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HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes¹

Toshiyuki Takaki^2,*, Michele P. Marron^2,3,, Clayton E. Mathews, Stephen T. Guttmann^*, Rita Bottino, Massimo Trucco, Teresa P. DiLorenzo^4,*, and David V. Serreze^4,

^* Department of Microbiology and Immunology and Department of Medicine (Division of Endocrinology), Albert Einstein College of Medicine, Bronx, NY 10461; The Jackson Laboratory, Bar Harbor, ME 04609; and Department of Pediatrics and Diabetes Institute, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213

	Abstract

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In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4⁺ and CD8⁺ T cell responses against pancreatic cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8⁺ T cell responses contributing to T1D development. CD8⁺ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

	Introduction

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Type 1 diabetes (T1D)⁵ in both humans and NOD mice is a multifactorial disease resulting from T cell-mediated autoimmune destruction of insulin-secreting pancreatic cells. Although T1D is a polygenic disease, the primary susceptibility component in both organisms is particular combinations of MHC genes (reviewed in Refs.1 and 2). Within the MHC, specific HLA-DQ and HLA-DR class II molecules provide a large component of T1D susceptibility in humans by mediating cell-autoreactive CD4⁺ T cell responses. Similarly, the H-2A^g7 class II variant provides an important component of T1D susceptibility in NOD mice. It is now clear that in addition to the pathogenic effects mediated by CD4⁺ T cells, T1D development also requires contributions from autoreactive MHC class I-restricted CD8⁺ T cells (3, 4, 5, 6, 7, 8). Interestingly, the H-2D^b and H-2K^d class I molecules encoded within the H2^g7 MHC haplotype of NOD mice are common variants also characterizing many strains lacking autoimmune proclivity. However, efficient T1D development in NOD mice is dependent on expression of the class I genes characterizing the H2^g7 haplotype (9, 10, 11, 12). The ability of the H2^g7-encoded class I variants to mediate the development of diabetogenic CD8⁺ T cell responses when expressed in NOD is due to interactive contributions from other susceptibility genes characterizing this strain (13, 14).

Association studies suggest certain MHC class I molecules, including the common variant HLA-A*0201 (hereafter designated HLA-A2.1), likely confer an additional risk factor for T1D development in humans when expressed in conjunction with particular class II susceptibility alleles, and perhaps other genes (15, 16, 17, 18, 19, 20, 21). Because it is difficult to directly assess the role of class I MHC molecules in T1D development in patients, we have used a "humanized" NOD mouse model to do so. Previously, we showed that T1D development is accelerated in NOD mice transgenically expressing the human HLA-A2.1 H chain (designated NOD.HLA-A2.1) (22). Furthermore, HLA-A2.1-restricted cell-autoreactive CD8⁺ T cells can be isolated from islets of young transgenic NOD mice. However, NOD.HLA-A2.1 mice also express the murine class I MHC molecules H-2K^d and H-2D^b, making it impossible to precisely define the ability of HLA-A2.1 alone to mediate autoreactive CD8⁺ T cell responses contributing to T1D development. To address this important issue, we used the chimeric monochain transgene construct designated HHD (23), which encodes human ₂-microglobulin (2m) covalently linked to the 1 and 2 domains of human HLA-A2.1, and the 3, transmembrane, and cytoplasmic domains of murine H-2D^b. We transgenically introduced the HHD construct into NOD mice and then crossed the transgenic mice to the NOD.2m^null strain to eliminate expression of murine class I MHC molecules. In HLA-transgenic models, the frequency of CD8⁺ T cells restricted to human class I MHC molecules is often low due to the poor interaction between murine CD8 and the 3 domain of human class I MHC molecules (24). The presence of a murine 3 domain in HHD molecules is designed to overcome this difficulty.

In this study, we show that NOD.2m^null.HHD mice are T1D-susceptible. This demonstrates that when expressed in the proper context, HLA-A2.1 alone can mediate sufficient cell-autoreactive CD8⁺ T cell responses to elicit T1D. Studies were also conducted to determine whether NOD.2m^null.HHD mice allow identification of candidate cell autoantigens that could be tested in future clinical studies as possible targets of pathogenic CD8⁺ T cells in HLA-A2.1-expressing T1D patients. CD8⁺ T cells isolated from NOD.2m^null.HHD mice were found to lyse human HLA-A2.1-positive islet cells. A subset of these T cells recognize the cell Ag islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), previously identified as an important target of CD8⁺ T cells in standard NOD mice (25, 26). We have mapped three novel epitopes of IGRP that are recognized by HLA-A2.1-restricted T cells in NOD.2m^null.HHD mice. Importantly, one of these antigenic peptides is conserved between mouse and human IGRP, and the other two are similar. Therefore, humanized NOD.2m^null.HHD mice can be used to identify HLA-A2.1-restricted T cells and cell autoantigens potentially relevant to human T1D.

	Materials and Methods

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Mice

NOD/LtDvs mice are maintained at The Jackson Laboratory by brother-sister mating. Currently, diabetes develops in 90% of female and 63% of male NOD/LtDvs mice by 30 wk of age. NOD.2m^null (3) and NOD.Rag1^null mice (27) have been previously described. The HHD transgene, provided by F. Lemonnier (Institut Pasteur, Paris, France), has been described (23). The transgene was injected directly into NOD zygotes, and founders carrying the HHD transgene were identified by PCR using forward primer 5'-CTTCATCGCAGTGGGCTAC-3' and reverse primer 5'-CGGTGAGTCTGTGAGTGGG-3'. Cell surface expression of transgenic HLA-A2.1 molecules in NOD.HHD mice was confirmed by flow cytometry using FITC-conjugated mAb CR11-351 (28), provided by V. Engelhard (University of Virginia, Charlottesville, VA). To eliminate expression of murine class I MHC molecules, the HHD transgene was transferred to the previously described NOD.2m^null background (3). The 2m^null mutation was fixed to homozygosity by backcrossing F₁ progeny to NOD.2m^null mice, then intercrossing to establish the NOD.2m^null.HHD line. The HHD transgene also was transferred to the NOD-scid.Emv30^null background (29) and fixed to homozygosity (designated NOD-scid.HHD mice). All mice were housed under specific pathogen-free conditions. All experiments involving mice were performed in compliance with federal laws and institutional guidelines and have been approved by an institutional animal care and use committee.

Assessment of T1D and insulitis development

T1D development was defined by glycosuric values of 3 as assessed with Ames Diastix (supplied by Miles Diagnostics). Rates of diabetes development in the indicated experimental groups were assessed for statistically significant differences by Kaplan-Meier life table analysis using Statview 4.5 (Abacus Concepts).

Insulitis levels were assessed in nondiabetic 8-wk-old female NOD and NOD.2m^null.HHD mice. Pancreata from these mice were fixed in Bouin’s solution, and sectioned at three nonoverlapping levels. Granulated cells were stained with aldehyde fuchsin and leukocytes with an H&E counterstain. Islets (at least 20 per mouse) were individually scored as follows: 0, no lesions; 1, peri-insular leukocytic aggregates, usually periductal infiltrates; 2, <25% islet destruction; 3, >25% islet destruction; and 4, complete islet destruction. An insulitis score for each mouse was obtained by dividing the total score for each pancreas by the number of islets examined. Data are presented as mean insulitis scores ± SEM for each strain.

Flow cytometric analysis of splenic leukocyte populations

Single-cell suspensions of splenocytes were analyzed by multicolor flow cytometric analysis. Total B lymphocytes were detected by staining with FITC-conjugated polyclonal anti-mouse Ig (Southern Biotechnology Associates). Total T lymphocytes were detected by staining with FITC-conjugated CD3-specific mAb 145-2C11. Total T cells were then further characterized for CD4 expression using the mAb GK1.5 conjugated to the red fluorescent tag Cy3.18-OSu (Cy3; Biological Detection Systems), or for CD8 expression with the mAb 53-6.72 conjugated to PE whose red fluorescence intensity can easily be distinguished from that of Cy3. CD8⁺ T cell populations were also analyzed by staining with PE-conjugated TCR -chain-specific mAb H57-597 and FITC-conjugated CD8-specific mAb 53-6.72. Expression of murine MHC class I molecules was assessed by staining with FITC-conjugated H-2K^d-specific mAb SF1-1.1, FITC-conjugated H-2D^b-specific mAb KH95, and FITC-conjugated pan murine class I MHC mAb M1/42. Expression of transgenic HLA-A2.1 molecules was assessed by staining with FITC-conjugated mAb BB7.2.

Cytotoxicity assays using intact islets as targets

Human pancreata that were not allocated for whole organ transplantation were obtained from organ procurement organizations (Center for Organ Recovery and Education, Pittsburgh, PA, and National Disease Research Interchange, Philadelphia, PA). Pancreata were harvested using standard multiorgan recovery techniques. Pancreatic islets were isolated using the semiautomated method described previously (30) with minor modifications. Pancreas dissociation was performed using multiple lots of Liberase (Liberase-HI; Roche). Enzymes were reconstituted and dissolved in cold (4°C) HBSS. Before being digested in a modified continuous digestion-filtration device, pancreata were intraductally injected with enzyme solution in a recirculation system designed by Rajotte and colleagues (31). Islets were purified with a COBE 2991 cell separator using discontinuous Euro-Ficoll gradients; the purity (islets/whole tissue) was assessed by dithizone staining, as previously described (32). Preliminary HLA typing was performed by the laboratory of the local donor hospital (HLA-A*02/A*68 sample) or the typing laboratory of the Allegheny General Hospital (Pittsburgh, PA) (HLA-A*11/A*13 sample). High-resolution HLA typing was performed using the PEL-FREEZ SSP UniTray PCR-based method for specific HLA class I alleles as per the manufacturer’s directions (Dynal Biotech).

Mouse islets were isolated from female NOD-scid.HHD and NOD.Rag1^null animals using a published collagenase inflation method (33). Islets were hand-picked for purity in HBSS under a dissecting microscope after isolation on a Histopaque 1119 (Sigma-Aldrich) gradient.

Cytotoxicity assays using intact islets as targets were performed as described (13, 34). Briefly, human, NOD-scid.HHD, and NOD.Rag1^null pancreatic islets (10 islets/well) were allowed to adhere in 96-well plates during a 10-day incubation at 37°C in low-glucose DMEM. Adherent, monolayered islets were then labeled with 5 µCi/well of ⁵¹Cr for 3 h at 37°C. Islets were washed and overlaid with 100 µl of medium containing various numbers of cultured islet-infiltrating T cells from NOD.2m^null.HHD mice. For establishing E:T ratios, each islet was assumed to contain 750 cells. A minimum of three wells were established for each E:T ratio. Spontaneous release controls consisted of nine wells of labeled islets from each donor cultured in the absence of T cells. Following a 20-h incubation at 37°C, the radioactivity in two fractions from each well was measured. The first fraction was the culture supernatant, and the second was obtained by solubilizing the remaining islets in 200 µl of 2% SDS. The percentage of ⁵¹Cr release for each well was calculated by the formula ((supernatant cpm)/(supernatant cpm + SDS lysate cpm)) x 100%. This allowed us to normalize for the fact that due to variation in the sizes of individual islets, the total levels of ⁵¹Cr incorporation in each well could differ greatly. In turn, the percent-specific cytotoxicity was calculated by subtracting the percent ⁵¹Cr release from islets cultured in medium alone (i.e., spontaneous release) from the release by each well of islets cultured with a given number of T cells. Percent-specific cytotoxicities against the different islet types were assessed for statistically significant differences by Bonferroni/Dunn analysis using SuperANOVA (Abacus Concepts).

IGRP peptide library and individual synthetic peptides

A peptide library containing all of the 8-, 9-, 10-, and 11-mer peptides that can be derived from murine IGRP was synthesized by Mimotopes using their proprietary Truncated PepSet technology. Each peptide mixture in the library contained four peptides with a common C terminus, but having a length of 8, 9, 10, or 11 residues. The four peptides in each mixture were present in approximately equimolar amounts. A library of 348 peptide mixtures was synthesized to cover the 355 aa of the IGRP protein. Concentrated peptide stocks (2.75 mM) were prepared in 50% acetonitrile/H₂O, and 40 µM (i.e., 10 µM for each peptide in the mixture) working stocks were obtained by serial dilution in PBS (pH 6.5). Murine (m) IGRP_206–214 (VYLKTNVFL), mIGRP_226–236 (RLFGIDLLWSV), mIGRP_227–236 (LFGIDLLWSV), mIGRP_228–236 (FGIDLLWSV), mIGRP_229–236 (GIDLLWSV), mIGRP_265–273 (VLFGLGFAI), mIGRP_337–345 (ALIPYCVHM), human (h) IGRP_228–236 (LNIDLLWSV), hIGRP_337–345 (AFIPYSVHM), and Flu-MP_58–66 (GILGFVFTL) peptides were synthesized by standard solid-phase methods using Fmoc chemistry in an automated peptide synthesizer (model 433A; Applied Biosystems), and their identities were confirmed by mass spectrometry. Concentrated stocks (10 mM) were prepared in DMSO, and 10 µM working stocks were obtained by dilution in PBS.

Propagation of islet-infiltrating T cells

Islet isolation by collagenase perfusion of the common bile duct was modified from a previously described protocol (35). Briefly, the bile duct was cannulated and the pancreas perfused with collagenase P (Roche). The inflated pancreas was removed and incubated at 37°C to digest exocrine tissue. Following dispersion of digested tissue and three washes with HBSS, islets were resuspended in HBSS containing DNase I (Worthington Biochemical) and handpicked using a siliconized micropipet under a dissecting microscope. Isolated islets were washed with 2% FBS in HBSS, resuspended in RPMI 1640 medium supplemented with 10% FBS (HyClone) and 50 U/ml recombinant human IL-2 (PeproTech), and cultured in 24-well tissue culture plates (50 islets/well) at 37°C, 5% CO₂ for 7 days. For the experiment depicted in Fig. 3C, where purified CD8⁺ T cells were used, 1.6 x 10⁷ cultured islet-infiltrating T cells were collected from four 11-wk-old female NOD.2m^null.HHD mice and incubated for 30 min with 40 µl of FITC-conjugated anti-CD8 Ab (53-6.7; BD Pharmingen). Then, T cells were washed three times and sorted using a MoFlo Fluorescence Activated Cell Sorter (DakoCytomation). After sorting, 97% of cells were CD8⁺ T cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Cultured islet-infiltrating T cells from NOD.2mnull.HHD mice recognize murine IGRP peptides. A, COS-7 cells were transiently transfected with varying concentrations of a murine IGRP cDNA expression construct and 1 µg/ml of an HHD (•), H-2Db (x), or H-2Kd () cDNA expression construct. Cultured islet-infiltrating T cells from 12-wk-old female NOD.2mnull.HHD mice were added, and T cell response was measured as IFN- release by ELISA. T cells did not respond to COS-7 cells cotransfected with the murine IGRP expression construct and pcDNA3.1, nor did they respond to COS-7 cells cotransfected with the HHD expression construct and pcDNA3.1 (data not shown). B, Islets from four 10-wk-old female NOD.2mnull.HHD mice; C, four 11-wk-old female NOD.2mnull.HHD mice; or D, five 12-wk-old female NOD.HHD mice were pooled to obtain sufficient numbers of T cells to screen the murine IGRP peptide library. Islets were cultured in the presence of IL-2, and islet-infiltrating T cells were harvested after 7 days. For the experiment depicted in C, CD8+ T cells were purified from the culture. Human T2 cells were used as APC and T cell response was measured by IFN- ELISPOT assay.

IFN- ELISPOT

ELISPOT plates (MAHA S45 10; Millipore) were precoated with anti-murine IFN- mAb (R4-6A2; BD Biosciences/BD Pharmingen) and blocked with 1% BSA (Fraction V; Sigma-Aldrich) in PBS. APC (mitomycin C-treated T2 cells; American Type Culture Collection) were added at 2 x 10⁴ cells/well and pulsed with 1 µM peptide. Cultured islet-infiltrating T cells were added at 2 x 10⁴ cells/well, and plates were incubated at 37°C for 40 h. IFN- secretion was detected with a second, biotinylated anti-murine IFN- mAb (XMG1.2; BD Pharmingen). Spots were developed using streptavidin-alkaline phosphatase (Zymed Laboratories) and 5-bromo-4-chloro-3-indolyl-phosphate/NBT chloride substrate (Sigma-Aldrich) and counted using an automated ELISPOT reader system (Autoimmun Diagnostika).

Transient transfection

COS-7 cells were transfected using a DEAE-dextran protocol as described (36). cDNA expression constructs (1 µg/ml) for class I MHC molecules (HHD/pcDNA3.1, H-2K^d/pRSV.5.neo, or H-2D^b/pcDNAI) were used along with varying concentrations of a murine IGRP cDNA expression construct (mIGRP/pcDNA3.1) or pcDNA3.1 alone as indicated in the legend to Fig. 3A. mIGRP/pcDNA3.1 was provided by P. Santamaria (University of Calgary, Calgary, Alberta, Canada), H-2D^b/pcDNAI by N. Shastri (University of California, Berkeley, CA), and H-2K^d/pRSV.5.neo by T. Hansen (Washington University Medical School, St. Louis, MO). Following coculture with cultured islet-infiltrating T cells, T cell response was measured as IFN- release by ELISA using capture (R4-6A2) and detecting (biotinylated XMG1.2) anti-murine IFN- mAbs purchased from BD Biosciences/BD Pharmingen. Plates were developed with streptavidin-conjugated HRP (Southern Biotechnology Associates) and tetramethyl benzidine (Pierce Biotechnology).

Peptide-binding assay

Peptide binding to the HLA-A2.1 molecule was determined as described (37). Briefly, T2 cells were incubated for 18 h at 26°C and washed in serum-free culture medium. Cells (2 x 10⁵) in 60 µl of serum-free medium were added to U-bottom 96-well culture plates with 20 µl of peptide solution or PBS and 20 µl of human 2m in PBS (final concentration 15 µg/ml; Sigma-Aldrich). The cells were incubated for 20 h at 37°C, 5% CO₂ in humidified air, then transferred into V-bottom 96-well culture plates and incubated in a water bath at 50°C for 3 min. The cells were washed twice in cold (4°C) PBS, stained with FITC-conjugated anti-HLA-A2 mAb BB7.2 (BD Biosciences/BD Pharmingen), and analyzed by flow cytometry. The up-regulation of HLA-A2.1 is reported as the fluorescence index, defined as the mean fluorescence intensity (MFI) of the experimental sample divided by the MFI in the absence of peptide.

	Results

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Accelerated T1D in the presence of transgenic HLA-A2.1 molecules

The HHD construct (23) was injected directly into NOD zygotes and a transgenic line was established. As previously observed in NOD.HLA-A2.1 mice (22), expression of the HHD transgene resulted in significant acceleration of T1D onset through 30 wk of age in NOD.HHD females compared with standard NOD controls (p = 0.02; Fig. 1).

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Transgenically expressed chimeric HLA-A2.1 class I MHC molecules contribute to development of T1D in NOD mice. Female mice were monitored weekly for development of T1D. NOD.HHD mice (; n = 19) had significantly accelerated onset of disease (p = 0.02) compared with nontransgenic littermates (; n = 27).

As in our previous study (22), acceleration of T1D in NOD.HHD mice could most simply be attributed to addition of HLA-A2.1-restricted CD8⁺ T cell responses to those elicited by murine class I MHC molecules. Thus, we tested whether HLA-A2.1 molecules could elicit CD8⁺ T cell responses sufficient for T1D development in the absence of murine MHC class I molecules. By crossing the HHD transgene into the NOD.2m^null stock (3), we eliminated expression of murine MHC class I molecules. Flow cytometric analysis of splenocytes confirmed NOD.2m^null.HHD mice express only human HLA-A2.1, and not murine MHC class I molecules (Fig. 2A, Table I). Although low-level surface expression of the H-2D^b H chain in the absence of 2m has been reported to occur in cells derived from certain mouse strains (38, 39), we saw no evidence for this in NOD.2m^null.HHD mice (Fig. 2A, Table I).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. HLA-A2.1 expression and development of HLA-A2.1-restricted CD8+ T cells and T1D in NOD.2mnull.HHD mice. Splenocytes from NOD (A and B, left panels) and NOD.2mnull.HHD mice (A and B, right panels) were analyzed by flow cytometry. A, NOD splenocytes express murine MHC class I molecules as indicated. In NOD.2mnull.HHD mice, the level of staining with the murine MHC class I-specific mAbs M1/42 (pan murine MHC class I) and KH95 (H-2Db-specific) is reduced to background levels, but transgenically expressed HLA-A2.1 molecules are readily detected using the mAb BB7.2. Broken line, isotype control; solid line, specific Ab. B, Splenocytes were stained with anti-TCR and anti-CD8 Abs. Although reduced compared with NOD controls, NOD.2mnull.HHD mice expressing only HLA-A2.1 are able to generate CD8+ T cells. Numbers given are mean percentage ± SEM (see Table I). C, NOD.2mnull.HHD mice (; n = 20) transgenically expressing HLA-A2.1 molecules were susceptible to T1D development while nontransgenic NOD.2mnull littermates (; n = 10), which lack class I MHC molecules, were completely resistant (p = 0.006).

View this table: [in this window] [in a new window]   Table I. Splenocyte composition of NOD.2mnull.HHD micea

.2m

.2m

Although female nontransgenic NOD.2m^null mice remained completely free of disease as previously reported (3, 4, 5, 6), 55% of HHD transgenic females developed T1D by 30 wk of age (p = 0.006; Fig. 2C). Pancreata from 8-wk-old NOD.2m^null.HHD and NOD female mice were examined histologically for insulitis levels (scale 0–4). This analysis indicated similar levels of leukocytic infiltration in NOD.2m^null.HHD (mean insulitis score, 2.26 ± 0.50; n = 5) and NOD islets (2.01 ± 0.27; n = 4).

NOD.2m^null.HHDmice generate T cells that can lyse HLA-A2.1-positive human pancreatic islets

T cells were propagated from the islets of NOD.2m^null.HHD mice as described in Materials and Methods. Comparable percentages of CD8⁺ T cells were observed in cultures derived from NOD.2m^null.HHD (68.96 ± 23.16%; n = 12) and NOD islets (67.27 ± 16.15%; n = 6). All the CD8⁺ T cells propagated from the islets of NOD.2m^null.HHD mice must be HLA-A2.1-restricted, because murine MHC class I molecules are not expressed in this strain. Thus, we asked whether NOD.2m^null.HHD T cell cultures would exhibit cytotoxicity against HLA-A2.1-positive human pancreatic islets.

When dissociated islet cells are used as targets in ⁵¹Cr-release cytotoxicity assays, they exhibit a high degree of spontaneous death that can complicate data interpretation. To overcome this problem, we recently developed an alternative assay in which intact islets are allowed to adhere and monolayer in a 96-well plate and are then used as targets (13, 34). Maintenance of cell-cell contact greatly enhances cell survival and leads to low spontaneous ⁵¹Cr-release values. Allowing the islets to adhere and monolayer increases the number of cells that are accessible to effector T cells. Using this assay, we measured the cytotoxic activity of NOD.2m^null.HHD T cell cultures against HLA-A2.1-positive or negative human pancreatic islets (Table II). As further controls, cytotoxic activity was also assessed against NOD-scid.HHD (H-2D^b, H-2K^d, and HLA-A2.1) and NOD.Rag1^null (H-2D^b, H-2K^d) islets. HLA-A2.1-restricted cytotoxicity against both human and mouse islets was observed. Although these results do not demonstrate the extent of overlap, they indicate that at least some subset of the antigenic peptides targeted by diabetogenic HLA-A2.1-restricted T cells are common to both mouse and human cells. Although cultured islet-infiltrating T cells from NOD.2m^null.HHD mice can recognize islet Ags, they were not able to recognize Con A blasts generated from HLA-A2-positive or HLA-A2-negative humans (data not shown). These collective results indicate that the cytotoxicity observed against HLA-A2.1-positive human islets is both HLA-A2.1-restricted and islet-specific.

View this table: [in this window] [in a new window]   Table II. Cultured islet-infiltrating T cells from NOD.2mnull.HHD mice show cytotoxic activity against HLA-A2.1-positive human pancreatic isletsa

IGRP peptides are targeted by HLA-A2.1-restricted T cells from NOD.2m^null.HHD mice

Residues 206–214 of IGRP (IGRP_206–214) represent the Ag recognized by the NOD-derived diabetogenic CD8⁺ T cell clone 8.3 (25). The pathogenicity of IGRP_206–214-reactive T cells is well-established (41, 42, 43, 44, 45), and 20–30% of CD8⁺ T cells in NOD pancreatic islets recognize this peptide (25). Previously, using a peptide-MHC tetramer reagent, we tested reactivity to IGRP_206–214 among islet-infiltrating T cells at various prodromal stages of T1D development (5, 7, 11, and 15 wk of age) in individual NOD mice (46). We detected an IGRP_206–214 response in 67–100% of mice in all age groups tested. This response was more frequent than that to the other autoantigens examined, i.e., insulin and dystrophia myotonica kinase (DMK). Importantly, the human IGRP open reading frame is identical in length to murine IGRP and is highly homologous (85% identity; 93% similarity), though a T cell response against IGRP in human T1D patients has not yet been described. For these reasons, we used NOD.2m^null.HHD mice to investigate IGRP as a possible source of HLA-A2.1-binding peptides targeted by diabetogenic CD8⁺ T cells.

To determine whether CD8⁺ T cells from NOD.2m^null.HHD mice recognize IGRP peptides, we transiently transfected COS-7 cells with varying concentrations of a full-length murine IGRP cDNA expression construct in combination with an HHD, H-2D^b, or H-2K^d cDNA expression construct. Transfected COS-7 cells were assessed for their ability to stimulate T cells propagated from the islets of NOD.2m^null.HHD mice as evidenced by IFN- secretion (Fig. 3A). T cell responses were only elicited in a dose-dependent manner when target cells were transfected with both the IGRP and HHD constructs. These results demonstrate that at least some portion of the HLA-A2.1-restricted islet-infiltrating T cells in NOD.2m^null.HHD mice recognize an IGRP-derived peptide(s). In addition, the inability of islet-infiltrating T cells from NOD.2m^null.HHD mice to recognize kidney-derived COS-7 cells transfected with the HHD construct alone further demonstrates that the response of these effectors against human HLA-A2.1-positive islets shown in Table II is cell type-specific.

To identify the IGRP epitopes recognized by CD8⁺ T cells from NOD.2m^null.HHD mice, we screened a peptide library containing all possible 8- to 11-mer sequences that can be derived from murine IGRP. The library consisted of peptide mixtures each containing four sequences with a common C terminus, but having a length of 8, 9, 10, or 11 residues. T cells were cultured from NOD.2m^null.HHD islets and their responsiveness was measured by IFN- ELISPOT assay using HLA-A2.1-expressing human T2 cells as APC. Responses were observed to peptide mixtures 229 and 338–340 (Fig. 3B). Islet-infiltrating T cell cultures contained, on average, 70% CD8⁺ T cells. However, to ensure we were not detecting CD4⁺ T cell responses, the peptide library was screened again using purified CD8⁺ T cells cultured from the islets of an independent group of NOD.2m^null.HHD mice. These CD8⁺ T cells responded to mixtures 229 and 266 (Fig. 3C), while purified islet-derived CD8⁺ T cells from a third group of mice responded to mixtures 229, 266, and 338 (data not shown).

Next, we screened the IGRP peptide library using T cells propagated from NOD.HHD mice that express endogenous H2^g7-encoded class I MHC molecules (H-2D^b and H-2K^d) in addition to the transgenic HLA-A2.1 variant (Fig. 3D). As expected, we detected a strong response to peptide mixtures 206–210, corresponding to the H-2K^d-binding IGRP_206–214 epitope. In contrast, T cells from NOD.2m^null.HHD mice do not respond to IGRP_206–214 (Fig. 3, B and C), consistent with the absence of H-2K^d expression in this strain. In addition to mixtures 206–210, T cells from NOD.HHD mice also released IFN- in response to peptide mixture 229. In separate screens using T cells from other NOD.HHD mice, responses to mixtures 266 and 338 were also observed (data not shown). These results support our hypothesis that acceleration of T1D in NOD.HHD mice can be attributed, at least in part, to addition of HLA-A2.1-restricted CD8⁺ T cell responses to those elicited by murine MHC class I molecules.

Screening of the peptide library using islet-infiltrating T cells from NOD.2m^null.HHD mice (Fig. 3, B and C) detected multiple positive peptide mixtures (229, 266, and 338–340). As mentioned, each peptide mixture consists of four sequences having a common C terminus and varying in length from 8 to 11 residues (Fig. 4A). In mixtures 266 and 338, the 9-mer peptides agreed most closely with the peptide-binding motif described for HLA-A2.1 (i.e., most commonly 9-mers having L or M at position 2 and V or L at position 9) (47). Thus, we predicted that the 9-mer peptides mIGRP_265–273 and mIGRP_337–345 were responsible for the activity observed with mixtures 266 and 338, respectively. In the case of peptide mixture 229, the 9-mer peptide did not have a preferred residue at position 2. To determine the antigenic target in this mixture, we synthesized all four of the peptides contained within it. T cell reactivity to the 9-mer peptides from mixtures 266 and 338, and to all four peptides from mixture 229, was then assayed (Fig. 4B). Each graph shows a separate experiment using islet-infiltrating T cells cultured from individual 12-wk-old female NOD.2m^null.HHD mice. In these experiments, one mouse showed a response to peptide mixtures 229 and 266, while the other showed a response to mixtures 266 and 338. The results clearly indicated the 9-mer peptides from mixtures 266 and 338 were antigenic. In the case of mixture 229, the 9-mer peptide was the minimal epitope. Recognition of the 10- and 11-mer peptides from this mixture suggests a proteolytic event generated the 9 mer during the assay period. Similarly, the response observed to mixtures 339 and 340 in the original screening likely reflects generation of the mIGRP_337–345 9-mer peptide from longer sequences in each mixture.

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Identification of three antigenic HLA-A2.1-binding peptides from murine IGRP. A, Schematic of three positive peptide mixtures (229, 266, and 338) from the murine IGRP peptide library. Bold letters indicate potential anchor residues based on the HLA-A2.1 peptide-binding motif (47 ). B, Individual peptides were synthesized, and their T cell stimulation ability () was compared with the original peptide mixtures (). Each graph shows a separate experiment using islet-infiltrating T cells cultured from individual 12-wk-old female NOD.2mnull.HHD mice and T2 cells as APC. C, T2 cells (HLA-A2.1) or RMA-S/Kd cells (H-2Db, H-2Kb, and H-2Kd) were used as APC to confirm the HLA-A2.1 restriction of the IGRP-reactive T cells.

Individual NOD.2m^null.HHD mice exhibit distinct patterns of CD8⁺ T cell reactivity to IGRP peptides

As shown in Fig. 3, B and C, and Fig. 4B, islet-infiltrating T cells exhibited different patterns of recognition to the three HLA-A2.1-binding IGRP peptides. To investigate this in more detail, we independently propagated T cells from the islets of 16 nondiabetic 12- to 13-wk-old female NOD.2m^null.HHD mice. Responses to the three peptides were measured by IFN- ELISPOT assay (Fig. 5A). As expected, the response to each IGRP epitope varied among mice; however, all exhibited a T cell response to at least one of the peptides. When the data are summarized as in Fig. 5B, it is clear that peptide 228–236 is the immunodominant HLA-A2.1-binding epitope of mIGRP.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Individual NOD.2mnull.HHD mice exhibit distinct patterns of CD8+ T cell reactivity to three HLA-A2.1-binding murine IGRP peptides. A, Islets were isolated from 16 12- to 13-wk-old female NOD.mnull.HHD mice and cultured individually in the presence of IL-2 for 7 days. Response of the resulting T cells to the three peptides was measured by IFN- ELISPOT assay using T2 cells as APC. Although some selection may occur during the culture period, we find that the rank order of prevalence of different T cell populations remains stable from days 0 to 7 of culture (our unpublished observations). Furthermore, islet preparations that are split in half on day 0 and analyzed independently on day 7 reveal identical rank orders of prevalence (our unpublished observations). B, Summary of quantification of ELISPOT assays performed on cultured islet-infiltrating T cells from the sixteen 12- to 13-wk-old female NOD.2mnull.HHD mice depicted in A.

Up to nearly one-tenth of all CD8⁺ T cells propagated from the islets of NOD.2m^null.HHD mice are IGRP-reactive. This robust response to IGRP suggested recognition of IGRP peptides may have contributed to the cytotoxicity against HLA-A2.1-positive human islet cells demonstrated in Table II. To investigate this possibility, we compared the sequences of the antigenic mIGRP peptides with the corresponding regions of the human protein (Fig. 6A). IGRP_265–273 was identical in both the murine and human molecules. The sequences of IGRP_228–236 and IGRP_337–345 differed in two amino acids each. Next, we compared the ability of the peptides to bind to HLA-A2.1 using an MHC stabilization assay (Fig. 6B). All three murine IGRP peptides demonstrated HLA-A2.1 binding, as expected. hIGRP_228–236 also bound well to HLA-A2.1. However, we were unable to detect any binding of hIGRP_337–345, perhaps due to the change of the anchor residue at position 2 from L to F (47). We then tested the ability of islet-infiltrating T cell cultures containing cells that recognized mIGRP_228–236 or mIGRP_337–345 to also recognize the corresponding hIGRP peptides. Probably due to its inability to bind HLA-A2.1, hIGRP_337–345 was not recognized by T cells responding to the murine sequence (data not shown). In contrast, three of the eight T cell cultures derived from 12-wk-old NOD.2m^null.HHD mice that exhibited reactivity to mIGRP_228–236 also clearly contained effectors capable of recognizing the human version of this peptide (Fig. 6C). There were no examples of cultures having reactivity to hIGRP_228–236, but not its murine counterpart. These collective results suggest that a subset of T cells in certain cultures that recognize mIGRP_228–236 are cross-reactive against the corresponding human peptide.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Characterization of the human counterparts of the HLA-A2.1-binding murine IGRP peptides. A, Schematic comparison of three HLA-A2.1-binding murine IGRP peptides with the corresponding peptides from human IGRP. Bold and underlined letters indicate amino acid differences between murine and human IGRP peptides. B, HLA-A2.1 stabilization assay for the three HLA-A2.1-binding murine IGRP peptides and their human counterparts. T2 cells were pulsed with the indicated peptides, stained with FITC-conjugated anti-HLA-A2 mAb BB7.2, and analyzed by flow cytometry. Each bar represents fluorescence index calculated as described in Materials and Methods. Flu-MP58–66 is an HLA-A2.1-binding influenza matrix protein (MP) peptide well characterized as a high-affinity HLA-A2.1 binder (37 ) and used as a positive control. C, mIGRP228–236 and hIGRP228–236 were compared regarding their ability to stimulate cultured islet-infiltrating T cells from NOD.2mnull.HHD mice using IFN- ELISPOT assay. Islets were isolated from eight 12-wk-old female NOD.2mnull.HHD mice and cultured individually in the presence of IL-2 for 7 days.

	Discussion

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T1D is clearly associated with specific class II MHC alleles (1, 2). However, association studies have revealed that particular class I MHC alleles, including HLA-A2.1, likely represent additional risk factors for T1D development (15, 16, 17, 18, 19, 20, 21). The frequency of HLA-A2.1 in Finnish children carrying high-risk class II MHC alleles who develop T1D is higher than in those remaining disease-free (15). A U.S.-based study indicated the HLA-A1/HLA-A2 genotype was associated with increased risk for developing islet autoimmunity (17). Also, among an independent group of U.S. children having high-risk class II alleles, HLA-A2 was found to be associated with development of autoantibodies and progression to T1D before 5 years of age (17). Thus, HLA-A2 may confer an increased risk for the development of anti-islet autoimmunity in early life. Although these epidemiological studies suggested HLA-A2.1-restricted CD8⁺ T cells might contribute to T1D pathogenesis, disease development by NOD.2m^null.HHD mice (Fig. 2C) provides critical experimental evidence that this is indeed the case.

Individual NOD mice exhibit unique patterns of CD8⁺ T cell reactivity to the three islet Ags IGRP, insulin, and DMK (46). Even within age-matched groups, individuals exhibit variability in the relative proportions of the three Ag-specific T cell populations in islet infiltrates. In this study, we observed similar diverse responses to different epitopes within the IGRP protein (Fig. 5), though peptide 228–236 was immunodominant overall. These results further confirm that individual NOD mice exhibit unique patterns of CD8⁺ T cell reactivity to cell Ags. As previously proposed (46), these data indicate several antigenic specificities contribute to cell destruction, and in any individual mouse the autoimmune response is dominated by T cells recognizing a unique subset of antigenic epitopes.

IGRP is an important target of pathogenic CD8⁺ T cells in NOD mice (25, 26, 46). An examination of islet infiltrates in individual NOD mice revealed T cells responding to IGRP_206–214/H-2K^d are present in nearly all mice examined, including those as young as 5 wk of age (46). Recently, additional IGRP epitopes targeted by CD8⁺ T cells in standard NOD mice were reported (26). We now show that IGRP antigenicity is not limited to peptides recognized by T cells restricted to murine MHC class I molecules, but extends also to those seen in the context of the human MHC class I molecule HLA-A2.1 (Fig. 3, B and C). Importantly, HLA-A2.1-restricted T cell responses to IGRP peptides can be detected in NOD.2m^null.HHD mice as young as 5 wk of age (data not shown). Our results provide candidate peptides that could allow the potential importance of IGRP in human T1D to be tested in future clinical studies. Of course, it is possible such future investigations could indicate that the candidate IGRP peptides identified in the current study are not targeted as frequently by pathogenic CD8⁺ T cells in HLA-A2.1-expressing T1D patients as is the case in NOD.2m^null.HHD mice. Thus, we have also begun to explore HLA-A2.1-restricted T cell responses to other cell Ags in NOD.2m^null.HHD mice. For example, our preliminary results indicate that multiple preproinsulin peptides are recognized by HLA-A2.1-restricted islet-infiltrating T cells in NOD.2m^null.HHD mice (T. Yamada, T. Takaki, M. Marron, D. Serreze, and T. DiLorenzo unpublished observations). These findings are consistent with recent reports suggesting the importance of insulin as a target autoantigen in the development of T1D in both NOD mice (48) and humans (49).

Knowledge of the target Ags of CD8⁺ T cells in human T1D patients is extremely limited. A peptide derived from islet amyloid polypeptide precursor protein (50) and several peptides derived from proinsulin (51) are the only ones reported to elicit a response by CD8⁺ T cells from patients without prior in vitro stimulation. Continued characterization of the Ags targeted by MHC class I-restricted T cells in human T1D patients is clearly necessary. Recently, van Endert and coworkers (52) identified peptides from human proinsulin that are immunogenic in HHD-expressing C57BL/6 mice, and these will undoubtedly be examined in the future for their relevance to human T1D. A limitation of human studies is that islet-infiltrating T cells are difficult to obtain. In contrast, application of the HHD transgenic strategy to the autoimmune-prone NOD mouse allows ready access to diabetogenic HLA-A2.1-restricted T cells and here has permitted identification of some of the cell peptides they recognize. We believe this to be the first case in which HHD-expressing mice were used to identify antigenic peptides bound to human MHC class I molecules that are targeted in a natural autoimmune disease process. Importantly, T cells isolated from NOD.2m^null.HHD mice can recognize human HLA-A2.1-positive islets (Table II). This indicates that murine and human HLA-A2.1-positive islets present common disease-relevant peptides. Multiple pathogen and tumor Ags recognized by HLA-A2.1-restricted T cells in HHD mice have been shown to be targets for HLA-A2.1-restricted T cells in patients (23, 53, 54, 55). Thus, human counterparts of the antigenic cell peptides identified through the use of NOD.2m^null.HHD mice are prime candidates for exploration in future clinical studies as targets of pathogenic T cells in T1D patients.

	Acknowledgments

 
We thank the staff of The Jackson Laboratory Scientific Services Microchemistry and Microinjection core facilities for assistance in preparation of the HHD construct and microinjection of NOD zygotes, and the staff of the Flow Cytometry core facility.

	Disclosures

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T. P. DiLorenzo has equity and other significant financial interests in and is a proposed consultant to a company that may have a commercial interest in the results of this research and technology. The authors have no other conflicting financial interests.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants DK69280 (to M.P.M.), DK64315 (to T.P.D.), DK52956 (to T.P.D.), DK51090 (to D.V.S.), DK46266 (to D.V.S.), AI56374 (to C.E.M.), Cancer Center Support Grant CA34196 (The Jackson Laboratory), and DK20541 (Albert Einstein College of Medicine’s Diabetes Research and Training Center), and by grants from the Juvenile Diabetes Research Foundation (to T.P.D., D.V.S., C.E.M., M.T.), and the Alexandrine and Alexander Sinsheimer Foundation (to T.P.D.). The flow cytometry facility at Albert Einstein College of Medicine is supported by National Institutes of Health Cancer Center Grant CA13330.

² T.T. and M.P.M. contributed equally to this work.

³ Current address: Department of Pediatrics, Division of Genetic and Translational Medicine, University of Alabama at Birmingham, Birmingham, AL 35294.

⁴ Address correspondence and reprint requests to Dr. David V. Serreze, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609; E-mail address: dvs{at}jax.org or Dr. Teresa P. DiLorenzo, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461; E-mail address: dilorenz{at}aecom.yu.edu

⁵ Abbreviations used in this paper: T1D, type 1 diabetes; 2m, ₂-microglobulin; IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein; m, murine; h, human; DMK, dystrophia myotonica kinase; MFI, mean fluorescence intensity.

Received for publication November 8, 2005. Accepted for publication December 22, 2005.

	References

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1. Serreze, D. V., E. H. Leiter. 2001. Genes and cellular requirements for autoimmune diabetes susceptibility in nonobese diabetic mice. Curr. Dir. Autoimmun. 4: 31-67. [Medline]
2. Todd, J. A., L. S. Wicker. 2001. Genetic protection from the inflammatory disease type 1 diabetes in humans and animal models. Immunity 15: 387-395. [Medline]
3. Serreze, D. V., E. H. Leiter, G. J. Christianson, D. Greiner, D. C. Roopenian. 1994. Major histocompatibility complex class I-deficient NOD-B2m^null mice are diabetes and insulitis resistant. Diabetes 43: 505-509. [Abstract]
4. Sumida, T., M. Furukawa, A. Sakamoto, T. Namekawa, T. Maeda, M. Zijlstra, I. Iwamoto, T. Koike, S. Yoshida, H. Tomioka, M. Taniguchi. 1994. Prevention of insulitis and diabetes in ₂-microglobulin-deficient non-obese diabetic mice. Int. Immunol. 6: 1445-1449. [Abstract/Free Full Text]
5. Wicker, L. S., E. H. Leiter, J. A. Todd, R. J. Renjilian, E. Peterson, P. A. Fischer, P. L. Podolin, M. Zijlstra, R. Jaenisch, L. B. Peterson. 1994. ₂-microglobulin-deficient NOD mice do not develop insulitis or diabetes. Diabetes 43: 500-504. [Abstract]
6. Katz, J., C. Benoist, D. Mathis. 1993. Major histocompatibility complex class I molecules are required for the development of insulitis in non-obese diabetic mice. Eur. J. Immunol. 23: 3358-3360. [Medline]
7. Christianson, S. W., L. D. Shultz, E. H. Leiter. 1993. Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice: relative contributions of CD4⁺ and CD8⁺ T lymphocytes from diabetic versus prediabetic NOD.NON-Thy 1^a donors. Diabetes 42: 44-55. [Abstract]
8. DiLorenzo, T. P., R. T. Graser, T. Ono, G. J. Christianson, H. D. Chapman, D. C. Roopenian, S. G. Nathenson, D. V. Serreze. 1998. Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor chain gene rearrangement. Proc. Natl. Acad. Sci. USA 95: 12538-12543. [Abstract/Free Full Text]
9. Hattori, M., E. Yamato, N. Itoh, H. Senpuku, T. Fujisawa, M. Yoshino, M. Fukuda, E. Matsumoto, T. Toyonaga, I. Nakagawa, et al 1999. Cutting edge: homologous recombination of the MHC class I K region defines new MHC-linked diabetogenic susceptibility gene(s) in nonobese diabetic mice. J. Immunol. 163: 1721-1724. [Abstract/Free Full Text]
10. Pomerleau, D. P., R. J. Bagley, D. V. Serreze, C. E. Mathews, E. H. Leiter. 2005. Major histocompatibility complex-linked diabetes susceptibility in NOD/Lt mice: subcongenic analysis localizes a component of Idd16 at the H2-D end of the diabetogenic H2^g7 complex. Diabetes 54: 1603-1606. [Abstract/Free Full Text]
11. Inoue, K., H. Ikegami, T. Fujisawa, S. Noso, K. Nojima, N. Babaya, M. Itoi-Babaya, S. Makimo, T. Ogihara. 2004. Allelic variation in class I K gene as candidate for a second component of MHC-linked susceptibility to type 1 diabetes in non-obese diabetic mice. Diabetologia 47: 739-747. [Medline]
12. Hattori, M., K. Hattori, T. Fujisawa, C. Owa, H. Senpuku, S. Noso, Y. Matsuda, T. Ijiri, T. Lund, E. Wakeland, R. Flavell. 2003. MHC class I K^d is diabetogenic in the NOD mouse. Diabetes 52: (Suppl. 1):A246
13. Choisy-Rossi, C.-M., T. M. Holl, M. A. Pierce, H. D. Chapman, D. V. Serreze. 2004. Enhanced pathogenicity of diabetogenic T cells escaping a non-MHC gene-controlled near death experience. J. Immunol. 173: 3791-3800. [Abstract/Free Full Text]
14. Hamilton-Williams, E. E., D. V. Serreze, B. Charlton, E. A. Johnson, M. P. Marron, A. Mullbacher, R. M. Slattery. 2001. Transgenic rescue implicates ₂-microglobulin as a diabetes susceptibility gene in nonobese diabetic (NOD) mice. Proc. Natl. Acad. Sci. USA 98: 11533-11538. [Abstract/Free Full Text]
15. Fennessy, M., K. Metcalfe, G. A. Hitman, M. Niven, P. A. Biro, J. Tuomilehto, E. Tuomilehto-Wolf. 1994. A gene in the HLA class I region contributes to susceptibility to IDDM in the Finnish population. Diabetologia 37: 937-944. [Medline]
16. Nejentsev, S., Z. Gombos, A. P. Laine, R. Veijola, M. Knip, O. Simell, O. Vaarala, H. K. Akerblom, J. Ilonen. 2000. Non-class II HLA gene associated with type 1 diabetes maps to the 240-kb region near HLA-B. Diabetes 49: 2217-2221. [Abstract]
17. Robles, D. T., G. S. Eisenbarth, T. Wang, H. A. Erlich, T. L. Bugawan, S. R. Babu, K. Barriga, J. M. Norris, M. Hoffman, G. Klingensmith, et al 2002. Identification of children with early onset and high incidence of anti-islet autoantibodies. Clin. Immunol. 102: 217-224. [Medline]
18. Honeyman, M. C., L. C. Harrison, B. Drummond, P. G. Colman, B. D. Tait. 1995. Analysis of families at risk for insulin-dependent diabetes mellitus reveals that HLA antigens influence progression to clinical disease. Mol. Med. 1: 576-582. [Medline]
19. Nakanishi, K., T. Kobayashi, T. Murase, T. Naruse, Y. Nose, H. Inoko. 1999. Human leukocyte antigen-A24 and -DQA1*0301 in Japanese insulin-dependent diabetes mellitus: independent contributions to susceptibility to the disease and additive contributions to acceleration of -cell destruction. J. Clin. Endocrinol. Metab. 84: 3721-3725. [Abstract/Free Full Text]
20. Noble, J. A., A. M. Valdes, T. L. Bugawan, R. J. Apple, G. Thomson, H. A. Erlich. 2002. The HLA class I A locus affects susceptibility to type 1 diabetes. Hum. Immunol. 63: 657-664. [Medline]
21. Tait, B. D., P. G. Colman, G. Morahan, L. Marchinovska, E. Dore, S. Gellert, M. C. Honeyman, K. Stephen, A. Loth. 2003. HLA genes associated with autoimmunity and progression to disease in type 1 diabetes. Tissue Antigens 61: 146-153. [Medline]
22. Marron, M. P., R. T. Graser, H. D. Chapman, D. V. Serreze. 2002. Functional evidence for the mediation of diabetogenic T cell responses by HLA-A2.1 MHC class I molecules through transgenic expression in NOD mice. Proc. Natl. Acad. Sci. USA 99: 13753-13758. [Abstract/Free Full Text]
23. Pascolo, S., N. Bervas, J. M. Ure, A. G. Smith, F. A. Lemonnier, B. Perarnau. 1997. HLA-A2.1-restricted education and cytolytic activity of CD8⁺ T lymphocytes from ₂ microglobulin (₂m) HLA-A2.1 monochain transgenic H-2D^b ₂m double knockout mice. J. Exp. Med. 185: 2043-2051. [Abstract/Free Full Text]
24. Irwin, M., W. Heath, L. Sherman. 1989. Species-restricted interactions between CD8 and the 3 domain of class I influence the magnitude of the xenogeneic response. J. Exp. Med. 170: 1091-1101. [Abstract/Free Full Text]
25. Lieberman, S. M., A. M. Evans, B. Han, T. Takaki, Y. Vinnitskaya, J. A. Caldwell, D. V. Serreze, J. Shabanowitz, D. F. Hunt, S. G. Nathenson, et al 2003. Identification of the cell antigen targeted by a prevalent population of pathogenic CD8⁺ T cells in autoimmune diabetes. Proc. Natl. Acad. Sci. USA 100: 8384-8388. [Abstract/Free Full Text]
26. Han, B., P. Serra, A. Amrani, J. Yamanouchi, A. F. Maree, L. Edelstein-Keshet, P. Santamaria. 2005. Prevention of diabetes by manipulation of anti-IGRP autoimmunity: high efficiency of a low-affinity peptide. Nat. Med. 11: 645-652. [Medline]
27. Shultz, L. D., P. A. Lang, S. W. Christianson, B. Gott, B. Lyons, S. Umeda, E. Leiter, R. Hesselton, E. J. Wagar, J. H. Leif, et al 2000. NOD/LtSz-Rag1^null mice: an immunodeficient and radioresistant model for engraftment of human hematolymphoid cells, HIV infection, and adoptive transfer of NOD mouse diabetogenic T cells. J. Immunol. 164: 2496-2507. [Abstract/Free Full Text]
28. Russo, C., A. K. Ng, M. A. Pellegrino, S. Ferrone. 1983. The monoclonal antibody CR11-351 discriminates HLA-A2 variants identified by T cells. Immunogenetics 18: 23-35. [Medline]
29. Serreze, D. V., E. H. Leiter, M. S. Hanson, S. W. Christianson, L. D. Shultz, R. M. Hesselton, D. L. Greiner. 1995. Emv30^null NOD-scid mice: an improved host for adoptive transfer of autoimmune diabetes and growth of human lymphohematopoietic cells. Diabetes 44: 1392-1398. [Abstract]
30. Ricordi, C., P. E. Lacy, D. W. Scharp. 1989. Automated islet isolation from human pancreas. Diabetes 38: (Suppl. 1):140-142.
31. Lakey, J. R., G. L. Warnock, A. M. Shapiro, G. S. Korbutt, Z. Ao, N. M. Kneteman, R. V. Rajotte. 1999. Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. Cell Transplant. 8: 285-292. [Medline]
32. Bottino, R., A. N. Balamurugan, S. Bertera, M. Pietropaolo, M. Trucco, J. D. Piganelli. 2002. Preservation of human islet cell functional mass by anti-oxidative action of a novel SOD mimic compound. Diabetes 51: 2561-2567. [Abstract/Free Full Text]
33. Ablamunits, V., D. Elias, I. R. Cohen. 1999. The pathogenicity of islet-infiltrating lymphocytes in the non-obese diabetic (NOD) mouse. Clin. Exp. Immunol. 115: 260-267. [Medline]
34. Takaki, T., S. M. Lieberman, T. M. Holl, B. Han, P. Santamaria, D. V. Serreze, T. P. DiLorenzo. 2004. Requirement for both H-2D^b and H-2K^d for the induction of diabetes by the promiscuous CD8⁺ T cell clonotype AI4. J. Immunol. 173: 2530-2541. [Abstract/Free Full Text]
35. Leiter, E. H.. 1997. The NOD mouse: a model for insulin-dependent diabetes mellitus. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds. Current Protocols in Immunology 15.19.11-15.19.23. John Wiley and Sons, Hoboken.
36. Karttunen, J., S. Sanderson, N. Shastri. 1992. Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc. Natl. Acad. Sci. USA 89: 6020-6024. [Abstract/Free Full Text]
37. Regner, M., M. H. Claesson, S. Bregenholt, M. Ropke. 1996. An improved method for the detection of peptide-induced upregulation of HLA-A2 molecules on TAP-deficient T2 cells. Exp. Clin. Immunogenet. 13: 30-35. [Medline]
38. Potter, T. A., C. Boyer, A. M. Verhulst, P. Golstein, T. V. Rajan. 1984. Expression of H-2D^b on the cell surface in the absence of detectable ₂-microglobulin. J. Exp. Med. 160: 317-322. [Abstract/Free Full Text]
39. Allen, H., J. Fraser, D. Flyer, S. Calvin, R. Flavell. 1986. ₂-microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H-2D^b or of a truncated H-2D^b. Proc. Natl. Acad