From the Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
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ABSTRACT |
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INTRODUCTION |
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Although it has been conclusively established that the factors responsible for the MHC-linked resistance to autoimmune diabetes reside in the bone marrow (511), the underlying mechanisms remain elusive. A number of early studies of congenic and transgenic NOD mice expressing antidiabetogenic MHC class II molecules found evidence for the existence of T-cell autoreactivity in the peripheral immune systems of these mice. These observations demonstrated that protective MHC class II molecules do not afford diabetes resistance by systematically deleting all autoreactive T-cell specificities (1117). However, they could not rule out a role for selective tolerance (i.e., of certain autoreactive T-cells). Studies of mice expressing a highly diabetogenic I-Ag7-restricted T-cell receptor (TCR) (4.1) later provided direct support for this possibility. We found that thymocytes expressing the 4.1-TCR undergo complete deletion in mice carrying a single copy of the antidiabetogenic haplotype H-2b in an I-Ab-dependent but superantigen-independent manner (18). Like the diabetes resistance afforded by protective MHC class II molecules in non-TCR-transgenic mice, the diabetes resistance afforded by the I-Ab-induced deletion of thymocytes in 4.1-TCR-transgenic mice was mediated by bone marrow-derived cells (18,19).
These observations suggested that in nontransgenic mice, protective MHC class II molecules might afford diabetes resistance by tolerizing certain highly pathogenic T-cell specificities. This hypothesis predicted that the 4.1-T-cell tolerogenic activity of I-Ab would be shared by other antidiabetogenic MHC class II molecules and that it would be mediated by dendritic cells, macrophages, and B-cells. Here, we have tested these predictions by investigating the ability of three different MHC class II molecules (I-Ek/I-Eßg7, I-Ad, and I-Ag7PD) and different antigen-presenting cell (APC) types to tolerize 4.1-CD4+ T-cells and/or to prevent 4.1-CD4+ T-cell-induced diabetes. We show that 4.1-CD4+ T-cells undergo different forms of tolerance in 4.1-TCR-transgenic NOD mice expressing I-E
k, I-Ad, or I-Ag7PD molecules. Surprisingly, T-cell tolerance in these mice is triggered by peptide/MHC class II expressed on dendritic cells and macrophages but not by peptide/MHC class II expressed on B-cells. These data provide but one explanation to the puzzling ability of structurally diverse MHC class II molecules to provide dominant resistance against autoimmune diabetes. We propose that protective MHC class II molecules afford diabetes resistance by presenting, on dendritic cells and macrophages, tolerogenic peptide(s) to a group of MHC-promiscuous and pathogenic 4.1-like CD4+ thymocytes that play a critical role in diabetogenesis.
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RESEARCH DESIGN AND METHODS |
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Diabetes.
Diabetes was monitored by measuring urine glucose levels with Diastix and a glucometer. Animals were considered diabetic after at least two consecutive readings of 3+.
Bone marrow chimeras.
Chimeras were generated following standard protocols (18). In brief, recipient mice were treated with two doses of 500 rads 3 h apart from a 137Cs source (Atomic Energy of Canada, Ottawa, ON) and then transfused with marrow cell suspensions (815 x 106 cells/mouse). Chimeric mice were killed 68 weeks after transplantation.
Antibodies and flow cytometry.
The monoclonal antibodies (mAbs) anti-CD8-PE (53-6.7), anti-CD4-fluorescein isothiocyanate (FITC) (RM4-5 or IM7), anti-Vß11-FITC (RR3-15), anti-I-Aßb-biotin (AF6-120.1), anti-I-Ek-biotin (17-3-3), anti-I-Aßk/g7-biotin (10-3.6), anti-CD45R/B220-biotin or -PerCP (RA36B2), anti-CD11c-biotin (HL3), and anti-CD11b-biotin (M1/70) were from PharMingen (San Diego, CA). Anti-L3T4 (CD4)-biotin (YTS191.1) was from Cedarlane (Hornby, ON). Streptavidin-PerCP was from Becton Dickinson (San Jose, CA). Thymocytes and splenocytes were analyzed with a FACScan.
Purification of splenic B-cells, thymic dendritic cells, splenic dendritic cells, and peritoneal macrophages.
B-cells were purified by positive selection with B220-coated microbeads using the MiniMACS system (Miltenyi Biotec, Auburn, CA). Purity was >95% CD19+ or B220+ cells. To purify dendritic cells, spleens or thymi (n = 10) were dissected in 10 ml of 100 units/ml collagenase type IV in Hanks buffered salt solution on ice and then incubated with 400 u/ml collagenase at 37°C for 30 min. Dendritic cells (CD11c+) were purified by two rounds of positive selection using anti-CD11c-coated microbeads (Miltenyi-Biotech). Purity was >95% CD11c+ cells. Peritoneal macrophages were collected by peritoneal lavage of mice injected intraperitoneally 3 days earlier with 1 ml of 3% thyoglicollate in PBS. The cells were >80% pure as determined by staining with anti-F4/80 and anti-CD11b mAbs.
Proliferation assays.
Splenocytes were depleted of CD8+ T-cells using anti-CD8 mAb-coated beads or used to prepare pure CD4+ T-cells using the MiniMACs system (Miltenyi Biotec). Pancreatic islets were isolated as described (18). CD4+ T-cells (2 x 104 cells) were incubated in duplicate with -irradiated (3,000 rad) NOD islet cells (105/well) in 96-well plates for 3 days at 37°C in 5% CO2, with or without rIL-2 (Takeda, Osaka, Japan). Alloreactivity of mature 4.1-T-cells was assessed by culturing 2 x 104 CD4+ T-cells from RAG-2-/- 4.1-NOD mice with 105 allogeneic dendritic cells, peritoneal macrophages, or B-cells. All cultures were pulsed with 1 µCi of [3H]-thymidine during the last 18 h of culture. Specific proliferation was calculated by subtracting background (counts per minute of cultures only containing islet cells or APCs and counts per minute of cultures of T-cells alone) from islet cell- or APC-induced proliferation (counts per minute of cultures containing T-cells and either islet cells or APCs).
Dulling assay.
Dulling assays were done by co-culturing 105 APCs with 105 thymocytes from RAG-2-deficient 4.1-NOD mice in triplicate in -bottomed 96-well plates at 37°C, 5% CO2, for 20 h. At the end of the incubation period, cells were stained with anti-CD4-PE, anti-CD8-FITC, and anti-CD11c-biotin/streptavidin-PerCP, anti-CD11b-FITC, or anti-B220-PerCP and analyzed by flow cytometry.
Cytokine profile of intra-islet 4.1-CD4+ T-cells and intra-cytoplasmic cytokine staining.
Freshly isolated islet and islet-associated T-cells (2 x 104) were activated with phorbol-myristate acetate (PMA) (10 ng/ml) and ionomycin (250 ng/ml) for 12 h at 37°C, 5% CO2. The supernatants were assayed for interleukin (IL)-2, IL-4, and -interferon (IFN-
) content by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Intracytoplasmic staining was done using the Cytofix/Cytoperm Plus (with GolgiStop) Kit (PharMingen) and the following mAbs: anti-IL2-PE (JES6-5H4), anti-IL4-PE (11B11), or anti-IFN-
-PE (XMG1.2).
In vivo bromodeoxyuridine labeling, histology, and immunopathology.
Mice were given two injections of 200 µl i.v. of a 4-mg/ml solution of 5-bromo-2'-deoxyuridine (BrdU; Calbiochem, La Jolla, CA) 4 h apart. The pancreata and spleens were collected 12 h later. Quantitation of the percentage of islet-infiltrating cells and splenocytes that incorporated BrdU was done on frozen tissue using a BrdU staining kit (Calbiochem). The degree of insulitis was determined on hematoxylin and eosin (H/E)-stained sections, using previously described criteria (18).
Statistics.
Statistical analyses were performed using the Mann-Whitney U and 2 tests.
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RESULTS |
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One-third of 4.1-NOD.I-Ek mice (7 of 21) had thymocyte and splenic cytofluorometric profiles that were compatible with complete negative selection of 4.1-thymocytes, as compared with other TCR-transgenic models (2730). These mice (herein referred to as "deleting") displayed a significant reduction in the percentage of CD4+CD8- thymocytes, a reduction in the percentage of CD4+CD8- thymocytes expressing the transgene-encoded Vß11 element, and an increase in the percentage of CD4-CD8- thymocytes when compared with 4.1-NOD mice (Table 1 and Fig. 1A and B, upper panels). In the spleen, deleting 4.1-NOD.I-E
k mice had significantly fewer CD4+ T-cells and CD4+Vß11+ T-cells than 4.1-NOD mice (Table 1 and Fig. 1A and B, lower panels). The few Vß11+CD4+ T-cells that matured in deleting mice expressed significantly lower levels of the transgenic TCRß chain on the cell surface than the Vß11+CD4+ T-cells that matured in 4.1-NOD mice (Table 1, Fig. 1A and B, and data not shown). This suggested that in deleting 4.1-NOD.I-E
k mice, most CD4+ T-cells were selected on endogenous TCR chains that had bypassed allelic exclusion. The remaining two-thirds of 4.1-NOD.I-E
k mice (14 of 21) displayed thymocyte and splenic profiles that were similar to those seen in 4.1-NOD mice (Table 1 and Fig. 1A and B), where 4.1-thymocytes undergo positive selection. Individual mice were classified as deleting if the values corresponding to the parameters described above were at least 2 SDs above or below the corresponding values in 4.1-NOD mice. Therefore, I-E
k/I-Eßg7 heterodimers can trigger massive deletion of 4.1-thymocytes, albeit only in a fraction of the mice.
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I-E-induced deletion of 4.1-thymocytes in vivo is induced by hematopoietic cells and is a stochastic process.
To investigate whether the I-Ek-induced deletion of 4.1-thymocytes was triggered by hematopoietic cells, we followed the fate of 4.1-CD4+ thymocytes in lethally irradiated NOD mice transfused with marrow from deleting or nondeleting 4.1-NOD.I-Ek mice. The phenotype of the bone marrow donors was a good, but imprecise, predictor of the phenotype seen in the chimeras. Altogether, 7 of 8 chimeras reconstituted with marrow from deleting donors deleted 4.1-thymocytes (P < 0.01 vs. 4.1-NOD
NOD chimeras), whereas 16 of 20 chimeras reconstituted with marrow from nondeleting donors displayed nondeleting flow cytometric profiles. Therefore, the phenotype of the chimeras is largely imprinted in the donor marrow, but marrow from single nondeleting or deleting donor mice can give rise to both nondeleting and deleting chimeras.
Deleting and nondeleting 4.1-NOD.I-Ek mice are diabetes resistant.
To determine the effects of thymocyte tolerance on 4.1-CD4+ T-cell-induced diabetes, we monitored 4.1-NOD.I-Ek mice for the development of diabetes (Table 2). As shown in Table 2, 4.1-NOD.I-E
k mice were nearly as diabetes-resistant as non-TCR-transgenic NOD.I-E
k mice, regardless of whether they were deleting or nondeleting.
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Deletion of 4.1-thymocytes by I-Ad molecules.
We next asked whether the ability of I-Ab (18) and I-E to induce 4.1-thymocyte tolerance was shared by other antidiabetogenic class II molecules (i.e., I-Ad). We crossed 4.1-NOD mice (expressing I-Ad and I-Aßg7) with I-Aßd-transgenic NOD mice (NOD.I-Ad) to produce 4.1-NOD.I-Ad mice (expressing I-A
d, I-Aßg7, and I-Aßd molecules). As shown in Table 1 and Fig. 4, these mice displayed cytofluorometric profiles compatible with negative selection of 4.1-thymocytes. When compared with 4.1-NOD mice, 4.1-NOD.I-Ad mice had significant reductions in the percentages of CD4+CD8- and Vß11+CD4+CD8- thymocytes and a significant increase in the percentage of CD4-CD8- thymocytes (Table 1 and Fig. 4A, left panels). Deletion was incomplete but was seen in all of the mice that were studied. As was the case for 4.1-NOD.I-E
k mice, deletion of 4.1-thymocytes in 4.1-NOD.I-Ad mice was not triggered by endogenous superantigens because non-TCR-transgenic NOD.I-Ad and NOD mice had comparable numbers of Vß11+CD4+ thymocytes and splenocytes (not shown). Studies of bone marrow chimeras indicated that I-Ad-induced deletion of 4.1-thymocytes was also triggered by bone marrow-derived APCs (eight of eight 4.1-NOD.I-Ad
NOD chimeras had a deleting phenotype versus none of seven chimeras generated with marrow from 4.1-NOD mice, P < 0.01).
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I-Ag7 molecules carrying proline and aspartic acid at I-Aß positions 56 and 57 (I-Ag7PD) are antidiabetogenic in I-Ag7PD-transgenic 4.1-NOD mice.
Comparison of the amino acid sequences of the class II molecules that can tolerize 4.1-thymocytes suggested an association between tolerogenic activity and the presence of certain residues between positions 56 and 67 of the different ß-chains. No other regions in I-A or I-Aß chains had sequence motifs that were either shared by most deleting class II molecules or absent from nondeleting class II molecules. This association was intriguing because this motif contains proline and aspartic acid at positions 56 and 57, which are epidemiologically associated with the ability of protective MHC molecules to afford diabetes resistance in both humans and mice (3,4).
To investigate the independent contribution of these two residues to the 4.1-tolerogenic activity of antidiabetogenic MHC class II molecules, we followed the fate of the 4.1-TCR in I-Ag7PD-transgenic 4.1-NOD mice (4.1-NOD.I-Ag7PD). Unlike I-Ag7 molecules, I-Ag7PD molecules carry proline and aspartic acid at I-Aß chain positions 56 and 57 and have a strong antidiabetogenic activity in NOD mice (3,4). Flow cytometry studies of 4.1-NOD.I-Ag7PD mice revealed that, unlike I-Ab, I-E, and I-Ad, I-Ag7PD did not cause massive deletion of 4.1-thymocytes (Table 1 and Fig. 4A, right panels). However, these mice had reduced CD4+-to-CD4-CD8- thymocyte ratios when compared with 4.1-NOD mice (Table 1 and Figs. 1 and 4), and their peripheral CD4+ T-cells were less responsive to antigenic stimulation than the peripheral CD4+ T-cells of 4.1-NOD mice (in the absence but not the presence of rIL-2) (Fig. 4B). Interestingly, 4.1-NOD.I-Ag7PD mice developed a significantly reduced incidence and a significantly delayed onset of diabetes when compared with 4.1-NOD mice (Table 2). The mice that did not develop diabetes developed insulitis. As was the case for 4.1-NOD.I-Ad and nondeleting 4.1-NOD.I-Ek mice, the insulitic T-cells of these mice were not immune-deviated (not shown). Therefore, the presence of proline and aspartic acid at I-Aß positions 56 and 57 has a protective effect against 4.1-CD4+ T-cell-induced diabetes through a mechanism that does not involve massive deletion of 4.1-thymocytes or their immune deviation.
I-Ag7PD, but not I-Ad or I-Ed, can mediate the positive selection of 4.1-thymocytes on thymic epithelial cells.
The above data show that different MHC class II molecules can tolerize a single, highly diabetogenic I-Ag7-restricted TCR. We have previously shown that one of these class II molecules, I-Ab, can trigger the deletion of 4.1-thymocytes in the thymic medulla but cannot restrict their positive selection in the thymic cortex (19). To determine whether the same was true for I-Ad, I-Ed, and I-Ag7PD, we investigated whether these molecules could mediate the positive selection of 4.1-thymocytes on thymic epithelial cells. We followed the fate of the 4.1-TCR in lethally irradiated NOD (I-Ag7), BALB/c (I-Ad and I-Ed), or BALB/c.I-Ag7PD mice (I-Ad/g7PD and I-Ed) that had been reconstituted with 4.1-NOD marrow. These chimeras expressed I-Ag7, I-Ag7PD, and/or I-Ad and I-Ed molecules on radioresistant thymic epithelial cells but only expressed I-Ag7 on the radiosensitive, bone marrow-derived APCs of the thymic medulla. As shown in Fig. 5A (upper right panel), thymi of 4.1-NOD BALB/c chimeras had profiles that were similar to those seen in MHC class II-deficient 4.1-NOD mice (4.1-I-A0/0 in Fig. 5A, lower right panel), where 4.1-thymocytes undergo massive developmental arrest. In contrast, thymi from 4.1-NOD
BALB/c.I-Ag7PD mice, which expressed I-Ag7PD, I-Ad, and I-Ed molecules on thymic epithelial cells and I-Ag7 on marrow-derived APCs, had profiles compatible with positive selection of the 4.1-TCR; 4.1-NOD
BALB/c.I-Ag7PD chimeras had more thymocytes (22 ± 4 vs. 5 ± 0.7 x 106; P < 0.003) and greater CD4+CD8+-to-CD4-CD8- thymocyte ratios than 4.1-NOD
BALB/c chimeras (3.2 ± 0.5 vs. 1 ± 0.3; P < 0.007) (Fig. 5A). As expected, splenic CD4+ T-cells from 4.1-NOD
BALB/c.I-Ag7PD chimeras proliferated almost as well as splenic CD4+ T-cells from 4.1-NOD mice in response to islet antigen, both in the presence and absence of rIL-2 (Fig. 5B). Therefore, 4.1-thymocytes can recognize I-Ag7PD (but not I-Ad or I-Ed) molecules on thymic epithelial cells. This demonstrates that, as was the case for I-Ab (19), deleting class II molecules can restrict the negative selection of 4.1-thymocytes in the medulla but not their positive selection in the thymic cortex.
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DISCUSSION |
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Expression of transgenic (and wild-type) I-Ek molecules in 4.1-NOD mice restored I-E expression and triggered the deletion of 4.1-CD4+ thymocytes in approximately one-third of the mice. The remaining two-thirds of mice positively selected 4.1-CD4+ T-cells, but these cells were unresponsive to antigenic stimulation in vitro. Experiments with bone marrow chimeras indicated that the deletion of 4.1-thymocytes in 4.1-NOD.I-E
k mice was a partially stochastic process that was mediated by bone marrow-derived cells. The mechanisms underlying the incomplete penetrance of this phenotype (deletion) are unclear. However, because the incomplete penetrance of the deleting phenotype seen in 4.1-NOD.I-E
k mice was also seen in 4.1-(NODxC58/j) F1 mice, it is unlikely that it can be accounted for by introduction of a genetic contaminant derived from the I-E
k donating strain. Furthermore, we have indirect evidence suggesting that it may be the result of differences in the number of APCs expressing I-E molecules (data not shown). It is also worth noting that whereas overexpression of an I-E
d transgene completely prevented diabetes in non-TCR-transgenic NOD mice, expression of this transgene at physiological levels did not completely abrogate diabetes susceptibility in NOD mice (17).
As expected, deletion of 4.1-thymocytes in deleting 4.1-NOD.I-Ek mice afforded the mice resistance to both insulitis and diabetes. Although nondeleting 4.1-NOD.I-E
k mice (bearing anergic 4.1-CD4+ T-cells) were also diabetes resistant, they developed insulitis. Surprisingly, the insulitis lesions of these mice were caused by actively proliferating 4.1-CD4+ T-cells. This indicated that 4.1-CD4+ T-cells maturing in nondeleting 4.1-NOD.I-E
k mice were only anergic to in vitro but not in vivo stimuli. The diabetes resistance of nondeleting 4.1-NOD.I-E
k mice was not caused by MHC-induced differentiation of 4.1-CD4+ T-cells into Th2 cells or by recruitment of regulatory Th2 cells to islets, because their islet-associated T-cells produced IL-2 and IFN-
but not IL-4. Furthermore, it was not caused by the inability of 4.1-CD4+ T-cells to recognize islet autoantigen, because 4.1-CD4+ T-cells from 4.1-NOD recognized NOD.I-E
k (I-Ag7+/I-E+) and NOD (I-Ag7+/I-E-) islets equally well (S.T., P.Se., A.A., J.V., P.Sa., unpublished observations). These data suggested that in nondeleting 4.1-NOD.I-E
k mice, the anergyzing stimulus was sufficiently strong to prevent the differentiation of 4.1-CD4+ T-cells into ß-cell-cytotoxic T-cells but not strong enough to prevent their proliferation in response to strong in vivo stimuli (i.e., autoantigen-loaded dendritic cells). The fact that nondeleting 4.1-NOD.I-E
k mice develop insulitis (unlike non-TCR-transgenic NOD.I-E
k mice) does not necessarily argue against a role for thymocyte tolerance as a mechanism for the MHC-linked resistance to diabetes in non-TCR-transgenic mice. The high frequency of 4.1-thymocytes in 4.1-TCR-transgenic mice may, in some mice, overwhelm the tolerogenic machinery that in non-TCR-transgenics would target a significantly smaller population of 4.1-like T-cells.
Although the overall three-dimensional structure of I-Ab and I-Ek molecules is similar, the amino acid sequences of their - and ß-chains are very different (36). This prompted us to investigate whether the ability of these two MHC molecules to tolerize 4.1-CD4+ thymocytes might be shared by other antidiabetogenic MHC class II molecules, such as I-Ad and I-Ag7PD. The 4.1-NOD.I-Ad mice displayed a phenotype that was similar to that seen in deleting 4.1-NOD.I-E
k mice, with two major differencesthe deleting phenotype of 4.1-NOD.I-Ad mice was incomplete, but its penetrance through the mouse population was complete. Unlike deleting 4.1-NOD.I-E
k mice, 4.1-NOD.I-Ad mice developed insulitis, and a small percentage of mice even developed diabetes. The mechanisms underlying these phenotypic differences are unknown but may be related to differences in the levels or timing of expression of I-E and I-A on thymic APCs. Subsequent studies of 4.1-NOD.I-Ag7PD mice suggested that this ability of protective class II molecules to prevent 4.1-CD4+ T-cell-induced diabetes was not unique to I-Ab, I-Ek, and I-Ad. The 4.1-NOD.I-Ag7PD mice displayed reduced CD4+-to-CD4-CD8- thymocyte ratios when compared with 4.1-NOD mice, and 4.1-CD4+ T-cells maturing in 4.1-NOD.I-Ag7PD mice consistently showed a slight reduction in proliferative responsiveness to antigen, which could be corrected by the addition of IL-2. The 4.1-NOD.I-Ag7PD mice developed moderate insulitis but a significantly reduced incidence (and delayed onset) of diabetes. It could be argued that the antidiabetogenic effect of the transgenic MHC class II molecules tested here was an artifact of transgenesis (i.e., by causing a reduction in the percentage of B-cells) (37). However, this is highly unlikely because 4.1-thymocyte tolerance is caused by both transgenic and wild-type MHC class II molecules. We cannot exclude the possibility that the different degrees of tolerance observed in this study reflect the effect of different levels of expression of the diverse transgenes in the relevant cell types. However, the fact that two different wild-type MHC class II haplotypes (H-2b and H-2k) have different degrees of tolerogenic activity on 4.1-thymocytes argues, in part, against this possibility.
The extensive MHC promiscuity of the 4.1-TCR during thymocyte development appears to result from its ability to recognize tolerogenic peptide/MHC class II complexes on dendritic cells and macrophages. The diabetogenic 4.1-TCR is not a classic alloreactive TCR, however, because it is only promiscuous for MHC molecules expressed on dendritic cells and macrophages, not on B-cells or thymic epithelial cells. It should also be pointed out that 4.1-thymocytes undergo neither positive nor negative selection in mice expressing deleting class II molecules (I-Ab [19], I-Ad, and I-E) exclusively on thymic epithelial cells. The only exception to this is the ability of the 4.1-TCR to undergo weak positive selection in chimeras whose thymic epithelial cells express I-Ag7PD (but not I-Ag7), as has been reported for another ß-cell-reactive TCR (BDC-2.5) (20). It is also clear that the MHC promiscuity of 4.1-thymocytes is peptide-specific, because H-2Ma-deficient thymic and splenic dendritic cells, which can almost exclusively express I-Ab molecules bound to CLIP, cannot trigger dulling of 4.1-CD4+CD8+ thymocytes. When taken together, these data suggest that the MHC-induced deletion of 4.1-thymocytes is mediated by one or more peptides that are selectively expressed in thymic bone marrow-derived APCs, as we had previously suggested for I-Ab (19).
Comparison of the amino acid sequences of the - and ß-chains of the nondeleting and deleting MHC class II molecules that we have tested so far did not reveal any obvious sequence homologies with the exception of I-Aß or I-Eß chain positions 5567. Deleting MHC class II molecules (I-Aq, I-Ad, I-Ab, and I-E) share a homologous 5667 motif that is absent in weakly tolerogenic (I-Ak and I-Ag7PD) or nontolerogenic (I-Ag7 and I-As) class II molecules. This sequence contains proline and aspartic acid at positions 56 and 57 (as opposed to histidine and serine in I-Ag7) and two insertions at positions 65 and 67. Because I-Ab, I-Ad, and I-Aq have an identical 5667 sequence but variable tolerogenic activity, the tolerogenicity of this motif may be modulated by other factors, such as other ß-chain residues, the nature of the
-chains, or the timing and levels of expression of the different MHC molecules. This hypothetical structure/function association is tantalizing, as the MHC-linked resistance to type 1 diabetes in both humans and mice is associated with polymorphisms at and around position 57 (3,4). The hypothetical contribution of residues at ß-chain positions 5667 to 4.1-thymocyte deletion is compatible with the recently described influence of amino acid substitutions at positions 56, 57, and 61 (different in I-Ag7 vs. I-Ad, I-Ak, and I-Ek) on structure (38,39). Our data are therefore compatible with a model in which structural differences conferred by polymorphisms between positions 56 and 67 would allow antidiabetogenic class II molecules to present tolerogenic peptides that cannot be presented at all by I-Ag7 and only inefficiently by I-Ak and I-Ag7PD (to certain highly diabetogenic T-cells).
In sum, our results lend support to the hypothesis that the MHC-associated resistance of non-TCR-transgenic mice (and perhaps humans, via DQB1*0602) to autoimmune diabetes might be mediated by thymocyte tolerance. Because it is highly unlikely that all I-Ag7-restricted diabetogenic effector T-cells in NOD mice will also recognize the "4.1-thymocyte-deleting" peptide(s) in the context of antidiabetogenic MHC class II molecules, we propose that 4.1-CD4+ T-cells are representative of a group of highly diabetogenic and MHC-promiscuous T-cells that, for as yet unknown reasons, would play a critical role in diabetogenesis. This would explain the presence of mildly insulitogenic, but not diabetogenic, T-cells in congenic NOD.H-2g7/b, NOD.H-2g7/q, or NOD.H-2g7/nb1 mice (14,40,41); I-Ad-transgenic NOD mice (16); NOD mice reconstituted with bone marrow from I-E-transgenic NOD mice (11); and I-Ak-transgenic NOD mice (13,42). This hypothesis does not rule out the involvement of other mechanisms of MHC-associated protection from diabetes (i.e., immunoregulation), especially because the phenomena described here was observed with a single TCR. However, it is compelling because the geography and timing of negative selection of diabetogenic thymocytes in 4.1-NOD mice are in agreement with the widely accepted notion that the factors underlying the MHC-linked resistance to diabetes reside in the bone marrow and are linked to polymorphisms at around I-A/I-Eß position 57 (3,4). Furthermore, it is attractive because it invokes a single mechanism by which multiple, structurally diverse MHC molecules can afford dominant resistance to a single autoimmune disease.
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ACKNOWLEDGMENTS |
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We thank H. McDevitt, O. Kanagawa, L. van Kaer, and L. Wicker for providing mice; T. Utsugi for providing Takeda rIL-2; S. Bou, M. Deuma, and S. Culp for technical assistance; Y. Yang and J. Elliott for helpful comments on the manuscript; and L. Bryant for flow cytometry.
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FOOTNOTES |
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Received for publication 17 May 2001 and accepted in revised form 24 October 2001.
APC, antigen-presenting cell; BrdU, 5-bromo-2'-deoxyuridine; CLIP, class II-associated invariant chain peptide; FITC, fluorescein isothiocyanate; H/E, hematoxylin and eosin; IFN-,
-interferon; IL, interleukin; mAb, monoclonal antibody; MHC, major histocompatibility complex; PMA, phorbol-myristate acetate; RAG-2, recombination activating gene-2; TCR, T-cell receptor.
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REFERENCES |
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