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* Institut National de la Santé et de la Recherche Médicale, Unité 561, and
Department of Pediatric Endocrinology, Groupe Hospitalier Cochin-Saint Vincent de Paul, Paris, France;
Centre National de la Recherche Scientifique, Unité Propre de Recherche 9021, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France;
Institut National de la Santé et de la Recherche Médicale, Unité 567, Institut Cochin, Paris, France; and
¶ Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell autoantigens, has been extensively studied as a model disease for development of autoimmunity (4, 5, 6, 7). Studies in the nonobese diabetic (NOD)3 mouse (4), a spontaneous model of type 1 diabetes, have identified proinsulin, glutamate decarboxylase, tyrosine phosphatase, and several other cell autoantigens as targets of autoimmunity in this model. Autoimmunity in human diabetes and in the NOD mouse results from a multigenic process in which numerous immune and cell defects associate to drive the diabetogenic process (5).
Several models have been established to address the issue of self tolerance to specific Ags expressed by cells in the absence of spontaneous autoimmunity. In a majority of models, transgenes expressed on cells have been used to study how these new self-Ags shape tolerance (8, 9). These studies have greatly enhanced our understanding of immune response regulation, but generalizing their results is limited by artifacts due to the transgenic process. For instance, variable thymic expression of the same transgene in different transgenic mouse lines was observed, leading to differences in the level of autoreactivity after infection with a virus expressing the same Ag (10). Transgenic expression of B7-1 in cells was able to induce an autoimmune reaction against a cell transgene but not against cell native Ags (11).
Proinsulin is thought to be a major autoantigen in type 1 diabetes for several reasons. Insulin (and proinsulin)-specific Abs and autoreactive T cells have been detected in NOD mice and in patients with diabetes or prediabetes. In young children at risk for developing the disease, insulin autoantibodies are detected first (12). Allelic variations in the variable number of tandem repeats region flanking the insulin gene are important determinants of the genetic susceptibility to diabetes (IDDM2) and influence cell insulin secretion and proinsulin expression in the thymus (13, 14, 15, 16). Protection from diabetes is obtained in the NOD mouse by injecting insulin, the insulin B-chain or B-chain peptides (17, 18, 19). Understanding how loss of tolerance to preproinsulin occurs and how IDDM2 regulates diabetes autoimmunity is therefore essential. In the NOD mouse, a progressive loss of tolerance to multiple autoantigens occurs, and deciphering the respective role of each of them is complex. Therefore, evaluating self tolerance to autoantigens such as preproinsulin in nonautoimmune animals allows a better understanding of the mechanisms involved.
Rodents carry two different preproinsulin genes on distinct chromosomes, encoding for two different proteins, both biologically active but differentially expressed in the islet and in the thymus (20, 21). Mice that are deficient for expression of preproinsulin-2 gene are fully viable due to the compensatory effect of the remaining gene (22). They provide a unique opportunity to study the role of this isoform in maintenance of immune tolerance to preproinsulin and to cells. Here, we show that mice deficient in preproinsulin-2 have an enhanced B and T cell reactivity to the missing preproinsulin isoform. Moreover, preproinsulin-2 expressing islets are the target of a nondestructive immune response when transplanted in preproinsulin-2-deficient mice.
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Materials and Methods |
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We used preproinsulin-2 knockout mice (22) (referred to as proins-2-/-), maintained on a pure 129/Sv/Pas/ICo (129) background and devoid of specific murine pathogens. Proins-2-/- mice did not contain the lacZ expression cassette (22) to avoid the interference of immune response to lacZ. Wild-type (wt) 129/Sv/Pas/ICo mice were obtained from Iffa Credo (l’Arbresle, France). Mice were bred as homozygous lines and typed by PCR on tail DNA. We used the following oligonucleotides for PCR amplification (436 bp): forward 5'-GGTGAGTTCTGCCACTGAATTC-3' and reverse 5'-GGCATCAGCAGCACAGAAGCAA-3'. All animal studies were approved by our institutional review board.
Preproinsulin-1 and -2 peptides, recombinant murine proinsulin-2
A set of sixteen 15-mer peptides spanning the sequence of preproinsulin-2 and overlapping by 10-aa residues and relevant homologous preproinsulin-1 peptides were synthesized as described (Ref. 23 ; Table I). In addition, we used two chimeric peptides derived from peptide II56–71: in II56–71–A64E, Ala64 residue was replaced by a glutamic acid residue, as in the preproinsulin-1 isoform; in II56–71–G71S, Gly71 residue was replaced by a serine residue, as in the preproinsulin-1 isoform. As control, we used the human -adrenergic receptor peptide h1AR34–57 known to be efficiently presented by I-Ab molecules (24). Mouse proinsulin-2 produced as a fusion protein with a (His) 6 coding segment immediately upstream of the proinsulin sequence was produced, purified, and checked for the presence of endotoxin as described (25).
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Individual adult mice (12–15 wk old) were immunized at the base of the tail with 50 µg of peptide or 100 µg of recombinant proinsulin-2 emulsified in CFA. Ten days later, draining lymph nodes and spleen were harvested, and cell suspensions were prepared in DMEM containing 5% FCS. Cell suspensions were incubated in triplicate in 96-well plates for 24 h at 5 x 105 cells/well (spleen) or 1 x 105 cells/well plus 5 x 105 irradiated spleen cells from unprimed mice as APCs (lymph nodes), with peptide at a final concentration of 20 µg/ml. Supernatants were harvested 24 h later for IL-2 measurements, and in some experiments culture supernatants obtained from a different parallel culture were harvested at 72 h for IFN-
, IL-4, and IL-10 measurements.
Cytokine measurements
IL-2 concentrations were measured by proliferation of the IL-2-dependent cell line CTLL-2 or by ELISA. Proliferation of CTLL-2 cells was measured by thymidine incorporation (in counts per minute) or expressed in units of IL-2, derived from a standard curve run at the same time using recombinant human IL-2 (PeproTech, Rocky Hill, NJ). For ELISA, we used Abs 1A12 (IL-2), AN18 (IFN-), 11B11 (IL-4), and JES5-2A5 (IL-10) as capture Abs and biotinylated 5H4 (IL-2), R4-6A2 (IFN-), BVD6 (IL-4), and SXC-1 (IL-10) as detection Abs (BD PharMingen, San Diego, CA). Briefly, 50 µl of standard recombinant mouse cytokine (R&D Systems, Minneapolis, MN) or culture supernatants were incubated in coated wells for 2 h, washed, and incubated for 1 h with 50 µl of detection Ab, combined with HRP-streptavidin (AMDEX; Amersham Pharmacia Biotech, Piscataway, NJ) and revealed with OPD substrate solution (Sigma-Aldrich, St. Louis, MO). OD490 was measured using an ELISA reader (MRX Microplate Reader; Dynatech Laboratories, Chantilly, VA). Results are expressed as concentrations of cytokine using recombinant mouse cytokine standards.
ELISPOT assays
Nitrocellulose plates (Millipore MAHA S45) were coated overnight at 37°C with relevant Abs (BD PharMingen: IL-2, 18161D; IL-4, 18191D; IFN-, 18181D), 10 mg/ml in PBS. After a washing step with PBS, 2 x 105 lymph nodes cells (LNCs)/well were dispensed, and peptides were added to a final concentration of 10 µg/ml. Control wells contained cells and medium or cells and Con A (2.5 µg/ml). After 18 h at 37°C in 5% CO2, cells were discarded, and biotin-coupled detection Abs (BD PharMingen: IL-2, 18172D; IL-4, 18042D; IFN-, 18112D) were added for 3 h, followed by streptavidin-conjugated alkaline phosphatase (ExtrAvidin-PA; Sigma-Aldrich) and an alkaline phosphatase conjugate substrate kit (Bio-Rad, Hercules, CA). Spots with diameters above 30 µm were counted with a computer-assisted device (Zeiss, Oberkochen, Germany).
Generation of preproinsulin-2-reactive hybridomas
Mice 12–15 wk old were immunized with peptides as described above. Ten days later, spleen cells and draining LNCs were cultured at 2 x 106/ml in medium with 20 µg/ml peptide. Four days later, blast cells were purified by Ficoll (Amersham Pharmacia Biotech) separation and fused with an hybridoma partner as described (23). After culture in selective medium, hybridomas were tested for reactivity with the immunizing peptide and selected positive hybridomas were cloned by limiting dilution. For testing, hybridoma cells (2 x 104/well) were cultured for 24 h with 5 x 105 irradiated spleen cells and peptide (10 µg/ml) or proinsulin-2 (30 µg/ml), supernatants were harvested, and IL-2 was measured by CTLL-2 proliferation assay. Results are expressed in counts per minute or as the ratio of IL-2 produced with peptide over background IL-2 production by the hybridoma alone. To test the reactivity of hybridomas to islet cells (2 x 104 cells/well), islets were prepared by collagenase digestion as described (26) and dispersed to single cells by treatment with EDTA (10-2 M, 5 min at 37°C) and dispase (0.7 mg/ml; 30 min at 37°C, Boehringer-Mannheim, Mannheim, Germany); islet cell viability was >90% by trypan blue exclusion.
Islet transplantation experiments
Islets of Langerhans were transplanted under the kidney capsule of proins-2-/- mice as described (26). Six weeks later, the pancreas and graft-bearing kidney were harvested for histology, and spleen cells were tested for their reactivity to proins-2 peptides as described above. In other experiments, proins-2-/- mice were immunized with peptide II46–60 or II56–71 in CFA as described above with three boosts in IFA given every 10 days. Two weeks after the last challenge, the animals were transplanted under the kidney capsule with wt 129 islets and analyzed as described above. Mice 6–10 wk old were used in all transplantation experiments. To quantify islet infiltration in kidney capsule transplants, we designed a scoring system where individual islets scored 0 if not infiltrated, 1 if <50% and 2 if 
50% of their periphery was surrounded by inflammatory cells. The slides were read by two observers after coding, and a minimum of 15 islets per graft were counted.
Insulin Ab microradioassay
Human insulin Abs were measured with a microradioassay adapted from (27), using competition with unlabeled insulin. 125I-insulin (Amersham) was incubated with serum with and without cold insulin for 3 days at 4°C. The samples were then incubated with a 50% protein A, 8% protein G-Sepharose mixture (Pharmacia, Peapack, NJ) in a MultiScreen 96-well filtration plate (Millipore, Bedford, MA). After incubation and washing, radioactivity was measured with a 96-well plate scintillation counter (TopCount; Packard Instrument, Meriden, CT). The result was calculated based on the difference between the well with and the well without cold insulin and was expressed as an index = 100 * [(sample 
cpm - negative control cpm)/(positive control cpm - negative control cpm)]. The positive control sample was obtained from a pool of positive NOD mice, and the upper limit of normal values observed in nonautoimmune mice and human sera was set to 1 as described (27).
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Results |
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To examine whether lack of expression of preproinsulin-2 alters the immune response to preproinsulin and to identify potential epitopes, we immunized individual proins-2-/- mice with peptides spanning the sequence of the deficient preproinsulin isoform. As control, age- and sex-matched wt 129 mice were immunized with the same peptides (Fig. 1). In proins-2-/- mice, 7 of 16 preproinsulin-2 peptides were identified that gave an IL-2 production at least 2-fold over background when lymphocytes were stimulated with the immunizing peptide. Among those peptides, the II56–71 peptide elicited a clear response in proins-2-/- but no response in wt mice. The response to the overlapping II50–65 peptide was barely detectable in proins-2-/- and not in wt mice and the other five peptides gave a similar response in proins-2-/- and wt mice. The difference observed with the II56–71 peptide was confirmed in a separate experiment in which five mice of each type were immunized (Fig. 2): the mean IL-2 concentration after stimulation with II56–71 peptide was 4.1 ± 2.2 IU/ml in proins-2-/- compared with 0.8 ± 0.5 IU/ml in wt mice (p < 0.03, Mann-Whitney test). An ELISPOT assay (Fig. 2b) confirmed the production of IL-2 by T cells from proins-2-/- but not wt mice immunized with II56–71 in response to the same peptide (p < 0.05, Mann-Whitney test). No response to the homologous I56–71 peptide was observed after immunization with II56–71 peptide (Fig. 2a) or after direct immunization (data not shown). As expected, wt and proins-2-/- mice responded to the control peptide h1AR34–57 (Fig. 1). Thus, expression of preproinsulin-2 in wt mice is associated with a loss of T cell response to II56–71 peptide.
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cellsTo further study the epitope specificity and Ag presentation of preproinsulin, we generated T cell hybridomas reactive with each of the seven peptides that gave an IL-2 production at least 2-fold over background in proins-2-/- mice (Table II). We used these hybridomas to evaluate whether preproinsulin-2 epitopes identified by peptide immunization were generated in vivo, through processing of endogenous preproinsulin-2. We tested the ability of islet cell preparation from wt (preproinsulin-2 positive) mice to stimulate these hybridomas from proins-2-/- mice (Table II; Fig. 3). None of the hybridomas reactive with II1–15, II14–30, or II26–41 peptides was stimulated by islet cell preparations. In contrast, 10 of 15 II46–60 and 14 of 15 II56–71-reactive hybridomas reacted with wt islet cells, indicating that their respective epitopes could be generated in vivo. There was no relationship between the level of reactivity of hybridomas with peptides and islets. As expected, preproinsulin-2-deficient islet cells from proins-2-/- mice did not stimulate II46–60-reactive and II56–71-reactive hybridomas (data not shown). In additional experiments, selected hybridomas were stimulated with recombinant mouse proinsulin-2 in the presence of irradiated spleen cells. Four hybridomas that reacted with islet cells (respectively, two directed against II46–60 and II56–71 peptides) also reacted with recombinant proinsulin-2. In contrast, two hybridomas against II46–60 peptide, not reacting with islets cells and two hybridomas against II1–15 (outside of proinsulin-2 sequence) were not activated by recombinant proinsulin-2.
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Generation of preproinsulin-2-reactive hybridomas from wt mice
We could detect a T cell polyclonal response to II46–60 but not to II56–71 in wt mice. Therefore, we evaluated whether we could generate hybridomas reacting against these two peptides from wt mice (Table II). We were unable to generate hybridomas against II56–71, confirming that immunogenicity of II56–71 peptide is only observed when preproinsulin-2 expression is deficient. In contrast, we successfully generated hybridomas against II46–60 peptide, raising the question of the presence of islet-autoreactive T cells in the peripheral repertoire of wt mice. As shown in Table II and Fig. 3, none of the II46–60 reactive hybridomas from wt mice responded to islet cells, contrasting with the results observed with II46–60-reactive hybridomas generated from proins-2-/- mice. This difference was not explained by different levels of reactivity to II46–60 peptide. This confirms that the II46–60-reactive T cells detected in proins-2-/- mice are a heterogeneous population and indicates that T cells recognizing islet cells are eliminated from the peripheral repertoire of wt mice.
Response of wt and proins-2-/- mice to recombinant proinsulin-2
T cells from proins-2-/- mice immunized with recombinant proinsulin-2 secreted IL-2 in response to proinsulin-2 and II46–60 but not II56–71 peptide (Fig. 4). The cytokine response was biased toward IFN- and IL-10 but not toward IL-4. In contrast, no response was detected from wt mice.
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Given most experimental models reported thus far, a single Ag expressed by islets is unlikely to elicit an immune response in vivo. We used an islet transplantation model to evaluate whether proins-2-/- mice would mount an immune response against preproinsulin-2-expressing islets (Fig. 5). When wt islets were transplanted in proins-2-/- mice, in four of eight animals a mild and localized inflammatory infiltrate could be detected on serial sections 6 wk after implantation (fIG. 5, b and c). When wt islets were transplanted in proins-2-/- mice who had received multiple challenges with peptide II56–71 or II46–60, peri-insulitis was observed in five of five and three of four mice, respectively (Fig. 5, d–g). In contrast, no cellular infiltrate was detected in two sets of controls, namely proins-2-/- islets grafted in proins-2-/- mice (n = 3) or wt islets grafted in wt mice (n = 5) (Fig. 5, h and i). Because II46–60 peptide is able to induce a response in both types of mice, we also evaluated the effect of immunization with this peptide on the outcome of wt islets grafted in wt mice. No infiltrate and Ab were detected (Fig. 5a) consistent with absence of response to the mature protein (islets and recombinant proinsulin-2) in T cells from wt mice. Skin grafts from wt to proins-2-/- mice were performed to check their histocompatibility and showed no sign of rejection after 3 mo (n = 3). We could not evaluate in vitro the response to preproinsulin-2 peptides in transplanted mice, due to a nonspecific IL-2 background release. In parallel, we measured the levels of circulating Abs to human insulin in the serum from transplanted mice taken 6 wk after grafting. Significant levels of insulin Abs were detected in 9 of 15 proins-2-/- recipients grafted with wt islets whether they were immunized with II46–60 (4 of 4), II56–71 (2 of 5) or not (3 of 6). Insulin Abs were not detected in control mice (0 of 12; Fig. 4a). Noticeably, human insulin was used as substrate and corresponds to residues II25–54 (B-chain) and II90–110 (A-chain) of mouse preproinsulin-2 sequence with several substitutions. II56–71 is located in the C peptide and II46–60 is located at the junction between the B-chain and C peptide. Therefore, our method might be insufficient to detect all Ab reactivity to mouse preproinsulin-2. The pancreatic islets of islet transplant recipients were devoid of cellular infiltrate in all cases. Together, these results indicate that grafted preproinsulin-2 expressing islets are able to induce a response in naive proins-2-/- mice. This response is enhanced by immunization against II46–60 and II56–71, suggesting that these peptides are part of the spectrum of response to the native preproinsulin-2.
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Discussion |
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cell-derived or recombinant mouse proinsulin-2. T cells responding to these epitopes were absent in wt mice expressing endogenous preproinsulin-2. Moreover, preproinsulin-2-expressing islet cells could initiate an immune response or represent the target of a pre-established immune response to preproinsulin-2. In contrast to results obtained in the NOD mouse (23), these experiments allow an appraisal of the mechanisms involved in self tolerance outside the context of spontaneous autoreactivity. Our results have implications for the mechanisms by which self Ags induce immune tolerance and more specifically for the role of insulin in shaping the immune repertoire in type 1 diabetes. We observed several profiles after preproinsulin-2 peptide immunization. First, with II46–60 peptide, a T cell response could be detected similarly in knockout and wt mice, and an epitope was generated from the mature protein by APCs. This would suggest that autoreactive T cells are present in the peripheral repertoire of wt mice. However, only II46–60 peptide-reactive hybridomas from proins-2-/- mice could react with islet cells. In contrast, all hybridomas from wt mice and some hybridomas from proins-2-/- mice did not react with islet cells. This identifies two populations of II46–60-specific T cells (islet-reactive and non-islet-reactive). Recombinant proinsulin-2 stimulated only islet-reactive II46–60-specific T cells (vs non-islet-reactive) and could prime an immune response in proins-2-/- mice. After immunization with recombinant proinsulin-2, a dominant response to II46–60 peptide was detected only in proins-2-/- mice. The response to II46–60 peptide induced in wt mice did not induce an islet graft infiltrate, in contrast to the findings in proins-2-/- mice. Taken together, these observations indicate that islet-reactive II46–60-specific T cells are functionally eliminated in wt mice. The differences underlying these two populations are currently unknown but might involve the epitope(s) recognized or specificities of TCRs. Second, with II56–71 peptide, the profile was different, because the response was detected in proins-2-/- but not wt mice. The corresponding epitope was generated from the mature protein by APCs. This also suggests that T cells reactive with this epitope are functionally eliminated in wt mice. Last, for peptides encoded in the signal peptide and B chain (II1–15, II14–30, and II26–41), we generated hybridomas but were unable to detect a response with islets. This indicates that these epitopes cannot be generated from the mature protein by APCs and probably have little in vivo relevance. Altogether, proins-2-/- mice allowed us to identify two naturally processed epitopes from mouse preproinsulin-2, and corresponding T cells could not be detected in wt mice.
Similar to in vitro observations, transplanted islets expressing preproinsulin-2 could initiate a B and T cell response, amplified by preimmunization by immunogenic peptides. However, within the 6-wk duration of our experiment, the islets were not rejected. Whether islets differing by a single Ag are recognized or destroyed by the immune system has been evaluated in previous transgenic models. For instance, transplanted islets expressing foreign I-E molecules are not destroyed by the immune system (28), although mouse islets expressing a human complement receptor can be the target of a destructive insulitis (26). Similarly, islets expressing a viral protein can be the target of a destructive insulitis only if the immune system is properly activated by virus infection or in the context of TCR-transgenic mice (10, 29). Altogether these models indicate that a destructive anti-islet immune response can target a single antigenic difference on islets but that additional costimulatory signals are generally needed to initiate such a response (30).
The discussion of the mechanism regulating normal tolerance to preproinsulin is relevant to type 1 diabetes. A first issue is the site of gene expression relevant to the regulation of self tolerance (31). Murine preproinsulin-2 gene is expressed in the thymus, although its cellular localization is still controversial (25, 32, 33, 34). Intrathymic dendritic cells have been proposed (33), but recent data suggest that medullary thymic epithelial cells are the primary thymic cells expressing peripheral Ags, in particular insulin (34, 35). In human fetal thymus, the insulin gene is expressed at the RNA and at the protein level (15, 16, 36). At the cellular level, preproinsulin transcripts and protein have been detected in APCs in the thymus and peripheral lymphoid organs (37). Moreover, variations in the levels of expression of the preproinsulin gene have been associated with susceptibility to type 1 diabetes in mouse and human. NOD mice with invalidated preproinsulin-2 gene develop accelerated diabetes and have increased insulin autoantibodies (38). In the human, diabetes susceptibility alleles of the insulin 5'-variable number of tandem repeats are associated with low thymic gene expression (15, 39). The postulated mechanism is that high levels of thymic expression are associated with deletion of autoreactive precursors and that failure to do so participates in the susceptibility to type 1 diabetes (40). Our experimental setting will allow further dissection of this mechanism, in particular through the establishment of thymus or bone marrow chimeras.
The use of knockout models in which the expression of self Ags is abolished allows the analysis of the mechanisms involved in T cell tolerance. Using a similar approach, Huseby et al. (41) showed that expression of myelin basic protein is associated with tolerance to the Ag through negative selection of Ag-specific thymocytes by bone marrow-derived cells presenting the Ag. In their model, myelin basic protein was not synthesized in APCs but was acquired from other cell types. In another model, B cells specific for desmoglein 3 could efficiently be generated in mice lacking this Ag and induce disease (pemphigus) when transferred to normal mice (42). Similarly, mice deficient in serum amyloid P component are intolerant, and thymic expression of serum amyloid P is sufficient to induce tolerance in bone marrow chimeras (34). These models indicate that different tolerance mechanisms might be involved with various somatically expressed autoantigens. Therefore, our model will allow a precise evaluation of these issues in the case of preproinsulin-2.
In conclusion, proinsulin-2 knockout mice display impaired tolerance to several proinsulin-2 epitopes. Islets expressing proinsulin-2 can prime mice to this Ag and can serve as a target to an established proinsulin-2 response. These findings point out the role of proinsulin-2 gene expression in the regulation of proinsulin-2 autoreactivity. Whether islet-derived proinsulin-2 or ectopically expressed Ag play a role as well as the exact mechanisms involved in these regulatory phenomena remain to be determined.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Claude Carel, Institut National de la Santé et de la Recherche Médicale Unité 561, Groupe Hospitalier Cochin-Saint Vincent de Paul, 82 avenue Denfert Rochereau, 75014 Paris, France. E-mail address: carel{at}paris5.inserm.fr
3 Abbreviations used in this paper: NOD, nonobese diabetic; LNC, lymph node cell; wt, wild type.
Received for publication November 20, 2002. Accepted for publication October 7, 2003.
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transcription. J. Clin. Invest. 95:628.
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leads to autoimmunity in transgenic mice. Proc. Natl. Acad. Sci. USA 91:5138.This article has been cited by other articles:
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I. Durinovic-Bello, E. Jelinek, M. Schlosser, T. Eiermann, B. O. Boehm, W. Karges, L. Marchand, and C. Polychronakos Class III Alleles at the Insulin VNTR Polymorphism Are Associated With Regulatory T-Cell Responses to Proinsulin Epitopes in HLA-DR4, DQ8 Individuals Diabetes, December 1, 2005; 54(suppl_2): S18 - S24. [Abstract] [Full Text] [PDF]
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) or proins2-/- mice (
) immunized with each peptide emulsified in CFA were incubated in vitro (5 x 105 cells/well) for 24 h alone or in the presence of the immunizing peptide (20 µg/ml); supernatants were harvested 24 h later, and IL-2 was measured by CTLL-2 proliferation assay (International Units per milliliter); each section represents a different peptide, and each line represents a single mouse. a–h, Peptides eliciting an IL-2 production >2 fold over background; i–l, some peptides eliciting an IL-2 production 
2-fold over background; IL-2 concentrations with peptides eliciting an IL-2 production 
, surrounded by inflammatory cells on <50% (
) or >50% (
) of their periphery; insulin autoantibody levels at the time of euthanasia are also shown on the top of the figure and expressed as index with positive results shown in bold (see Materials and Methods for details); b and c, typical histology in proins-2-/- mice transplanted with wt islets; d and e, typical histology in proins-2-/- mice immunized with II56–71 peptide and transplanted with wt islets; f and g, typical histology in proins-2-/- mice immunized with II46–60 peptide and transplanted with wt islets; h and i, typical histology in proins-2-/- mice transplanted with proins-2-/- islets; white arrows, grafted islets; black arrows, inflammatory cells; white arrowheads, kidney cortex; b, d, f, and h, x10; c, e, g, and i, x40.