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*
Departments of Pediatrics and Immunology, University of Toronto, The Hospital For Sick Children, Research Institute, Toronto, Ontario, Canada; and
Department of Pediatrics, University of Pittsburgh, Children’s Hospital, Pittsburgh, PA 15213
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß cell autoimmunity in humans and diabetes-prone rodents targets a similar spectrum of autoantigens that include insulin/proinsulin and several proteins of ß cell and neuronal origin (1). These "diabetes-Ags" were defined in serological studies where the acquisition of multiple autoantibody specificities was strongly associated with progression of pre-diabetes to overt disease (e.g., Refs. 4, 5, 6). Less is known of the natural history of T cell autoreactivity (7), although patients with recent onset IDDM have autoreactive T cells that recognize glutamate decarboxylase of 65 kDa (GAD65) (8, 9, 10), GAD67 (11, 12, 13), IA-2 (14, 15), insulin/proinsulin (16, 17, 18), islet cell cytoplasmic autoantibody (ICA) 69 (19, 20, 21), and other self Ags (8, 22, 23). However, the measurement of T cell autoreactivity in IDDM has overall been difficult and has not yet attained a level of clinical usefulness. Data presented here demonstrate that autoreactive T cells can be routinely measured and that diabetes onset is characterized by multiple T cell autoreactivities as well as multiple autoantibodies.
We previously developed a serum-free assay system for proliferative T cell responses that allowed detection of autoreactive T cells undergoing anergy upon cognate activation, characterized by normal induction of IL-2 receptor expression but insufficient IL-2 production (20, 24). Here we asked how common are such cells, what is their Ag specificity, and how do they relate to disease course before, at, and after onset of overt IDDM. Based on lengthy pilot studies (24, 25), because of the small response amplitudes routinely observed, and as a basis for a long-term prospective study of pre-diabetes progression, we designed a large, blinded study protocol with stringent validation criteria for the assay system.
Our assay system could detect T cells autoreactive to all of the 10 diabetes-relevant test Ags employed here, although prevalences of positive responses differed. Diabetes onset was characterized by multiple autoreactivities, which were very rare in MHC-matched siblings (sibs) and absent in healthy controls. In patients and the smaller subset of sibs that had autoreactive T cells, most responses to heat shock protein (HSP) 60, GAD65, ICA69, diabetes-associated T cell epitope in ICA69 (Tep69), BSA, and diabetes-associated T cell epitope in BSA (ABBOS) were anergic and required exogenous IL-2. In contrast, T cells specific for proinsulin, IA2, and tetanus toxoid were not anergic. Diabetic children maintained anergic and nonanergic T cell autoimmunity over a 2-yr prospective follow-up period, and patients with longstanding IDDM (3–26 yr) still maintained multiple anergic and nonanergic T cell pools. T cell anergy may contribute to difficulties in their routine detection. We document a greatly enhanced resistance to IL-2 deprivation and apoptosis in patient T cells that may contribute to the persistence of anergic T cell pools and autoreactive T cells in general.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consecutive patients with IDDM, diagnosed at the Children’s
Hospital of Pittsburgh (n = 148, age 10 ± 4.18,
range 1–18 years, 142 white, 6 black) were recruited with informed
consent. Two thirds were analyzed in the first week of diagnosis and
the remainder within 2 mo. Subsequently, 109 index cases provided
2.8 ± 0.8 (range 2–5) consecutive follow-up samples for 22 mo
after onset. Siblings of index cases (n = 51) were
matched for age (age 10.6 ± 4.29, range 1–18, p
= 0.35 vs patient ages) and recruited into a control cohort. These
siblings were chosen to provide age- and MHC-matched controls that
shared environment and a proportion of the autoimmune predisposing
genes with patients, but have a long term disease risk of only 5–10%
(26, 27). As shown in Table I
, siblings and patients had similar MHC
class II (DQ) alleles. Five siblings had GAD65- and five had had ICA,
but no risk-associated DQ alleles. Patients with longstanding IDDM were
recruited from Registry records (n = 31, age 19.2
± 11.6 yr, disease duration 9.2 ± 5.1, range 3–26 yr, age at
onset 9.4 ± 5.2 yr). Forty unrelated, healthy volunteers with no
family history of diabetes were tested on one to four occasions (age
40 ± 9 yr, range 19–55).
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PCR-based DQ tissue typing followed standard procedures (26, 28, 29). ICA were measured by indirect immunohistochemistry on cryopreserved human organ donor as well as rat pancreas sections (26). The measurement of GAD65 and IA-2 autoantibodies employed standard liquid phase radio-immunoassays (5), and insulin autoantibodies were measured with the Kronus kit (Kronus, Sheffield, U.K.). These assays have shown excellent sensitivity and specificity in multiple proficiency workshops.
Blood samples for T cell assays received preservative-free heparin and
were shipped to Toronto by overnight courier in Styrofoam isolators
without ice. Until reaching an enrollment of 92 new onset patients
(62% of the study population), the status of sample donors (patient,
sib, healthy control) was blinded to the Toronto lab. To test
intraassay variability, samples from 12 subjects were split in
Pittsburgh and shipped with different identifications ("blind
duplicates series"). Samples usually had a cell viability of 
95%.
Blood samples from 22 newly diagnosed Toronto patients and 33 of their
first degree relatives were analyzed fresh and after overnight storage
at room temperature. We previously reported that neither prevalences
of positive T cell responses (p values 0.3,
Fisher’s exact test) nor mean amplitudes of responses to the various
test Ags were different between Pittsburgh and Toronto patients with or
without overnight storage (p > 0.2)
(25).
T cell proliferation assay
Mononuclear cells were enriched on Ficoll-Hypaque gradients, and
1 x 105 cells per flat-bottom microculture
well were incubated in 200 µl serum-free Hybrimax 2897 medium (Sigma,
St. Louis, MO) with or without 0.005–10 µg/ml of the test Ags (Table II). Ags (50 µl in medium) were added
to replicate dry wells and allowed to adhere before addition of other
culture ingredients and cells. Optimal Ag doses were reasonably
consistent from responder to responder, and we omitted full Ag dose
responses when cell yields were limiting. To detect anergic T cells,
parallel sets of cultures received 10 U recombinant human IL-2 in
addition to test Ags, as previously described (20). Unless
indicated otherwise, we will refer to results from IL-2-supplemented
responses throughout this paper. After 6 days, cultures were pulsed
overnight with 1 µCi [3H]thymidine,
harvested, and submitted to scintillation counting. Data are presented
as average cpm or mean stimulation indices (SI, cpm test ÷ cpm
unstimulated culture) (20). Addition of IL-2 raised
background [3H]TdR incorporation on average
2-fold, thus lowering SIs by that factor.
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T cell test Ags
Eleven diabetes-associated and control Ags/peptides were
routinely used (* in Table II
). In addition, three self and five
control Ags/peptides were tested in subsets of study subjects as
discussed in the text. All data on HSP60 were obtained with peptide p
227 (31). In pilot studies, two Escherichia
coli-expressed preparations of GAD65 gave unacceptable variability
and considerable numbers of positive responses in healthy controls.
Based on pilot data (24) as well as seven published
reports (10, 32, 33, 34, 35, 36, 37), we used GAD65 peptide 524–543, and
all GAD65 data presented were obtained with this peptide. A
baculovirus-expressed preparation of GAD65 has since become available,
and 18 of 20 GAD65–524-reactive patients showed positive, anergic
responses to the protein as well.
Data analysis and statistics
Our T cell assay validation criteria defined that 1) responses should show consistent Ag dose kinetics, 2) blinded and unblinded data sets should delineate similar distributions (e.g. % positives/negatives) in the different cohorts, and 3) the blind duplicate series should show less than 20% intraassay variation. In addition, and similar to the practice in IDDM autoimmune serology (e.g., Ref. 38), our main functional validation criterion required that a positive/negative discriminator allow distinction of patient from control cohorts. OVA and/or OVA peptide p157 were used as negative control Ag/peptide. Proliferative responses to a given Ag/peptide with a stimulation index 4 SDs above mean OVA/OVA157 peptide responses were deemed positive. Proliferation in OVA- or OVA157-stimulated cultures was similar to that in unstimulated cultures (p > 0.2). Tetanus toxoid provided a control Ag expected to give positive responses in the majority of samples.
Mann-Whitney tests were used to compare numeric results. Significance
was set at 5%, all p values were two-tailed. Since data
distribution was often skewed, nonparametric regression data are
reported where applicable (Spearman, corrected for ties). Fisher’s
exact test was employed to analyze tables, using Katz’ approximation
to calculate relative risks (RR). For larger tables, we performed
2 tests with Yates correction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Results from well over nine thousand replicate T cell assays were evaluated in this report. Thymidine incorporation in cultures with positive responses showed similar replicate variation within ± 11% of the mean. This figure is within the validation criteria and suggests that the numbers of islet reactive T cells was not limiting (39).
Positive T cell responses to diabetes-associated Ags/peptides followed
distinct Ag dose kinetics (Fig. 1).
Reasons for our choice of proinsulin over insulin as test Ag included
results of pilot experiments where insulin often showed erratic,
U-shaped dose responses in patients as well as some controls (not
shown). Diabetes-associated test Ags/peptides (Fig. 1) had defined dose
optima, usually between 0.1–1 µg/ml. Since responders to HSP60 were
rare, a full dose-response curve was available from only four
HSP60-reactive patients (not shown). Fig. 1 (top
panel) also illustrates, with proinsulin as an example, that
responders and nonresponders were clearly distinguished. On a molar
basis, insolubilized protein Ags were processed and presented with
100-fold higher efficiency than peptides (see Materials and
Methods). There were no positive responses to human hemoglobin,
cytochrome c, actin, or four control peptides derived from
regions of homology between ICA69 and BSA outside of the Tep69/ABBOS
epitopes (Table II) (20, 21).
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). This generated two sets of 96
replicate assays/set. Mean response amplitudes in the sample pairs were
highly correlated (r = 0.97, p <
0.0001), and the intraassay variation was ± 6.7%, well within
the validation criteria.
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). The prevalence of positive tetanus
responses was similar in all three groups, but healthy controls had
higher mean tetanus response amplitudes than patients or sibs
(p < 0.001). Positive responses to each of the
diabetes Ags were far more frequent in patients than in siblings
(p < 0.0001, Fig. 3), which in turn had more positive
responses than healthy population controls (p =
0.0009). Responses to the diabetes Ags were rare in healthy volunteers:
seven had self-reactive T cells recognizing a single islet Ag. In
addition, there were some positive responses to the one environmental
Ag used, BSA (n = 4) or its ABBOS epitope
(n = 1) (20). Normal controls were
reexamined one to four times over the ensuing 22 mo, and only two
positive responses remained, whereas three new responses appeared
transiently. This contrasts with results from the patient follow-up
study, which indicate that response patterns are stable within 2 yr of
prospective follow-up (see below).
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). Nevertheless, definition of
positive responses as 4 SDs above background proliferation delineated
significant differences in the prevalence of positive responses to a
given test Ag between patients, low risk sibs, and population controls
(Fig. 3, Table III). Overall, the assay system appears to be valid by
our criteria. T cell pools recognizing diabetes-relevant Ags 1)
circulate in peripheral blood of most diabetic individuals, 2) are
detectable in some of their relatives, but 3) rarely and transiently in
healthy population controls. Effects of exogenous IL-2
Most (78–91%) positive responses to GAD65, ICA69, Tep69, BSA,
ABBOS, and HSP60 Ags/peptides were observed only in the presence of
added IL-2, consistent with the presence of anergy (Fig. 4). In contrast, responses to proinsulin,
IA-2, and tetanus toxoid showed essentially no IL-2 effect (Fig. 4).
The remaining 10 test Ags/peptides (Table II) failed to elicit
responses in the presence or absence of added IL-2. Anergic and
nonanergic response patterns were the same in patients and sibs
(including those from fresh Toronto samples reported earlier (24, 25)). Anergic responses were thus Ag, but not donor or disease
course specific, and they covered several but not all diabetes Ags even
in subjects with a low risk to develop the disease. This is reminiscent
of NOD mice where a broad tendency to sustain anergic T cell pools has
been described (40).
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, anergic responses could be rescued
not only by addition of IL-2, but also by costimulation with either
proinsulin, IA2, or tetanus. Proliferation in cultures stimulated by
OVA or with other "anergic" Ags showed no rescue effect. Thus, in a
host with both anergic and nonanergic T cells, the latter may rescue
the former if they happen to encounter their respective Ag at similar
times and locales, such as a given islet. However, rescue can also be
provided by other bystanders, not primarily related to diabetes, in
this case tetanus toxoid-activated T cells (Table IV). Bystander
mechanisms have previously been implied in diabetes development
(41).
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As the main control cohort for patient data analysis, we used
siblings with a low risk to develop diabetes, matched for MHC (Table I), age, and environment. Except for tetanus, OVA, and HSP60, patients
had T cell responses to the test Ags in 85% (Table III), three
quarters targeting multiple (at least four) test Ags. Since 82% of
patients had multiple (3) autoantibodies, no correlation could be
delineated between any autoantibody and any (anergic or
nonanergic) T cell responses measured. In contrast, only four siblings
(7.8%) responded to multiple diabetes Ags (Fig. 3), whereas two thirds
had no or one positive T cell response (p values
<0.0001, Table III). None of the healthy controls had multiple T cell
responses.
We were able to compare several peptides and isoforms of one
autoantigen, ICA69. Patient responses to the full-length 
-, the
C-terminal truncated ß isoforms of ICA69, and to Tep69 peptide
(21) were concordant (r = 0.84, data not
shown), suggesting the absence of important target epitopes outside of
Tep69. We have previously suggested that mimicry between an epitope
present in dietary cow milk and ICA69 may play a role in diabetic
autoimmunity and demonstrated, in both patients and NOD mice, T cells
that show antigenic mimicry between the BSA-derived ABBOS peptide and
its ICA69 homologue, Tep69 (20, 21, 42). In the present
study, responses to ABBOS and ICA69/Tep69 were highly concordant in
patients, but not low risk siblings (Table V, p = 0.001, RR =
32.8). Mimicry thus appears to be a marker of disease risk, despite the
fact that these cells are anergic by our criteria. To test this
possibility, we have begun to analyze first degree relatives of our
index cases that have a high risk to develop overt disease, based on
the presence of autoantibodies and risk-associated but not protective
DQ alleles (26, 27). Only 1 of 27 tested high risk
subjects with Tep69-reactive T cells had undetectable ABBOS responses
(p = 0.012 vs sibs, p = 0.4 vs
patients). The maintenance of these cross-reactive T cell pools thus is
associated with progressive pre-diabetes and high disease
risk.
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Autoimmunity after diabetes onset
Blood samples (n = 297) from 109 index cases were
analyzed on two to five occasions over a period of 22 mo after onset
(Fig. 5A). In 64.2% of these
patients, positive and negative response patterns to a given test Ag
remained the same throughout the observation period, 17% showed
additional responses to one (10%) or more (7%) test Ags, and 13%
lost reactivity to one or more. Only two patients (1.8%) had very
variable patterns, and five showed intermittent changes that returned
to onset patterns. A trend for lower prevalences of T cell responses
early after onset did not reach significance (p
> 0.2). The fact that proinsulin responses were at best intermittently
affected by insulin therapy would tend to confirm reports that target
epitope(s) lie outside of the mature insulin molecule
(43). Anergic T cells did not behave differently from
nonanergic T cells recognizing proinsulin or IA2.
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B). Surprisingly, 35% recognized almost
all test Ags, 54% recognized half or more, and only 16% had no or one
responses. Humoral autoimmunity was active as well, with 63% of
patients still positive for ICA. For example, a patient with 22 years
disease duration and undetectable levels of C-peptide for many years,
had ICA of 320 JDF units and T cells responding to all test Ags but
HSP60. There was no correlation between humoral or cellular
autoreactivities or the duration of disease. Anergic and nonanergic T
cell pools again behaved similarly, suggesting that T cell anergy was
independent of disease status and duration. Abnormalities of diabetic T cells
While the basis of anergy to some of the Ags measured here is
unknown, the reversal by IL-2 indicates that IL-2 deprivation plays a
critical role. The appearance of anergic T cells in even young siblings
with low disease risk and minimal signs of autoimmunity, and the
persistence of these cells even decades after disease onset, implied
the possibility of an underlying abnormality in T cell function, as has
been demonstrated in nonobese diabetic (NOD) mice (e.g., Refs.
40, 44, 45). Lymphocytes from patients, sibs, or controls
were stimulated with anti-CD3 in the presence or absence of graded
amounts of anti-IL-2. When measured in an apoptosis assay,
anti-IL-2 induced cell death in a dose-dependent fashion as
expected (not shown). However (Fig. 6),
patient and some of the sib T cells required dramatically (20- to
100-fold) higher anti-IL-2 doses than controls. Measuring
[3H]TdR incorporation gave the same results
(not shown). These data indicate that diabetic T cells can survive even
severe IL-2 deprivation, which kills activated normal T cells. In
this sense, T cell function in diabetes is highly IL-2 sensitive,
enhancing susceptibility to bystander effects and synergizing with the
reluctance to die toward persistence of anergic cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Assay validation was an important part of this study, and we conclude that all validation criteria were met. The stability of T cell response patterns in index cases over the 2 yr of prospective follow-up support this conclusion.
Previously reported observations and pilot experiments for the present
study established that the responses measured here involved MHC class
II-restricted CD4+ T cells that respond to
cognate activation with IL-2 receptor expression and recruitment of
p56lck (20, 25, 46, 47). The
absolute response amplitudes observed were small, as has been observed
by others (13, 16). In preliminary experiments, we also
measured cytokine production in proinsulin- or IA2-stimulated cultures.
IL-4, IL-10 and IFN-
responses were found in 5 of 11 patients
studied, but the ratios of IFN-, IL-4, and/or IL-10 varied. Two
patients had neither proliferative nor cytokine responses, and four
produced mainly IFN-. Further cytokine studies could be of
mechanistic interest and help to distinguish relatives with high or low
disease risk.
Our definition of positive responses was based on proliferation in
cultures containing a control protein or peptide OVA/OVA157. Using
thymidine incorporation either in unstimulated cultures or in
MHC-matched siblings or in unrelated controls unresponsive to the test
Ags would not have altered the principal study conclusions but added
variability. Thus, the data analysis strategy was optimal to
distinguish T cell responses of patients, low risk sibs, and controls.
This is a practice commonly used to validate autoantibody assays (e.g.,
Refs. 38, 48). Our definition of positive responses as
an SI 4 SDs above control values has statistical power and
accommodates intraassay variation; hard cut-off rules for SI values
such as 2 or 3 are arbitrary and would have reduced the strength of
patient-sib distinctions.
In our hands, a serum-free culture system appears to exaggerate or unmask the requirement for exogenous IL-2 in responses to some diabetes Ags. Among published reports of GAD65, ICA69, or HSP60 responses (8, 9, 10, 11, 12, 13, 19), only ours used a serum-free culture system and identified anergic T cells (20, 21, 46). Serum supplements may in some way contribute to responses observed, perhaps allowing low level IL-2 production by recently activated, circulating bystander T cells that rescues some anergic lymphocytes; the IL-2 deprivation studies emphasize how little IL-2 is needed by patient T cells.
The Ag restriction of anergy was robust. It will be necessary to determine whether these Ags activate T cells in a peculiar way, perhaps through unusual MHC binding (49, 50), which is biased toward anergy in a precarious fashion (51). Inherent T cell abnormalities may contribute to anergic responses; signaling abnormalities have been associated with abnormal anergy in NOD mice (52).
Where tested, protein and peptide (epitope) responses followed the same
patterns. This could imply that anergic T cell responses reflect
peculiarities of the sensitization process to these Ags, which in the
case of the BSA/ABBOS
ICA69/Tep69 mimicry pairs of Ags includes
exposure through the oral route (53, 54). We recently
tested a full-length GAD65 preparation and found that responses nearly
always (18 of 20 responders) required exogenous IL-2, like the GAD-524
peptide used throughout the study. Responses to the study GAD peptide
thus are likely typical for responses to GAD65 in general. Preliminary
data from a collaborative study with A. Notkins (National Institutes of
Health, Bethesda, MD) identified several peptide target epitopes in
IA2, and all these responses were nonanergic (A Notkins, unpublished
observations).
Autoimmunity continues unabated in many patients long after disease onset. This confirms clinical observations (55, 56, 57) and presents a challenge for transplantation and gene therapy approaches in diabetes. Humoral autoimmunity was previously reported to continue in C-peptide-negative patients (58, 59). It is unclear what sustains this autoimmunity. Relevant islet Ags might be supplied through continued regeneration of ß cell precursors, sufficient to maintain autoimmune repertoires, even if these ß cells die before functional maturity. If correct, then immune intervention even at a late stage of disease could have promise if it was effective.
We were surprised that anergic T cells persisted as well and as long as nonanergic cells. Our observations delineate two mechanisms that could contribute to persistence: an ability to resist cell death even under severe IL-2 deprivation and rescue from apoptotic ablation through IL-2 from bystander lymphocytes. The molecular mechanisms of death resistance are unknown, but abnormal expression of members of the bcl-2 family of proteins has been described in NOD mice (44, 45) and needs to be examined in patient T cells, as does the role of other cytokines, such as Type I IFNs (60). High resistance of autoreactive T cells to cell death may explain the difficulties to break autoimmunity through immunosuppressive therapies (59).
In an extension of these studies to a large cohort of first degree relatives with varying levels of diabetes risk (H.-M. Dosch et al., manuscript in preparation), high long-term disease risk was associated with multiple T cell autoreactivities, while multiple autoantibodies appeared only closer to overt disease. Among these autoreactivities, nonanergic responses to proinsulin and IA2 were strongly associated with disease risk, while multiple anergic T cell autoreactivities were also found in subjects with moderate disease risk. However, data presented here directly associated targeting of the ABBOS/Tep69 mimicry epitopes with disease risk. This could imply that such anergic cells actually serve as effector cells. If these cells enter into insulitis lesions, the chance to encounter relevant APCs as well as be rescued by nearby, nonanergic T cells specific for proinsulin or IA2 may be considerable and allow expression of effector function. The need for coincidence of cognate activation of both classes of T cells would add a stochastic element to islet destruction and perhaps relate to the long periods of time required for completion of ß cell destruction and overt disease.
The previously reported anergy of diabetes-associated T cell pools (20, 46, 61) is unexpectedly broad. And unexpectedly, anergy is not a functional property that changes over the course of pre-diabetes and overt disease, but it rather appears to be a characteristic of some of the major autoantigens targeted in IDDM. Equally unexpected was the long persistence of autoimmune T cells up to 26 years after onset of overt disease, data consistent with the clinical experience of a rapid emergence of autoimmunity postislet transplantation. However, it remains difficult to envision the Ag sources that maintain, for example, proinsulin-reactive T cells, or for that matter, GAD65-specific T cells when even mature ß cells express barely detectable levels. Collectively, the data presented here argue that validated assays of diabetes-associated T cell abnormalities offer important new insights and questions in diabetic autoimmunity.
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Acknowledgments |
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and critical help throughout the project. We gratefully
acknowledge the excellent efforts of the General Clinical Research
Center nurses, J. Gay, K. Riley, and S.
Pietropaolo. Dr. M. Trucco provided advice and support
throughout the project. We are also grateful to Drs. D. Daneman
and D. Wherrett (Hospital for Sick Children, Toronto) for providing
fresh patient and FDR samples during development and validation of the
T cell assay system. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. H.-Michael Dosch, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8. E-mail address:
3 Current address: Department of Internal Medicine, University of Ulm, Ulm, Germany.
4 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; ABBOS, diabetes-associated T cell epitope in BSA (see Table II); FDR, first degree relative of index case with IDD; GAD65, glutamate decarboxylase of 65 kDa; ICA, islet cell cytoplasmic autoantibodies; PI, proinsulin; RR, relative risk; SI, stimulation index (mean cpm test:mean cpm control); Tep69, diabetes-associated T cell epitope in ICA69 (see Table II); sibs, siblings; HSP, heat shock protein; NOD, nonobese diabetic.
Received for publication July 27, 1999. Accepted for publication October 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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kerblom. 1996. Islet cell autoimmunity and progression to insulin-dependent diabetes mellitus in genetically high- and low-risk siblings of diabetic children. The Childhood Diabetes in Finland (DiMe) Study Group. Eur. J. Clin. Invest. 26:640.[Medline]
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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]
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S. Winer, L. Gunaratnam, I. Astsatourov, R. K. Cheung, V. Kubiak, W. Karges, D. Hammond-McKibben, R. Gaedigk, D. Graziano, M. Trucco, et al. Peptide Dose, MHC Affinity, and Target Self-Antigen Expression Are Critical for Effective Immunotherapy of Nonobese Diabetic Mouse Prediabetes J. Immunol., October 1, 2000; 165(7): 4086 - 4094. [Abstract] [Full Text] [PDF]
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