. J. Immunol. 29:345.[Medline]
  53. Genain, C. P., K. Abel, N. Belmar, F. Villanger, D. P. Rosenberg, C. Linington, F. S. Raine, S. L. Hauser. 1996. Late complications of immune deviation therapy in a non-human primate. Science 274:2054.[Abstract/Free Full Text]
  54. Bellmann, K., H. Kolb, S. Rastegar, P. Jee, F. W. Scott. 1998. Potential risk of oral insulin with adjuvant for the prevention of type 1 diabetes: a protocol effective in NOD mice exacerbates disease in BB rats. Diabetologia 41:844.[Medline]
  55. Blanas, E., F. R. Carbone, J. Allison, J. F. Miller, W. R. Heath. 1996. Induction of autoimmune diabetes by oral administration of autoantigen. Science 274:1707.[Abstract/Free Full Text]
  56. Falk, K., O. Rotzschke, L. Santambrogio, M. E. Dorf, C. Brosnan, J. L. Strominger. 2000. Induction and suppression of an autoimmune disease by oligomerized T cell epitopes. enhanced in vivo potency of encephalitogenic peptides. J. Exp. Med. 191:717.[Abstract/Free Full Text]
  57. Qin, H. Y., B. Singh. 1997. BCG vaccination prevents insulin-dependent diabetes mellitus (IDDM) in NOD mice after disease acceleration with cyclophosphamide. J. Autoimmun. 10:271.[Medline]
  58. Hayashi, R. J., D. Y. Loh, O. Kanagawa, F. Wang. 1998. Differences between responses of naive and activated T cells to anergy induction. J. Immunol. 160:33.[Abstract/Free Full Text]
  59. Karges, W., M. Pietropaolo, C. Ackerley, H. M. Dosch. 1996. Gene expression of islet cell antigen p69 (ICAp69) in man, mouse and rat. Diabetes 45:513.[Abstract]
  60. Laufer, T. M., L. Fan, L. H. Glimcher. 1999. Self-reactive T cells selected on thymic cortical epithelium are polyclonal and are pathogenic in vivo. J. Immunol. 162:5078.[Abstract/Free Full Text]
  61. Alting, A. C., R. J. G. M. Meijer, E. C. H. van Beresteijn. 1997. Incomplete elimination of the ABBOS epitope of bovine serum albumin under simulated gastrointestinal conditions of infants. Diab. Care 20:875.[Abstract]
  62. Bachmaier, K., N. Neu, L. M. de la Maza, S. Pal, A. Hessel, J. M. Penninger. 1999. Chlamydia infections and heart disease linked through antigenic mimicry. Science 283:1335.[Abstract/Free Full Text]
  63. Gautam, A. M., R. Liblau, G. Chelvanagam, L. Steinman, T. Boston. 1998. A viral peptide with limited homology to a self peptide can induce clinical signs of experimental autoimmune encephalomyelitis. J. Immunol. 176:60.
  64. Hammond-McKibben, D., H.-M. Dosch. 1997. Cow milk, BSA and IDDM: can we settle the controversies?. Diab. Care 20:897.[Medline]
  65. Malkani, S., D. Nompleggi, J. W. Hansen, D. L. Greiner, J. P. Mordes, A. A. Rossini. 1997. Dietary cow’s milk protein does not alter the frequency of diabetes in the BB rat. Diabetes 46:1133.[Abstract]



This article has been cited by other articles:


Home page
 DiabetesHome page
H. Tsui, Y. Chan, L. Tang, S. Winer, R. K. Cheung, G. Paltser, T. Selvanantham, A. R. Elford, J. R. Ellis, D. J. Becker, et al.
Targeting of Pancreatic Glia in Type 1 Diabetes
Diabetes, April 1, 2008; 57(4): 918 - 928.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. P. Friday, S. L. Pietropaolo, J. Profozich, M. Trucco, and M. Pietropaolo
Alternative Core Promoters Regulate Tissue-specific Transcription from the Autoimmune Diabetes-related ICA1 (ICA69) Gene Locus
J. Biol. Chem., January 3, 2003; 278(2): 853 - 863.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Winer, I. Astsaturov, R. Gaedigk, D. Hammond-McKibben, M. Pilon, A. Song, V. Kubiak, W. Karges, E. Arpaia, C. McKerlie, et al.
ICA69null Nonobese Diabetic Mice Develop Diabetes, but Resist Disease Acceleration by Cyclophosphamide
J. Immunol., January 1, 2002; 168(1): 475 - 482.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Winer, I. Astsaturov, R. K. Cheung, K. Schrade, L. Gunaratnam, D. D. Wood, M. A. Moscarello, P. O'Connor, C. McKerlie, D. J. Becker, et al.
T Cells of Multiple Sclerosis Patients Target a Common Environmental Peptide that Causes Encephalitis in Mice
J. Immunol., April 1, 2001; 166(7): 4751 - 4756.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Winer, I. Astsaturov, R. K. Cheung, L. Gunaratnam, V. Kubiak, M. A. Cortez, M. Moscarello, P. W. O'Connor, C. McKerlie, D. J. Becker, et al.
Type I Diabetes and Multiple Sclerosis Patients Target Islet Plus Central Nervous System Autoantigens; Nonimmunized Nonobese Diabetic Mice Can Develop Autoimmune Encephalitis
J. Immunol., February 15, 2001; 166(4): 2831 - 2841.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Winer, S.
Right arrow Articles by Dosch, H.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Winer, S.
Right arrow Articles by Dosch, H.-M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS