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


     
 

The Journal of Immunology, 2008, 180, 3250 -3259
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Serreze, D. V.
Right arrow Articles by Marron, M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Serreze, D. V.
Right arrow Articles by Marron, M. P.

Through Regulation of TCR Expression Levels, an Idd7 Region Gene(s) Interactively Contributes to the Impaired Thymic Deletion of Autoreactive Diabetogenic CD8+ T Cells in Nonobese Diabetic Mice1

David V. Serreze2,*, Caroline Morgane Choisy-Rossi*, Alexandra E. Grier*, T. Mathew Holl*, Harold D. Chapman*, J. Reed Gahagan*, Melissa A. Osborne*, Weidong Zhang*, Benjamin L. King*, Aaron Brown{dagger}, Derry Roopenian* and Michele P. Marron{ddagger}

* The Jackson Laboratory, Bar Harbor, ME 04609; {dagger} Bar Harbor Biotechology Incorporated, Trenton, ME 04605; and {ddagger} Department of Pediatrics, Division of Genetic and Translational Medicine, University of Alabama, Birmingham, AL 35233


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
When expressed in NOD, but not C57BL/6 (B6) genetic background mice, the common class I variants encoded by the H2g7 MHC haplotype aberrantly lose the ability to mediate the thymic deletion of autoreactive CD8+ T cells contributing to type 1 diabetes (T1D). This indicated some subset of the T1D susceptibility (Idd) genes located outside the MHC of NOD mice interactively impair the negative selection of diabetogenic CD8+ T cells. In this study, using both linkage and congenic strain analyses, we demonstrate contributions from a polymorphic gene(s) in the previously described Idd7 locus on the proximal portion of Chromosome 7 predominantly, but not exclusively, determines the extent to which H2g7 class I molecules can mediate the thymic deletion of diabetogenic CD8+ T cells as illustrated using the AI4 TCR transgenic system. The polymorphic Idd7 region gene(s) appears to control events that respectively result in high vs low expression of the AI4 clonotypic TCR {alpha}-chain on developing thymocytes in B6.H2g7 and NOD background mice. This expression difference likely lowers levels of the clonotypic AI4 TCR in NOD, but not B6.H2g7 thymocytes, below the threshold presumably necessary to induce a signaling response sufficient to trigger negative selection upon Ag engagement. These findings provide further insight to how susceptibility genes, both within and outside the MHC, may interact to elicit autoreactive T cell responses mediating T1D development in both NOD mice and human patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type 1 diabetes (T1D)3 in both humans and NOD mice results from T cell mediated autoimmune destruction of insulin producing pancreatic β cells (reviewed in Refs. 1, 2, 3). While controlled by multiple susceptibility (Idd) genes, particular MHC haplotypes provide the primary risk factor for T1D development in both genera (1, 2, 3). Within the MHC, specific combinations of HLA-DQ and DR class II alleles provide a large component of T1D susceptibility in humans (4). A particularly strong class II contributor to T1D in humans is the unusual DQ8 molecule that is distinguished from most other variants by the presence of amino acid residues other than proline and aspartic acid at positions 56 and 57 of the β-chain subunit (4). Similarly, T1D development in NOD mice is also dependent upon the unusual MHC class II variants characterizing their H2g7 MHC haplotype. Specifically, this entails a need for NOD APCs to homozygously express the unusual H2-Ag7 MHC class II gene product (homologue of human DQ8) in the absence of H2-E (homologue of human DR) molecules (5, 6, 7, 8, 9).

Given the critical contributions of certain class II alleles, it is not surprising that autoreactive CD4 T cell responses they control are essential for T1D development in NOD mice, and also presumably in humans. However, it is now clear that while representing quite common variants shared by many strains lacking autoimmune proclivity, the Kd and/or Db class I molecules encoded by the H2g7 haplotype mediate autoreactive CD8+ T cell responses that are also essential to T1D development in NOD mice (10, 11). Indeed, there is evidence that T1D development in NOD mice is dependent upon expression of the particular class I variants characterizing the H2g7 haplotype (12, 13). Likewise, both clinical studies and transgenic mouse analyses have indicated certain common human HLA class I molecules such as HLA-A2.1 can in some circumstances mediate diabetogenic CD8+ T cell responses (14, 15, 16, 17, 18, 19, 20, 21, 22). We previously found that the impaired ability of H2g7 class I molecules to mediate the intrathymic deletion of diabetogenic CD8+ T cells when expressed in NOD mice results from interactive contributions from genes outside of the MHC (23). Several genetic resources allowed us to reach this conclusion. One of these is a NOD stock transgenically expressing the TCR from the H2g7 class I restricted CD8+ T cell clone AI4 (NOD.AI4) that contributes to the earliest phases of autoimmune β cell destruction leading to T1D (24, 25). Another was a stock of C57BL/6 mice congenic for both the H2g7 MHC and the AI4 TCR transgenes (B6.H2g7.AI4) (23). AI4 transgenic T cells undergo intrathymic negative selection much more efficiently in B6.H2g7 than NOD genetic background mice (23). This difference in negative selection efficiency is a T cell intrinsic trait.

In this study, we mapped non-MHC quantitative trait loci (QTL) contributing to the differing efficiency of intrathymic negative selection of AI4 T cells in NOD and B6.H2g7 mice. We previously found the development of endogenously derived TCR {alpha}-chains is more efficiently blocked in B6.H2g7.AI4 than NOD.AI4 mice due to differing efficiencies of transgene induced allelic exclusion (23). This difference in allelic exclusion efficiency dilutes expression of the clonotypic AI4 TCR in NOD, but not B6.H2g7 thymocytes, below the threshold level presumably necessary to induce a signaling response sufficient to trigger negative selection upon Ag engagement. Thus, we also determined whether any QTL contributing to poor vs efficient intrathymic deletion of AI4 T cells in NOD and B6.H2g7 background mice might do so by regulating events resulting in differential expression levels of the clonotypic TCR.


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

NOD/ShiLtDvs mice are maintained at The Jackson Laboratory by brother-sister mating. NOD mice separately carrying directly introduced transgenes subsequently fixed to homozygosity that encode the TCR {alpha} (V{alpha}8)- or β (Vβ2)-chain of the β cell autoreactive CD8+ T cell clone AI4 have been previously described (25). A C57BL/6 background stock congenic for a NOD-derived chromosome (Chr.) 17 interval delineated by the flanking markers D17Mit21 (33.6 Mb) and D17Mit10 (~49Mb) which encompasses the H2g7 MHC haplotype (B6.H2g7) is maintained at the N8 backcross generation (26). The AI4 {alpha}- and β-chain transgenes were congenically transferred to the B6.H2g7 background. At the N7 backcross generation an intercross strategy was used to separately fix the AI4 {alpha}- and β-chain transgenes to homozygosity in B6.H2g7 background mice (23). A genome wide microsatellite screen was performed on the N7 B6.H2g7 carriers of the AI4 {alpha}- or β-chain transgenes. With the noted exceptions, all markers were found to be homozygous for B6 origin alleles in the B6.H2g7 carriers of the AI4 {alpha}- or β-chain transgenes. Exceptions included a NOD-derived segment of Chr. 1 delineated by the flanking markers D1Mit451 (155.9 Mb) and D1Mit459 (187.3 Mb) that cosegreated with the AI4 TCR {alpha}-chain transgene transferred to the B6.H2g7 background. A NOD-derived segment of Chr. 3 delineated by the flanking markers D3Mit40 (87 Mb) and D3Mit100 (97 Mb) cosegregated with the AI4 TCR β-chain transgene congenically transferred to the B6.H2g7 background. When needed, NOD or B6.H2g7 mice expressing both the AI4 {alpha}- and β-chains were generated by intercrossing appropriate homozygous carriers of the individual TCR transgenes. An N11 stock of NOD mice congenic for a segment of B6-derived Chr. 7 delineated by the flanking markers Gpib (33.9Mb) and D7Mit346 (58.6Mb) (here designated NOD.Chr7B6) has also been previously described (27).

F2 intercross strategy

NOD and B6.H2g7 mice individually carrying the AI4 {alpha}- or β-chain transgene in a homozygous state were intercrossed. This resulted in two separate sets of F1 progeny that were also separately homozygous for either the AI4 {alpha}- or β-chain transgene. However, because they only expressed one component of the transgenic AI4 TCR, this avoided the possible complication of either type of F1 progeny rapidly developing T1D making it difficult to use them for further breeding. F1 carriers of either the AI4 {alpha}- or β-chain transgenes were then intercrossed to produce 295 F2 progeny. Because of this breeding strategy all of the F2 progeny were heterozygous for both the AI4 {alpha}- and β-chain transgene. At 5–6 wk of age, all 295 F2 progeny were assessed by flow cytometry as described below for numbers of CD4/CD8 double positive (DP) thymocytes that coexpressed the AI4 transgenic TCR. Genomic DNA was also prepared from all F2 progeny to allow for analyses of single nucleotide polymorphisms (SNP) linked to the levels of AI4 TCR expressing DP thymocytes.

Flow cytometry

Thymocytes or splenic leukocytes from 5- to 6-wk-old mice were enumerated and characterized by multicolor FACS analyses. Data were collected using either FACSCalibur or FACScan (BD Biosciences) instrumentation using the CellQuest 4.0 analysis system. The FACScan has been fitted with a Cytek five color upgrade (BD Biosciences). Expression of the transgenic AI4 TCR {alpha}- and β-chain was detected by respective use of red fluorescent PE- or green fluorescent FITC-conjugated Abs specific for V{alpha}8 (B21.14) or Vβ2 (B20.6) elements. Three color flow cytometric analyses were performed to determine the proportions of CD4/CD8 DP thymocytes that expressed the AI4 TCR based on positive staining by the V{alpha}8 specific Ab. We felt it was most appropriate to use V{alpha}8 expression to delineate AI4 clonotypic T cells based on our previous observation of much lower transgene induced allelic exclusion of endogenous TCR {alpha}- than β-chain genes (23). V{alpha}8 positive cells were defined as those with mean fluorescence intensity (MFI) of Ab staining exceeding that of unstained control cells. CD8 expression was detected with the mAb 53-6.72 conjugated to a blue fluorescent allophycocyanin (AP) tag. CD4 expression was detected with the mAb GK1.5 conjugated with FITC- or AP-Cy7. Splenic T cells expressing the transgenic AI4 TCR were detected by staining with the PE-labeled TCR V{alpha}8-specific Ab mixed with the AP-labeled CD8 Ab. The expression level of the transgenic AI4 {alpha}-chain was also assessed by flow cytometry on DP thymocytes or CD8+ splenic T cells from the indicated mice by the MFI of V{alpha}8 Ab staining. Overall expression levels of TCR{alpha}β complexes were assessed with the FITC-conjugated H57–597 mAb. To insure that the availability of staining Abs was not a limiting factor contributing to possible false conclusions about strain differences in TCR expression patterns, for all of the analyses the sample tubes contained an equal number (1 x 106) of thymocytes or splenic leukocytes from the examined mice.

Genotyping and linkage analyses methods

The (NODxB6.H2g7)F2 progeny with the 64 highest and lowest numbers of AI4 TCR expressing DP thymocyes were genotyped as previously described (28) for 132 polymorphic SNPs at ~20Mb intervals spanning the entire genome (all marker positions based on NCBI build 36 of the mouse genome). This number of mice were chosen for genotyping because they represent F2 segregants with approximately the highest and lowest quartile of DP AI4 thymocytes, and also for the logistical reason that when including parental and F1 control samples this allowed for complete usage of all wells in the SNP typing plates. Linkage markers for genes controlling the negative selection efficiency of AI4 T cells were identified as previously described by Sen and Churchill (29) using the Pseudomarker 2.02 software package. This algorithm calculated linkage not only on the basis of the genotyped SNPs, but also through the incorporation of additional pseudomarkers generated on each chromosome at ~4Mb spacing with 128 imputations. All QTL initially detected using the Pseudomarker 2.02 algorithm, were subsequently verified through use of the previously described R/QTL 1.04–53 alternative software system (30). Initially, a genome-wide scan based on a single QTL model was conducted at each marker. One thousand permutations were performed to determine the thresholds for selection of candidate QTL (31). Four thresholds: 1, 5, 10, and 63% were generated. A total of 1% was considered as the threshold for a significant QTL, whereas 63% was considered as the threshold for a suggestive QTL (32).

Genome-wide pairwise scans were also performed on the data from the single QTL scan. All possible pairs of markers were tested for association with the efficiency of AI4 T cell negative selection. The likelihood from the full model (marker pair and the interaction between them) and the null model (no genetic effect) was compared and logarithm of odds (LOD) scores calculated. In addition, LOD scores from comparing the likelihood from the full model and the additive model (with only the main effects of markers and no interaction) were calculated for detection of possible QTL*QTL interactions.

Candidate QTL and possible QTL*QTL interactions identified from the single QTL or pairwise scans were fitted into multiple regression models. Terms were dropped sequentially until all of the terms in the model were significant at 1% (for QTL main effect) and 0.1% level (for QTL*QTL interaction effect). By doing so, a final model was generated, and variations of the phenotype explained by the QTL in the model were estimated. p values for QTL in the final regression model were also calculated. Posterior probability density (PPD) distributions of each QTL detected in the final regression model were also determined. A 95% confidence interval for the chromosomal position of each QTL was calculated based on the PPD.

Monitoring of T1D development

T1D development in the indicated mice was defined by glycosuric values of >3, as assessed with Ames Diastix (Bayer).

IFN-{gamma} ELISPOT assay

CD8+ T cells were purified from the spleens of 5–6 wk-old NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice by the previously described magnetic bead based negative selection approach (33). The ability of these T cells to respond to stimulation by the previously described AI4 mimotope peptide (YFIENYLEL) (34) at concentrations ranging from 0 to 100 nM was assessed in an IFN-{gamma} ELISPOT assay. MultiScreen HTS plates (Millipore) were used for the analyses. Capture and detection Abs as well as colorimetric reagents were provided as part of the Cytokine ELISPOT pair kit from BD Biosciences. Quintuple replicates of 2 x 104 CD8+ T cells from each strain were cultured with 5 x 104 syngeneic irradiated (2000R) splenocytes as a source of APC plus the indicated concentration of mimotope peptide for 60 h at 37°C. After development, spots were counted using an automated ELISPOT reader system (Cellular Technology). Data are presented are mean number of spots ± SEM/104 CD8+ T cells in each of the quintuple replicates.

Determination of TCR V{alpha} gene usage and expression levels

CD8+ T cells were purified from the spleens of 5- to 6-wk-old NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice as described above. The CD8+ T cell samples were lysed immediately in RLT buffer from an RNeasy Mini kit (Qiagen). Total RNA was isolated according to manufacturer’s protocols (Qiagen) including the optional DNase treatment step, and quality was assessed using an Agilent 2100 Bioanalyzer instrument and RNA 6000 Nano LabChip assay. A total of 500 ng of RNA was then reverse transcribed with random decamers and M-MLV RT using the Message Sensor RT kit (Ambion). Expression levels of RNA transcripts derived from gene rearrangement events incorporating members from any of 20 TCR V{alpha} families were determined using the previously described primers (24) and quantitative real-time PCR analyses methods (35) using an ABI 7900 thermocycler (Applied Biosystems) and normalized to that of the β-actin housekeeping gene. TCR {alpha}- and β-actin mRNA transcript levels were assessed in two biological replicate samples per strain each prepared from pooled purified CD8+ T cells from three to five mice. Three technical replicates of each sample were analyzed. A potential transcript was scored as absent if not detected through 40 PCR amplification cycles. Data are presented as 1/mean normalized cycle threshold value ± the SD for each biologically replicated sample. This was done so a larger value would depict higher expression levels of the RNA transcript in question.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mapping of non-MHC QTL contributing to development of diabetogenic CD8+ T cells

We previously found that in contrast to NOD mice, in the MHC matched B6.H2g7 stock, diabetogenic CD8+ T cells transgenically expressing the AI4 TCR undergo far more efficient negative selection at the DP stage of thymocyte development (23). Thus, genes outside the MHC polymorphically differing between NOD and B6 mice regulate the extent to which diabetogenic CD8+ T cells are negatively selected. As an initial step to map such genes we determined how the development of DP thymocytes transgenically expressing the AI4 TCR segregated in 295 (NODxB6.H2g7)F2 progeny. In the F2 segregants the number of AI4 TCR expressing DP thymocytes was widely distributed ranging from the low to high levels respectively characterizing the B6.H2g7 (4.0 ± 0.8 x 106; n = 22) and NOD (9.1 ± 1.7 x 107; n = 13) parental background strains (Fig. 1A). This segregation pattern indicates the development of AI4 T cells is genetically controlled by multiple QTL outside of the MHC. To map the underlying QTL, the F2 segregants characterized by the 64 highest and lowest numbers of AI4 TCR expressing DP thymocytes were genotyped for 132 SNP markers polymorphic between the NOD and B6 strains and distributed at ~20Mb intervals across the genome.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 1. Multiple genes contribute to impaired thymic deletion of the diabetogenic AI4 CD8+ T cell clonotype in NOD mice, but the primary contributor resides on the proximal end of Chr. 7. A, Numerical distribution of DP thymocytes expressing the AI4 TCR in 295 (NODxB6.H2g7)F2 progeny at 5–6 wk of age. Numbers of AI4 DP thymocytes characterizing the parental NOD.AI4 and B6.H2g7.AI4 parental stocks at an equivalent age are also noted. B, LOD score analyses of SNP markers linked to efficiency of intrathymic AI4 T cell deletion in (NODxB6.H2g7)F2 progeny. Horizontal lines depict LOD scores indicative of 1, 5, 10, and 63% QTL support thresholds. The 1 and 63% threshold were respectively considered to indicate a significant and suggestive QTL.

 
An initial one-dimensional genome wide scan indicated that as evidenced by a highly significant LOD score of ~8.0, the strongest NOD-derived QTL inhibiting the intrathymic deletion of AI4 T cells was delineated by markers on the proximal end of Chr. 7 (Fig. 1B). This initial scan also provided evidence for a NOD-derived QTL in the middle to distal region of Chr. 12 that exerted significant effects on the inhibition of AI4 T cell negative selection (Fig. 1B). However, although statistically supported, the existence of this QTL should be interpreted with caution due to our subsequent finding after the cross was completed of a segment of homozygous NOD-derived genomic material spanning a region from ~69–96 Mb on Chr. 12 in the B6.H2g7 carriers of the AI4 TCR β-chain transgene (SNP marker rs3655558 NOD like, SNP marker rs3692977 reverting to B6 like). It is possible this region of genetic contamination introduced during backcrossing of the AI4 TCR β-chain transgene (integration site on Chr. 3) to the B6.H2g7 background influenced the apparent presence of the Chr. 12 QTL. NOD origin markers on Chr. 4, 13, and 17 that exhibited suggestive linkage with impaired thymic deletion of AI4 T cells were also identified in the initial one-dimensional genome wide scan (Fig. 1B). No pairwise interactions were detected between any of the significant or suggestive QTL for thymic AI4 T cell development.

Multiple regression analysis was subsequently conducted to determine the proportion of the variance in thymic AI4 T cell development that was controlled by each significant and suggestive QTL. The most significantly acting QTL on Chr. 7 and 12 respectively accounted for 10.5 and 3.5% of the variance in thymic AI4 T cell development among the F2 segregants (Table I). More weakly acting suggestive QTL on Chr. 4, 13, and 17 could respectively account for 1.9, 2.6, and 1.7% of the variance in thymic AI4 T cell levels (Table I). These collective results indicated that additional genes outside the two significant and three suggestive QTL regions identified also contribute to the regulation of thymic AI4 T cell development, but individually exert trait variance effects even weaker than the 1.7% level which could be detected in our F2 analyses.


View this table:
[in this window]
[in a new window]

  		Table I. Analysis of Variance on the QTL detected

 
Multiple regression analysis also allowed us to determine through PPD calculations the 95% confidence interval for the location of the two significant and three suggestive QTL regulating differential intrathymic AI4 T cell deletion in NOD and B6.H2g7 mice. The 95% confidence interval for the Chr. 7 QTL that had the most significant effect in regulating thymic AI4 T cell development spanned a region from 21.2 to 43.3 Mb (Fig. 2A). Interestingly, this support interval overlaps markers delineating the previously defined Idd7 locus partially regulating susceptibility to overt T1D (36, 37). The 95% confidence interval for the second potentially significant QTL for regulating thymic AI4 T cell levels ranged from 60.4 to 117.4 Mb on Chr. 12 (Fig. 2B). This rather large positional support interval, and the distribution of LOD/PPD scores across it, suggests if this Chr. 12 QTL is truly valid, it may contain more than one polymorphic gene controlling the efficiency of thymic AI4 T cell deletion. The 95% confidence intervals for the positions of the suggestive QTL on Chr. 4, 13, and 17 are all quite large (76–100 Mb in size), most likely due to the fact that if these regions do contain genes regulating the efficiency of intrathymic deletion of AI4 T cells, their individual effects are quite small. Of potential interest, the broad support intervals for the suggestive QTL on Chr. 4 and 13, respectively, encompasses markers delineating the previously described Idd9/11 and Idd14 loci, each partially regulating susceptibility to overt T1D (37, 38, 39). In contrast, the peak LOD/PPD scores for the Chr. 17 QTL suggestively contributing to thymic levels of AI4 T cells mapped quite distally (~57–66 Mb) from the linked MHC (Idd1) and Idd16 loci (~32–46 Mb) known to each partially regulate susceptibility to overt T1D (13).


Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 2. PPD analyses of the 95% confidence intervals for positions of significant QTL on Chr. 7 (A) and 12 (B) regulating efficiency of intrathymic AI4 T cell deletion in (NODxB6.H2g7)F2 progeny.

 
Congenic analyses confirm Idd7 region genes strongly regulate thymic development of diabetogenic CD8+ T cells

We were fortunate that institutional colleagues had previously developed a NOD stock congenic for a B6-derived segment of Chr. 7 which encompassed the support interval for the QTL most strongly regulating thymic development of AI4 T cells in our F2 segregation analysis (27). Thus, we were able to readily assess whether this congenic segment of B6-derived Chr. 7 in isolation could correct the impaired thymic deletion of AI4 TCR transgenic T cells that characterizes fully NOD genetic background mice. The B6-derived Chr. 7 congenic interval (flanking markers Gpib-33.9 Mb and D7Mit346-58.6 Mb) was fixed to homozygosity in NOD background mice expressing both the AI4 {alpha}- and β-chain transgenes (here designated NOD.Chr7B6.AI4 mice). Numbers of DP thymocytes expressing the TCR V{alpha}8 element characterizing the transgenic AI4 clonotype were then compared in NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice. As previously reported (23), both the proportion of DP thymocytes, and absolute numbers of such cells expressing the V{alpha}8 element characterizing the AI4 TCR were significantly lower in B6.H2g7 than NOD genetic background mice (Fig. 3, A and B). The proportion of DP thymocytes was only moderately less in NOD.Chr7B6.AI4 than NOD.AI4 mice (Fig. 3A). However, overall thymocyte numbers were much lower in NOD.Chr7B6.AI4 (5.01 ± 0.33 x 107) than NOD.AI4 mice (1.81 ± 0.11 x 108). As a result, the numbers of AI4 DP thymocytes in the NOD.Chr7B6.AI4 stock were also significantly less than in fully NOD genetic background mice (Fig. 3B). However, levels of AI4 DP thymocytes in NOD.Chr7B6.AI4 mice remained significantly higher than in the B6.H2g7 background stock (Fig. 3B). These collective results indicate a polymorphic Idd7 region gene(s) on Chr. 7 is a major, but not sole contributor to the differing efficiency of intrathymic AI4 T cell deletion in NOD and B6.H2g7 mice.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 3. Congenic analyses confirm a polymorphic Idd7 region gene(s) on Chr. 7 is a major, but not sole contributor to the differing efficiency of intrathymic AI4 T cell deletion in NOD and B6.H2g7 mice. NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice were assessed at 5–6 wk of age by flow cytometry for numbers of DP thymocytes expressing the AI4 TCR. A, Left-hand panels, Proportions of DP and SP thymocyte populations in NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice. Numbers depict mean proportions ± SEM of the various thymocyte subpopulations in each strain. Right-hand panels, DP thymocytes from NOD (white histogram), NOD.Chr7B6 (gray histogram), or B6.H2g7 (black histogram) background mice staining positive by the Ab recognizing the TCR V{alpha}8 element characterizing the AI4 clonotype. Positive cells were scored as those characterized by a higher MFI of V{alpha}8 Ab binding than the background level characterizing unstained control cells (histograms depicted by thick dark lines). The dashed vertical lines in the right-hand panels represent the cutoff level above, which a cell was considered to be positive for TCR V{alpha}8 expression. B, Total numbers determined by the criteria described above of TCR V{alpha}8 expressing DP thymocytes in NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice. *, Significantly less than in NOD.AI4 mice (p ≤ 0.0039, Wilcoxon rank sum test). **, Significantly greater than in B6.H2g7.AI4, but less than in NOD.AI4 mice (p = 0.0039, Wilcoxon rank sum test).

 
The Idd7 region controls expression levels of the transgenic AI4 TCR {alpha}-chain

Thymocytes normally express fewer TCR molecules than mature T cells. As a result, at equivalent concentrations, some peptides can activate mature T cells, but do not induce the degree of signaling required to trigger the negative selection of clonotype matched DP thymocytes due to their lower levels of TCR expression (40, 41). Overall levels of TCR{alpha}β expression are equivalent on CD8+ T cells from NOD.AI4 and B6.H2g7.AI4 mice (23). The transgenic AI4 TCR β-chain is also expressed at equivalent levels in NOD and B6.H2g7 mice (23). However, due to less efficient allelic exclusion of endogenously-derived TCR {alpha}-chains, expression of the transgenic AI4 TCR {alpha}-chain is diluted to much lower levels on NOD than B6.H2g7 DP thymocytes (23). Similar differences in allelic exclusion efficiency also results in the {alpha}-chain of a H2-Db restricted LCMV reactive transgenic TCR being expressed at lower levels in NOD than B6 background mice (23). Thus, the lower expression level of the transgenic AI4 TCR {alpha}-chain on NOD vs B6.H2g7 background T cells is not a clonotype specific phenomenon. Due to the differing efficiency of {alpha}-chain allelic exclusion, the number of TCR molecules with AI4 antigenic specificity is likely to be lower on NOD than B6.H2g7 DP thymocytes. We hypothesized such differences in clonotypic TCR expression could reciprocally determine the extent to which Ag engagement drives the negative selection of AI4 DP thymocytes in NOD and B6.H2g7 mice. Hence, we determined whether the major QTL on Chr. 7 regulating the extent of intrathymic AI4 T cell deletion might functionally do so by controlling the level of clonotypic TCR expression.

There were no differences in overall levels of AI4 β-chain expression on DP thymocytes or splenic CD8+ T cells from NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice (data not shown). In contrast, as previously observed (23) the transgenic AI4 TCR {alpha}-chain was expressed at significantly higher levels on DP thymocytes from B6.H2g7.AI4 than NOD.AI4 mice (Fig. 4, A and B). Interestingly, the expression level of the AI4 TCR {alpha}-chain on DP thymocytes from NOD.Chr7B6.AI4 mice was also greater than on similar cells from the NOD.AI4 strain, but equivalent to those of B6.H2g7.AI4 origin (Fig. 4, A and B). These findings suggest the Chr. 7 QTL controlling differential efficiency of intrathymic AI4 T cell deletion in NOD.AI4 and B6.H2g7.AI4 mice may do so by regulating expression levels of the clonotypic TCR {alpha}-chain.


Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 4. The ability of a B6-derived Idd7 region congenic interval to enhance the efficiency of intrathymic AI4 T cell deletion in NOD background mice is associated with elevated expression levels of the clonotypic TCR {alpha}-chain. DP thymocytes from 5- to 6-wk-old NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice were assessed by flow cytometry for expression levels of the V{alpha}8 element used by the AI4 TCR. A, Representative flow cytometric profiles of TCR V{alpha}8 expression levels on gated DP thymocytes from NOD.AI4 (white histogram), B6.H2g7.AI4 (black histogram), and NOD.Chr7B6.AI4 (gray histogram) mice. Note that because NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice varied greatly in total numbers of DP thymocytes, a strain comparison of V{alpha}8 expression levels by these cells required the collection of differing numbers of events (B). Summary data of the MFI ± SEM of V{alpha}8 Ab staining of DP thymocytes from the three strains. *, Significantly greater than in NOD.AI4 mice (p = 0.0039, Wilcoxon rank sum test).

 
The Idd7 region does not control AI4 clonotypic TCR expression levels by regulating the development of additional {alpha}-chains

Fewer somatic gene rearrangement events involving endogenous TCR V{alpha} elements appear to contribute to the higher expression levels of the transgenic AI4 TCR {alpha}-chain by DP thymocytes and splenic CD8+ T cells from B6.H2g7 than NOD background mice (23). Thus, we tested whether a similar feature accounted for the differing levels of the AI4 TCR {alpha}-chain expression in NOD.AI4 and NOD.Chr7B6.AI4 mice. As seen in DP thymocytes, and discussed in more detail later, the AI4 TCR {alpha}-chain is also expressed at higher levels on peripheral CD8+ T cells from both B6.H2g7 and NOD.Chr7B6, than NOD background mice. Thus, we used quantitative real-time PCR analyses to determine whether splenic CD8+ T cells from NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice differed in levels of RNA transcripts derived from somatic gene rearrangement events incorporating endogenous TCR V{alpha} elements. We did not feel it was appropriate to carry out such gene expression analyses in thymii from NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice because they differ dramatically in the number of cells at the DP stage of development. As previously described, lower levels of RNA transcripts using multiple endogenous TCR V{alpha} elements (e.g., V{alpha}1, 2, 4, 5, 6, 10, 13, 14, 15, 17, 18, 19, 20) were observed in B6.H2g7.AI4 than NOD.AI4 CD8+ T cells (Fig. 5A, left panel). However, NOD.AI4 and NOD.Chr7B6.AI4 CD8+ T cells did not differ in levels of RNA transcripts using endogenous TCR V{alpha} elements (Fig. 5A, left panel). This indicated that polymorphic genes outside the investigated region of Chr. 7 contribute to the differential levels of RNA transcripts using endogenous TCR V{alpha} elements in CD8+ T cells from NOD.AI4 and B6.H2g7.AI4 mice. In contrast, differing levels of RNA transcripts incorporating endogenous TCR V{alpha} elements could not explain the higher surface expression of the transgenic AI4 TCR {alpha}-chain on CD8+ T cells from NOD.Chr7B6 than NOD background mice.


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 5. An Idd7 region gene(s) does not control AI4 clonotypic TCR expression levels by regulating the development of additional {alpha}-chains. A, Two biological replicates of purified splenic CD8+ T cells from 5- to 6-wk-old NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice were assessed by quantitative real-time PCR analyses for levels of RNA transcripts incorporating various TCR V{alpha} family members. Note a separate right-hand panel depicts levels of mRNA transcripts incorporating the V{alpha}8 element used by the transgenic AI4 TCR. Expression levels of mRNA transcripts incorporating the V{alpha}8 element were significantly higher in CD8+ T cells of B6.H2g7 origin than from either NOD or NOD.Chr7B6 background mice (p = 0.0000080 and 0.0000514, respectively). Levels of mRNA transcripts using V{alpha}8 were also significantly higher in CD8+ T cells from NOD.Chr7B6.AI4 than NOD.AI4 mice (p = 0.0000979). Similar results were obtained in a second experiment. B, MFI ± SEM of V{alpha}8 Ab staining of Vβ2 coexpressing DP thymocytes from NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice. *, Significantly greater than in NOD.AI4 mice (p = 0.0209, Wilcoxon rank sum test). **, Significantly greater than in NOD.AI4, but less than in B6.H2g7.AI4 mice (p = 0.0209, Wilcoxon rank sum test). C, MFI ± SEM of V{alpha}8 Ab staining of Vβ2 coexpressing CD8+ splenic T cells from NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice. *, Significantly greater than in NOD.AI4 mice (p < 0.05, Wilcoxon rank sum test). **, Significantly greater than in NOD.AI4, but less than in B6.H2g7.AI4 mice (p = 0.0433, Wilcoxon rank sum test).

 
We also assessed whether variation in expression levels of the AI4 clonotypic TCR between NOD, B6.H2g7, and NOD.Chr7B6 background mice could at least be partly attributed to quantitative differences in transgenic {alpha}-chain encoding mRNA transcripts. It should be noted the AI4 TCR transgenes were congenically transferred from the NOD strain to the B6.H2g7 and NOD.Chr7B6 background stocks. Thus, variations in transgene copy number or insertion sites could not account for any differences found in levels of AI4 clonotypic TCR expression in NOD, B6.H2g7, and NOD.Chr7B6 background mice. Levels of mRNA transcripts using the V{alpha}8 component characterizing the AI4 TCR were much higher (2.3-fold; p = 0.0000080) in CD8+ T cells from B6.H2g7 than NOD background mice (Fig. 5A, right panel). Levels of mRNA transcripts using the V{alpha}8 component were also significantly higher (p = 0.0000979) in CD8+ T cells from NOD.Chr7B6.AI4 than NOD.AI4 mice (Fig. 5A, right panel). The magnitude of the difference in mRNA transcripts using V{alpha}8 was 1.6-fold higher in CD8+ T cells from NOD.Chr7B6.AI4 than NOD.AI4 mice. These collective results indicate the mechanism by which a polymorphic Idd7 region gene(s) determines the extent of AI4 clonotypic TCR expression at least partially entails the quantitative regulation of {alpha}-chain mRNA levels, without excluding the possibility of other contributory factors.

Previous studies have demonstrated that in addition to potentially occurring at the gene rearrangement level, the extent to which a particular TCR {alpha}-chain is expressed on the surface of T cells can also be dictated by its ability to compete for pairing with available TCR β-chains (42, 43). Thus, we tested whether the frequency of AI4 TCR {alpha}- and β-chain pairing differed on DP thymocytes and splenic CD8+ T cells from NOD, B6.H2g7, and NOD.Chr7B6 background mice. This was done by comparing the MFI of V{alpha}8 Ab staining of DP thymocytes and CD8+ splenic T cells that coexpressed the Vβ2 element characterizing the AI4 TCR. As shown in Fig. 5B, V{alpha}8 was expressed at a significantly higher level on Vβ2 positive DP thymocytes from B6.H2g7.AI4 than NOD.AI4 mice. Although not to the extent observed in the B6.H2g7 background stock, V{alpha}8 was also expressed at a significantly higher level by Vβ2 positive DP thymocytes from NOD.Chr7B6.AI4 than NOD.AI4 mice (Fig. 5B). A similar V{alpha}8 expression pattern characterized Vβ2 positive splenic CD8+ T cells from NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice (Fig. 5C). Although further study is required to determine the extent to which it results from transcriptional vs posttranscriptional processes, these results do demonstrate that the efficiency of AI4 TCR {alpha}- and β-chain pairing is at least partly regulated by an Idd7 region gene(s).

Idd7 region genes do not regulate the ability of β cell autoreactive CD8+ T cells in the periphery to induce T1D

We previously reported that the higher level of clonotypic TCR expression which is correlated with more efficient intrathymic deletion of AI4 T cells in B6.H2g7 than NOD mice may be a "two edged sword process" because this feature was also associated with enhanced pathogenicity of the limited number of such effectors that did reach the periphery in the former strain (23). This was partly reflected by more rapid onset of T1D in B6.H2g7.AI4 than NOD.AI4 mice (23). The greater capacity of peripheral AI4 T cells of B6.H2g7 than NOD origin to induce T1D was accompanied by increased functional activity in response to Ag stimulation (23). Thus, we assessed whether the B6 origin Idd7 region congenic interval contributing to higher levels of AI4 TCR expression and more efficient thymic deletion of this clonotype than in fully NOD background mice, also enhanced the pathogenicity of such effectors that did reach the periphery.

As previously observed, numbers of peripheral AI4 T cells were significantly lower in B6.H2g7 than NOD background mice (Fig. 6A). However, unexpectedly, despite being present at significantly lower levels in the thymus, the numbers of peripheral AI4 T cells in the NOD.Chr7B6 stock were actually somewhat higher than observed in fully NOD background mice. Therefore, although genes in the Idd7 region control the efficiency of intrathymic deletion of diabetogenic CD8+ T cells, perhaps through the reciprocal regulation of clonotypic TCR expression levels, other factors common to the NOD and NOD.Chr7B6 strains determine the extent to which such effectors can expand and/or survive in the periphery. As discussed earlier, AI4 T cells from the B6.H2g7 and NOD.Chr7B6 stocks express the clonotypic TCR at a higher level than those from fully NOD background mice. Thus, we initially tested whether their higher levels of TCR expression correlated with an ability of AI4 T cells from B6.H2g7 and NOD.Chr7B6 mice to respond more vigorously upon antigenic peptide stimulation than those of NOD origin. As shown in Fig. 6B, ELISPOT analyses confirmed this to be the case. Based on these results we then hypothesized that like those in the B6.H2g7 background strain, AI4 T cells from the NOD.Chr7B6 stock would also induce a more rapid onset of T1D than those of fully NOD origin. Compared with standard nontransgenic NOD mice, T1D did develop more rapidly in all three of the AI4 stocks (Fig. 6C). Also as previously observed, T1D onset was significantly more rapid in B6.H2g7.AI4 than NOD.AI4 mice. However, despite their quite different levels of clonotypic TCR expression and responsiveness to antigenic stimulation, the rate of T1D development was equivalent in NOD.AI4 and NOD.Chr7B6.AI4 mice. These collective results indicated that while fewer in number than in NOD background mice, the basis for the greatly enhanced pathogenic activity of the peripheral AI4 T cells that do develop in the B6.H2g7 stock cannot be solely attributed to their higher level of Idd7 locus induced clonotypic TCR expression. However, Idd7 region control of processes resulting in different levels of clonotypic TCR expression remains a viable mechanistic explanation for the variable extent to which diabetogenic CD8+ T cells undergo intrathymic deletion in NOD and B6.H2g7 background mice.


Figure 6
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 6. Idd7 region genes do not regulate the pathogenicity of diabetogenic CD8+ T cells in the periphery. A, Numbers of splenic CD8+ T cells expressing the transgenic AI4 TCR in 5–6 wk old NOD, NOD.Chr7B6, and B6.H2g7 background mice. *, Significantly less than in NOD.AI4 mice (p = 0.0039, Wilcoxon rank sum test). **, Significantly greater than in NOD.AI4 mice (p = 0.0065, Wilcoxon rank sum test). (B) Comparative response of purified splenic CD8+ T cells from NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice to AI4 mimotope stimulation as assessed by IFN-{gamma} ELISPOT analyses. Response of CD8+ T cells from NOD.AI4 mice was significantly less than those of B6.H2g7.AI4 or NOD.Chr7B6.AI4 origin (p < 0.001 by ANOVA). Response of CD8+ T cells from NOD.Chr7B6.AI4 mice was significantly less than those from the B6.H2g7.AI4 strain (p = 0.0191 by ANOVA). C, Kinetics of T1D development in NOD, NOD.AI4, B6.H2g7.AI4, and NOD.Chr7B6.AI4 mice. Compared with standard NOD mice, T1D development was significantly accelerated in all three AI4 TCR transgenic stocks (p ≤ 0.03, Kaplan Meier life table analysis). T1D development was significantly more rapid in B6.H2g7.AI4 mice compared with both the NOD.AI4 and NOD.Chr7B6.AI4 stocks (p ≤ 0.0001, Kaplan-Meier life table analysis). The NOD.AI4 and NOD.Chr7B6.AI4 stocks did not differ in rates of T1D development.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In a previous study (23), we found that unlike the case in a B6 background stock, when expressed in NOD mice the common class I variants (Kd and Db) encoded within the H2g7 MHC haplotype aberrantly lose the ability to mediate the intrathymic deletion of autoreactive diabetogenic CD8+ T cells. In this study, using the AI4 TCR transgenic system, we have determined through both linkage and congenic strain analyses, that interactive contributions from a polymorphic gene(s) in the previously described Idd7 locus on the proximal portion of Chr. 7 (36, 37) predominantly determines the extent to which H2g7 class I molecules can mediate the thymic deletion of diabetogenic CD8+ T cells. Other genes possibly contributing to this phenotype, but in a much weaker fashion, mapped to sites on Chr. 4 (Idd9/11 region), 12, 13 (Idd14 region), and 17. The ability of the Idd7 region gene(s) to contribute to less efficient intrathymic deletion of AI4 T cells in NOD than B6.H2g7 background mice is associated with processes that may be transcriptional, and also possibly posttranscriptional in nature, which result in lower expression levels of the clonotypic TCR {alpha}-chain in the former strain. As a result, expression levels of the clonotypic AI4 TCR in NOD, but not B6.H2g7 thymocytes, may fall below the threshold necessary to induce a signaling response sufficient to trigger negative selection upon Ag engagement.

Other investigators have mapped genetic loci interactively contributing to the impaired ability of NOD mice to mediate the intrathymic deletion of CD4+ T cells that recognize natural or neo-self Ags expressed in pancreatic β cells (44, 45, 46). One of these studies found that genes strongly contributing to the differential thymic development in NOD and B6.H2g7 mice of the diabetogenic BDC2.5 CD4+ T cell clonotype mapped to sites on Chr. 1 and 3 (46). The Chr. 3 QTL regulating thymic development of BDC2.5 T cells largely overlapped the ~80–88 Mb location of the previously defined Idd17 locus (46, 47). As noted in the Materials and Methods, the AI4 TCR β-chain transgene congenically transferred to the B6.H2g7 background was accompanied by linked NOD-derived genomic material on Chr. 3 spanning from ~87–97 Mb. This would have resulted in the displacement, and hence an inability to detect in our study, any B6-derived gene between 87 and 97 Mb on Chr. 3 that might also limit the thymic development of disease relevant effectors in the CD8+ compartment such as the AI4 clonotype. The AI4 TCR {alpha}-chain transgene congenically transferred to the B6.H2g7 background is flanked by NOD-derived genomic material on Chr. 1 spanning from ~156–187 Mb. However, the Chr. 1 QTL regulating thymic development of BDC2.5 CD4+ T cells mapped somewhat telomeric to the Idd5 complex at ~90Mb (46). Thus, if the B6-derived genes on Chr. 1 that promotes the negative selection of diabetogenic BDC2.5 CD4+ T cells also enable elimination of the AI4 CD8+ clonotype, then this effect should have been observed in our cross. The impaired ability of NOD compared with B10 background mice to mediate the thymic deletion of CD4+ T cells specific for a neo-self Ag transgenically expressed in β cells has also been reported to be largely controlled by a gene(s) mapping to the Idd13 locus region on Chr. 2, with a prime candidate being Bim due its known role in apoptosis regulation (44, 45). The fact these previously reported Chr. 1 and 2 effects were not observed in our current analyses suggest the possibility that the genes controlling the negative selection of diabetogenic CD4+ and CD8+ T cells are not completely overlapping. It should also be remembered that during thymocyte differentiation, transgenic TCR molecules can appear on the cell surface at an earlier stage of development than those of endogenous origin, and CD8 expression precedes that of CD4 (48, 49). These factors could also contribute to the negative selection of diabetogenic T cells in the CD4 and CD8 compartments being differentially regulated.

Although the non-MHC genetic loci we found to regulate the thymic deletion of diabetogenic AI4 CD8+ T cells did not completely overlap with those previously reported to control development of pathogenic effectors in the CD4 compartment, there were some possible similarities. In the present study, a polymorphic gene(s) on the proximal end of Chr. 7 exerted the strongest effect in controlling the efficiency of intrathymic AI4 T cell deletion. Suggestive effects on Chr. 7 were also noted in earlier studies mapping genes controlling the extent to which diabetogenic CD4 T cells undergo thymic deletion (44, 45, 46). Linkage analyses suggested the Chr. 7 genes controlling thymic development of diabetogenic CD4 T cells mapped distally to the Idd7 region identified in our current study to control the extent to which pathogenic AI4 CD8+ T cells undergo negative selection. However, unlike our AI4 based analyses, the position of Chr. 7 genes possibly controlling the extent to which diabetogenic CD4 T cells undergo negative selection were not more finely delineated through the use of congenic strains. Without congenic mapping of Chr. 7 genes controlling the negative selection efficiency of diabetogenic CD4 T cells, it is difficult to ascertain the extent to which they may possibly overlap with those regulating the thymic development of pathogenic effectors in the CD8+ compartment. Also similar to what we observed in the current study for AI4 CD8+ T cells, genes on Chr. 4 and 13 have been reported to have a suggestive effect on controlling the thymic deletion of diabetogenic effectors in the CD4 compartment (46).

Another issue is why do the significantly lower number of AI4 T cells that escape thymic deletion in B6.H2g7 than NOD background mice then go on to induce a much more rapid onset of T1D? We previously conjectured this is because expression levels of the clonotypic TCR is significantly higher on AI4 T cells reaching the periphery in B6.H2g7 than NOD mice (23). This explanation now appears to be at best only partially correct based on the finding that higher expression levels of the clonotypic TCR do not also result in faster T1D development in NOD.Chr7B6.AI4 than NOD.AI4 mice. However, it is possible that while achieving statistical significance, the difference in clonotypic TCR expression may not be sufficiently large enough to render AI4 T cells more pathogenic in NOD.Chr7B6 than NOD background mice. An additional possible explanation for more rapid T1D development in B6.H2g7.AI4 than either NOD.AI4 or NOD.Chr7B6.AI4 mice is that because transgene induced allelic exclusion efficiency is most pronounced in the former strain it also has the least capacity to generate regulatory T cells (Treg) expressing endogenously derived TCR elements. The frequency of endogenous TCR {alpha}-chain gene rearrangement events appears not to differ between NOD.AI4 and NOD.Chr7B6.AI4 mice which could result in these two strains being characterized by an equivalent level of Treg, and hence T1D development. Such a possible relationship between the plasticity of TCR {alpha}-chain expression and Treg development will be the subject of future investigations.

In conclusion, we have found that when expressed in NOD mice the common class I variants encoded by the H2g7 MHC haplotype aberrantly lose an ability to mediate the thymic deletion of autoreactive diabetogenic CD8+ T cells primarily, but not exclusively, due to strong interactive contributions of a gene(s) within the previously identified Idd7 locus. It seems likely that this Idd7 region gene controls events that determine whether the clonotypic TCR of diabetogenic CD8+ T cells is expressed at a level necessary to induce a sufficiently high signaling response to trigger negative selection following intrathymic antigenic ligand engagement. This finding provides further insight to how susceptibility genes both within and outside the MHC may interact to elicit development of the pancreatic β cell autoreactive T cells that mediate T1D development in both NOD mice and human patients.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 DK51090 and DK46266, Cancer Center Support Grant CA34196, and grants from the Juvenile Diabetes Research Foundation. Back

2 Address correspondence and reprint requests to Dr. David V. Serreze, Senior Staff Scientist, The Jackson Laboratory, Bar Harbor, ME 04609. E-mail address: dave.serreze{at}jax.org Back

3 Abbreviations used in this paper: T1D, type 1 diabetes; DP, double positive; SNP, single nucleotide polymorphisms; AP, allophycocyanin; QTL, quantitative trait loci; MFI, mean fluorescence intensity; PPD, posterior probability density; LOD, logarithm of odds; Chr., chromosome. Back

Received for publication September 12, 2007. Accepted for publication December 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Anderson, M. S., J. A. Bluestone. 2005. The NOD mouse: a model of immune dysregulation. Annu. Rev. Immunol. 23: 447-485. [Medline]
  2. Serreze, D. V., E. H. Leiter. 2001. Genes and pathways underlying autoimmune diabetes in NOD mice. M. G. von Herrath, ed. Molecular Pathology of Insulin Dependent Diabetes Mellitus 31-67. Karger Press, New York.
  3. 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]
  4. Nepom, G. T., W. W. Kwok. 1998. Molecular basis for HLA-DQ associations with IDDM. Diabetes 47: 1177-1184. [Abstract]
  5. Hanson, M. S., M. Cetkovic-Cvrlje, V. K. Ramiya, M. A. Atkinson, N. K. MacLaren, B. Singh, J. F. Elliott, D. V. Serreze, E. H. Leiter. 1996. Quantitative thresholds of MHC class II I-E expressed on hematopoietically derived APC in transgenic NOD/Lt mice determine level of diabetes resistance and indicate mechanism of protection. J. Immunol. 157: 1279-1287. [Abstract]
  6. Lund, T., L. O’Reilly, P. Hutchings, O. Kanagawa, E. Simpson, R. Gravely, P. Chandler, J. Dyson, J. K. Picard, A. Edwards, et al 1990. Prevention of insulin-dependent diabetes mellitus in non-obese diabetic mice by transgenes encoding modified I-A β-chain or normal I-E {alpha}-chain. Nature 345: 727-729. [Medline]
  7. Miyazaki, T., M. Uno, M. Uehira, H. Kikutani, T. Kishimoto, M. Kimoto, H. Nishimoto, J. Miyazaki, K. Yamamura. 1990. Direct evidence for the contribution of the unique I-Anod to the development of insulitis in non-obese diabetic mice. Nature 345: 722-724. [Medline]
  8. Singer, S. M., R. Tisch, X.-D. Yang, H. O. McDevitt. 1993. An Abd transgene prevents diabetes in nonobese diabetic mice by inducing regulatory T cells. Proc. Natl. Acad. Sci. USA 90: 9566-9570. [Abstract/Free Full Text]
  9. Slattery, R. M., L. Kjer-Nielsen, J. Allison, B. Charlton, T. Mandel, J. F. Miller. 1990. Prevention of diabetes in non-obese diabetic I-Ak transgenic mice. Nature 345: 724-726. [Medline]
  10. Serreze, D. V., E. H. Leiter, G. J. Christianson, D. Greiner, D. C. Roopenian. 1994. MHC class I deficient NOD-B2mnull mice are diabetes and insulitis resistant. Diabetes 43: 505-509. [Abstract]
  11. 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. β2-microglobulin-deficient NOD mice do not develop insulitis or diabetes. Diabetes 43: 500-504. [Abstract]
  12. Hattori, M., E. Yamato, N. Itoh, H. Senpuku, T. Fujisawa, M. Yoshino, M. Fukuda, E. Matsumoto, T. Toyonaga, I. Nakagawa, et al 1999. 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]
  13. Pomerleau, D. P., R. J. Bagley, D. V. Serreze, C. E. Mathews, E. H. Leiter. 2005. Major histocompatibility complex-linked diabetes susceptibility in NOD mice: subcongenic analysis localizes a component of Idd16 at the H2-D end of the diabetogenic H2g7 complex. Diabetes 54: 1603-1606. [Abstract/Free Full Text]
  14. Hassainya, Y., F. Garcia-Pons, R. Kratzer, V. Lindo, F. Greer, F. Lemonnier, G. Niedermann, P. M. van Endert. 2005. Identification of naturally processed HLA-A2-restricted proinsulin epitopes by reverse immunology. Diabetes 54: 2053-2059. [Abstract/Free Full Text]
  15. Marron, M. P., R. T. Graser, H. D. Chapman, D. V. Serreze. 2002. Functional evidence for the mediation of diabetogenic T cell responses by human 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]
  16. Ouyang, Q., N. E. Standifer, H. Qin, P. A. Gottlieb, C. B. Verchere, G. T. Nepom, R. Tan, C. Panagiotopoulos. 2006. Recognition of HLA-class I restricted β-cell epitopes in type 1 diabetes. Diabetes 55: 3068-3074. [Abstract/Free Full Text]
  17. Pinske, G. G. M., O. H. M. Tysma, C. A. M. Bergen, M. G. D. Kester, F. Ossendorp, P. A. van Veelen, B. Keymeulen, D. Pipeleers, J. W. Drijfhout, B. O. Roep. 2005. Autoreactive CD8 T cells associated with β cell destruction in type 1 diabetes. Proc. Natl. Acad. Sci. USA 102: 18425-18430. [Abstract/Free Full Text]
  18. Robles, D. T., G. S. Eisenbarth, T. Wang, H. A. Erlich, T. L. Bugawan, S. R. Babu, K. Barriga, J. 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]
  19. Standifer, N. E., Q. Ouyang, C. Panagiotopoulos, C. B. Verchere, R. Tan, C. J. Greenbaum, C. Pihoker, G. T. Nepom. 2006. Identification of novel HLA-A*0201-restricted epitopes in recent onset type 1 diabetic subjects and antibody positive relatives. Diabetes 55: 3061-3067. [Abstract/Free Full Text]
  20. Takaki, T., M. P. Marron, C. E. Mathews, S. T. Guttman, R. Bottino, M. Trucco, T. P. DiLorenzo, D. V. Serreze. 2006. HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes. J. Immunol. 176: 3257-3265. [Abstract/Free Full Text]
  21. Undlien, D. E., B. A. Lie, E. Thorsby. 2001. HLA complex genes in type 1 diabetes and other autoimmune diseases: which genes are involved?. Trends Genet. 17: 93-100. [Medline]
  22. Mallone, R., E. Martinuzzi, P. Blancou, G. Novelli, G. Afonzo, M. Dolz, G. Bruno, L. Chaillous, L. Chatenoud, J.-M. Bach, P. M. van Endert. 2007. CD8+ T cell responses identify β-cell autoimmunity in human type 1 diabetes. Diabetes 56: 613-621. [Abstract/Free Full Text]
  23. 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]
  24. DiLorenzo, T. P., R. T. Graser, T. Ono, G. J. Christianson, H. D. Chapman, D. C. Roopenian, S. G. Nathenson, D. V. Serreze. 1998. MHC class I-restricted T cells are required for all but the end stages of diabetes development in NOD mice and utilize a prevalent T cell receptor {alpha} chain gene rearrangement. Proc. Natl. Acad. Sci. USA 95: 12538-12543. [Abstract/Free Full Text]
  25. Graser, R. T., T. P. DiLorenzo, F. Wang, G. J. Christianson, H. D. Chapman, D. C. Roopenian, S. G. Nathenson, D. V. Serreze. 2000. Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions. J. Immunol. 164: 3913-3918. [Abstract/Free Full Text]
  26. Yui, M. A., K. Muralidharan, B. Moreno-Altamirano, G. Perrin, K. Chestnut, E. K. Wakeland. 1996. Production of congenic mouse strains carrying NOD-derived diabetogenic genetic intervals: an approach for the genetic dissection of complex traits. Mamm. Genome 7: 331-334. [Medline]
  27. Chen, J., P. C. Reifsnyder, F. Scheuplein, W. H. Schott, M. Mileikovsky, S. Soodeen-Karamath, A. Nagy, H.-M. Dosch, J. Ellis, F. Koch-Nolte, E. H. Leiter. 2005. "Agouti NOD": identification of a CBA-derived Idd locus on chromosome 7 and its use for chimera production with NOD embryonic stem cells. Mamm. Genome 16: 775-783. [Medline]
  28. Myakishev, M., Y. Khripin, S. Hu, D. H. Hamer. 2001. High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers. Genome Res. 11: 163-169. [Abstract/Free Full Text]
  29. Sen, S., G. A. Churchill. 2001. A statistical framework for quantitative trait mapping. Genetics 159: 371-387. [Abstract/Free Full Text]
  30. Broman, K. W., H. Wu, S. Sen, G. A. Churchill. 2003. R/qtl: QTL mapping in experimental crosses. Bioinformatics 19: 889-890. [Abstract/Free Full Text]
  31. Doerge, R. W., G. A. Churchill. 1996. Permutation tests for multiple loci affecting a quantitative character. Genetics 142: 285-294. [Abstract]
  32. Lander, E., L. Kruglyak. 1995. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat. Genet. 11: 241-247. [Medline]
  33. Serreze, D. V., E. A. Johnson, H. D. Chapman, R. T. Graser, M. P. Marron, T. P. DiLorenzo, P. Silveira, Y. Yoshimura, S. G. Nathenson, S. Joyce. 2001. Autoreactive diabetogenic T-cells in NOD mice can efficiently expand from a greatly reduced precursor pool. Diabetes 50: 1992-2000. [Abstract/Free Full Text]
  34. Serreze, D. V., T. M. Holl, M. P. Marron, R. T. Graser, E. A. Johnson, C. Choisy-Rossi, R. M. Slattery, S. M. Lieberman, T. P. DiLorenzo. 2004. MHC class II molecules play a role in the selection of autoreactive class I restricted CD8 T cells that are essential contributors to type 1 diabetes development in NOD mice. J. Immunol. 172: 871-879. [Abstract/Free Full Text]
  35. Akilesh, S., D. J. Shaffer, D. C. Roopenian. 2003. Customized molecular phenotyping by quantitative gene expression and pattern recognition analysis. Genome Res. 13: 1719-1727. [Abstract/Free Full Text]
  36. Ghosh, S., S. M. Palmer, N. R. Rodrigues, H. J. Cordell, C. M. Hearne, R. J. Cornall, J. B. Prins, P. McShane, G. M. Lathrop, L. B. Peterson, et al 1993. Polygenic control of autoimmune diabetes in nonobese diabetic mice. Nat. Genet. 4: 404-409. [Medline]
  37. McAleer, M. A., P. C. Reifsnyder, S. M. Palmer, M. Prochazka, J. M. Love, J. B. Copeman, E. E. Powell, N. R. Rodrigues, J.-B. Prins, D. V. Serreze, et al 1995. Crosses of NOD mice with the related NON strain: a polygenic threshold model for type I diabetes. Diabetes 44: 1186-1195. [Abstract]
  38. Brodnicki, T. C., A. L. Fletcher, D. G. Pellicci, S. P. Berzins, P. J. McClive, F. Quirk, K. E. Webster, H. S. Scott, R. L. Boyd, D. I. Godfrey, G. Morahan. 2005. Localization of Idd11 is not associated with thymus and NKT cell abnormalities in NOD mice. Diabetes 54: 3453-3457. [Abstract/Free Full Text]
  39. Lyons, P. A., W. W. Hancock, P. Denny, C. J. Lord, N. J. Hill, N. Armitage, T. Siegmund, J. A. Todd, M. S. Phillipps, J. F. Hess, et al 2000. The NOD Idd9 genetic interval influences the pathogenicity of insulitis and contains molecular variants of Cd30, Tnfr2, and Cd137. Immunity 13: 107-115. [Medline]
  40. McNeil, L. K., B. D. Evavold. 2002. Dissociation of peripheral T cell responses from thymocyte negative selection by weak agonists supports a spare receptor model of T cell activation. Proc. Natl. Acad. Sci. USA 99: 4520-4525. [Abstract/Free Full Text]
  41. McNeil, L. K., B. D. Evavold. 2003. TCR reserve: a novel principal of CD4 T cell activation by weak ligands. J. Immunol. 170: 1224-1230. [Abstract/Free Full Text]
  42. Alam, S. M., N. R. J. Gascoigne. 1998. Posttranslational regulation of TCR V{alpha} allelic exclusion during T cell differentiation. J. Immunol. 160: 3883-3890. [Abstract/Free Full Text]
  43. Huang, C.-Y., O. Kanagawa. 2001. Ordered and coordinated rearrangement of the TCR {alpha} locus: role of secondary rearrangement in thymic selection. J. Immunol. 166: 2597-2601. [Abstract/Free Full Text]
  44. Liston, A., S. Lesage, D. H. D. Gray, L. A. O’Reilly, A. Strasser, A. M. Fahrer, R. L. Boyd, J. Wilson, A. G. Baxter, E. M. Gallo, et al 2004. Generalized resistance to thymic deletion in the NOD mouse: a polygenic trait characterized by defective induction of Bim. Immunity 21: 817-830. [Medline]
  45. Liston, A., K. Hardy, Y. Pittelkow, S. R. Wilson, L. E. Makaroff, A. M. Fahrer, C. C. Goodnow. 2007. Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling. Genome Biol. 8: R12[Medline]
  46. Zucchelli, S., P. Holler, T. Yamagata, M. Roy, C. Benoist, D. Mathis. 2005. Defective central tolerance induction in NOD mice: genomics and genetics. Immunity 22: 385-396. [Medline]
  47. Lyons, P. A., N. Armitage, C. J. Lord, M. S. Phillips, J. A. Todd, L. B. Peterson, L. S. Wicker. 2001. Mapping by genetic interaction: high-resolution congenic mapping of the type 1 diabetes loci Idd10 and Idd18 in the NOD mouse. Diabetes 50: 2633-2637. [Abstract/Free Full Text]
  48. Baldwin, T. A., M. M. Sandau, S. Jameson, K. A. Hogquist. 2005. The timing of TCR{alpha} expression critically influences T cell development and selection. J. Exp. Med. 202: 111-121. [Abstract/Free Full Text]
  49. Starr, T. K., S. Jameson, K. A. Hogquist. 2003. Positive and negative T cell selection. Annu. Rev. Immunol. 21: 139-176. [Medline]




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

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS