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Autoantigen-Independent Deletion of Diabetogenic CD4⁺ Thymocytes by Protective MHC Class II Molecules¹

Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada

	Abstract

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Some MHC class II genes provide dominant resistance to certain autoimmune diseases via mechanisms that remain unclear. We have shown that thymocytes bearing a highly diabetogenic, I-A^g7-restricted ß-cell-reactive TCR (4.1-TCR) undergo negative selection in diabetes-resistant H-2^g7/x mice by engaging several different antidiabetogenic MHC class II molecules on thymic (but not peripheral) hemopoietic cells, independently of endogenous superantigens. Here we have investigated 1) whether this TCR can also engage protective MHC class II molecules (I-A^b) on cortical thymic epithelial cells in the absence of diabetogenic (I-A^g7) molecules, and 2) whether deletion of 4.1-CD4⁺ thymocytes in I-A^b-expressing mice might result from the ability of I-A^b molecules to present the target ß-cell autoantigen of the 4.1-TCR. We show that, unlike I-A^g7 molecules, I-A^b molecules can restrict neither the positive selection of 4.1-CD4⁺ thymocytes in the thymic cortex nor the presentation of their target autoantigen in the periphery. Deletion of 4.1-CD4⁺ thymocytes by I-A^b molecules in the thymic medulla, however, is a peptide-specific process, since it can be triggered by hemopoietic cells expressing heterogeneous peptide/I-A^b complexes, but not by hemopoietic cells expressing single peptide/I-A^b complexes. Thus, unlike MHC-autoreactive or alloreactive TCRs, which can engage deleting MHC molecules in the thymic cortex, thymic medulla, and peripheral APCs, the 4.1-TCR can only engage deleting MHC molecules (I-A^b) in the thymic medulla. We therefore conclude that this form of MHC-induced protection from diabetes is based on the presentation of an anatomically restricted, nonautoantigenic peptide to highly diabetogenic thymocytes.

	Introduction

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Genetic susceptibility and resistance to autoimmunity are primarily determined by highly polymorphic genes of the class II region of the MHC (1). Much of what is currently known about the MHC-linked susceptibility and resistance to autoimmunity has been learned from studies of insulin-dependent diabetes mellitus (IDDM),³ a spontaneous autoimmune disease that results from selective destruction of the insulin-producing pancreatic ß-cells by T lymphocytes (2, 3). The diabetes-prone nonobese diabetic (NOD) mouse, which spontaneously develops a form of diabetes resembling human IDDM, is homozygous for a unique MHC haplotype (H-2^g7) that carries a nonproductive I-E gene and encodes an I-A^d/I-Aß^g7 heterodimer with unique molecular structure and biochemical behavior (4, 5). Studies of NOD mice expressing non-NOD H-2 haplotypes or MHC transgenes have shown that MHC class II molecules play a direct role in providing either susceptibility or resistance to spontaneous IDDM (2, 6). The mechanisms underlying the associations between MHC class II polymorphism and autoimmune disorders such as IDDM, however, remain unclear.

MHC molecules play a crucial role in instructing T cells maturing in the thymus how to discriminate between self and nonself and in presenting Ags to mature T cells in the periphery (7, 8). During T cell development, CD4⁺CD8⁺ thymocytes bearing TCRs capable of recognizing self peptide/MHC complexes on cortical thymic epithelial cells above certain affinity/avidity thresholds mature into CD4⁺CD8^- or CD4^-CD8⁺ cells and exit the thymic cortex toward the thymic medulla (positive selection) (9, 10, 11, 12, 13, 14, 15, 16). High affinity/avidity engagement of self peptide/MHC complexes by positively selected thymocytes, usually on bone marrow-derived APCs of the thymic medulla, leads to thymocyte death (negative selection) or functional unresponsiveness (anergy) (14, 15, 16, 17, 18, 19, 20, 21, 22). These processes ensure that the only T cells exiting the thymus are those capable of recognizing foreign, but not self, Ags in the context of self MHC.

It has been hypothesized that the ability of specific MHC molecules to afford autoimmune disease susceptibility or resistance is a function of their ability to promote the thymic selection or deletion of pathogenic T cells, respectively (23, 24, 25, 26). The observation that murine and human prodiabetogenic MHC class II molecules form unstable complexes on the surface of APCs has provided circumstantial support to this theory (5, 27); however, direct evidence has been lacking until recently. We found that thymocytes bearing a diabetogenic, I-A^g7-restricted, ß-cell-reactive TCR (4.1) undergo negative selection in H-2^g7/b-, H-2^g7/k-, H-2^g7/q-, and H-2^g7/nb1-4.1-NOD mice by engaging antidiabetogenic MHC class II molecules on bone marrow-derived APCs of the thymic medulla independently of superantigens (28). Like the diabetes resistance afforded by protective MHC class II molecules in non-TCR transgenic mice (29), the diabetes resistance afforded by the MHC-induced deletion of thymocytes in 4.1-TCR transgenic mice was found to reside in the bone marrow (28). These results provided a compelling explanation of how protective MHC class II alleles carried on one haplotype can override the susceptibility to autoimmune diabetes provided by pathogenic alleles carried on a second haplotype.

The TCR:peptide/MHC interactions that drive the positive selection of thymocytes in the thymic cortex are usually less specific than those driving their subsequent negative selection in the thymic medulla (22, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). As a result, MHC molecules restricting the negative selection of thymocytes bearing MHC-autoreactive or MHC-alloreactive TCRs in the thymic medulla can also usually restrict the thymocytes’ prior positive selection in the thymic cortex (22, 32, 36, 37); I-A^b-autoreactive TCRs, for example, undergo unopposed positive selection in thymi expressing I-A^b molecules only in the cortex, but undergo sequential positive and negative selection in thymi expressing I-A^b molecules in both cortex and medulla (22). We thus hypothesized that when expressed only on cortical thymic epithelial cells, MHC molecules capable of triggering the deletion of 4.1-thymocytes in the thymic medulla (i.e., I-A^b) would actually be able to promote the positive selection of these thymocytes. Since the 4.1-TCR does not cross-react with these MHC molecules on peripheral APCs, and thymocyte deletion is almost invariably target Ag or superantigen dependent, we also hypothesized that negative selection of 4.1-thymocytes in 4.1-NOD.H-2^g7/b mice would be triggered by ß-cell autoantigen/I-A^b complexes ferried from the periphery to the thymic medulla by bone marrow-derived APCs.

Here, we have tested these hypotheses 1) by following the fate of the 4.1-TCR in 4.1-TCR transgenic mice selectively expressing either a protective MHC transgene (I-A^b) on cortical thymic epithelial cells or wild-type (nontransgenic) I-A^b molecules on radioresistant thymic epithelial cells, 2) by investigating whether I-A^b molecules can present the 4.1-TCR’s target autoantigen to 4.1-CD4⁺ T cells, and 3) by investigating whether 4.1-thymocytes can engage I-A^b molecules bound to single peptides on bone marrow-derived APCs. We have found that I-A^b molecules delete 4.1-thymocytes in the thymic medulla in a peptide-dependent manner, but to our surprise can restrict neither the positive selection of 4.1-thymocytes in the thymic cortex nor the presentation of their target autoantigen in the periphery. Pathogenic MHC class II molecules (I-A^g7), on the other hand, can efficiently restrict the positive selection of 4.1-CD4⁺ T cells in the thymus and their autoantigen-driven activation in the periphery. Thus, unlike MHC-autoreactive or alloreactive TCRs, which can engage deleting MHC molecules in the thymic cortex, thymic medulla, and peripheral APCs, the 4.1-TCR can only engage deleting MHC molecules (I-A^b) in the thymic medulla. These observations establish the existence of a mechanism of MHC-induced protection from autoimmunity that is based on the presentation of an anatomically restricted, nonautoantigen-derived peptide to highly pathogenic TCRs.

	Materials and Methods

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Mice

NOD/Lt mice were purchased from The Jackson Laboratory (Bar Harbor, ME). RAG-2^-/- NOD have been described previously (42). C57BL/6.I-Aß^b- mice were purchased from Taconic Laboratories (Germantown, NY). C57BL/10.H-2^g7 mice were provided by L. Wicker and L. Peterson (Merck Research Laboratories, Rahway, NJ). 4.1-NOD mice, expressing a transgenic, I-A^g7-restricted, ß-cell-reactive TCR derived from a CD4⁺ T cell clone (NY4.1) that was isolated from pancreatic islets of a diabetic NOD mouse have been described previously (28). 4.1-NOD (4.1-TCR-transgenic, I-A^d/I-Aß^g7) mice were outcrossed with K14-C57BL/6.I-Aß^b- mice (K14-I-Aß^b-transgenic, I-A^b+/I-Aß^b-; a gift from T. Laufer and L. Glimcher, Harvard School of Public Health, Harvard University, Boston, MA) (22) or wild-type C57BL/6 mice (I-A^b/I-Aß^b; The Jackson Laboratory), to generate 4.1/K14-I-A^g7/0 (4.1-TCR⁺, K14-I-Aß^b+, I-A^d/b, I-Aß^g7/b-) and 4.1-I-A^g7/b mice (4.1-TCR⁺, I-A^d/b, and I-Aß^g7/b), respectively. 4.1/K14-I-A^g7/0 mice were then intercrossed to generate I-A^g7, 4.1-I-A^g7/g7, 4.1-I-A^0/0, 4.1-I-A^g7/0, K14-I-A^g7/0, K14-I-A^0/0, and 4.1/K14-I-A^0/0 mice (see Table 1 for MHC genotypes and phenotypes of each mouse strain). 4.1-NOD mice were also crossed with A^bE_pli^0/0 mice (I-A^b, I-Aß^b-, li^0/0 mice expressing the I-Aß^b-E_p-transgene; a gift from P. Marrack, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO) (32) or H-2 M^-/- mice (I-A^b, I-Aß^b, H-2 M^0/0; a gift from L. Van Kaer, Vanderbilt University School of Medicine, Nashville, TN) (43). The 4.1-TCR transgenic F1 mice resulting from these crosses were intercrossed to generate 4.1-A^bE_pli^0/0 and 4.1-H-2 M^0/0 mice, respectively. The mice were screened for inheritance of transgenes (4.1, 4.1ß, K14-I-Aß^b, A^bE_p), mutated genes (I-Aß^b, li, H-2M), and MHC genotypes by PCR of tail DNA. The sequences of the primers used for genotyping were as follows: 4.1, 5'-CAGCAGGTGGAGCAGCTTCC-3' and 5'-GACGTATTTGTAGTTTCTTAC-3' (300 bp); 4.1ß, 5'-AATGCTGGTGTCATCCAAACACC-3' and 5'-GTACAAGGTGTTTTGACCCTG-3' (300bp); K14-I-Aß^b and A^bE_p, 5'-TACCGCGCGGTGACCGAGCTG-3' and 5'-AGGTGTAGACCTCTCCCCGCC-3' (400 bp); li, 5'-GACTTAAGACCATAGTGTTCCCC-3' and 5'-GGAGACTGCTGACGTCACATC-3' (450 bp); H-2M, 5'-CAGAAGTCAGGAGCTGTGCTG-3' and 5'-CATGCTGCCTCCACTTTGACG-3' (250 bp); I-Aß, 5'-CCTAGAACAGCCCAATGTCGC-3' and 5'-GGTGTAGACCTCTCCCTGAT-3' (250 bp); mutated I-Aß^b, 5'-TGTCAAGACCGACCTGTCCG-3' and 5'-CCAACGCTATGTCCTGATAGCGGT-3' (550 bp). All PCRs were performed for 30 cycles (1 min at 94°C, 1 min at 55°C, 1 min at 72°C). All mice were housed under specific pathogen-free conditions and were studied at 6–9 wk of age.

The anti-CD8 mAb-secreting hybridoma 53-6.7 was obtained from the American Type Culture Collection (Manassas, VA). The hybridoma 30-2, secreting the anti-I-A^b/CLIP mAb, was a gift from A. Rudensky (University of Washington, Seattle, WA). The mAbs anti-Lyt-2 (CD8)-phycoerythrin (53-6.7), anti-L3T4-FITC (IM7), anti-L3T4-biotin (CD4; H129.19), anti-Vß11-FITC (RR3-15), anti-H-2K^d-FITC (SF1-1.1), and anti-I-A^b-biotin (AF6-120.1) were all purchased from PharMingen (San Diego, CA). Streptavidin-PerCP was obtained from Becton Dickinson (San Jose, CA) and was used to reveal biotin-coupled staining. Thymocytes and splenocytes (RBC-depleted) were analyzed by three-color flow cytometry using a FACScan (Becton Dickinson) (28).

Proliferation assays

Splenocytes were depleted of CD8⁺ T cells using anti-CD8 mAb (53-6.7)-coated magnetic beads (44). Splenocytes were also used to prepare pure CD4⁺ T cells using the MACS microbead purification system (Miltenyi Biotec, Auburn, CA). Pancreatic islets were isolated by collagenase digestion of the pancreas, purified by Ficoll density purification and hand picking, and disrupted into single cell suspensions by trypsin treatment (44). CD4⁺ T cells were adjusted to 2 x 10⁴ cells/100 µl of complete medium (RPMI 1640 medium containing 10% heat-inactivated FBS (Life Technologies, Grand Island, NY), 50 U/ml penicillin, 50 µg/ml streptomycin (Flow Laboratories, McLean, VA), and 50 µM 2-ME (Sigma, St. Louis, MO)) and incubated in triplicate with gamma-irradiated (3000 rad) islet cells (10⁵/well) in 96-well tissue culture plates for 3 days at 37°C in 5% CO₂. Irradiated NOD splenocytes (10⁵/well) were used as feeder cells in some experiments. Cultures were pulsed with 1 µCi of [³H]thymidine during the last 18 h of culture, harvested, and counted. Specific proliferation was calculated by subtracting background proliferation (counts per minute of cultures only containing islet cells with/without feeders and counts per minute of cultures of T cells alone) from islet cell-induced proliferation (counts per minute of cultures containing T cells and islet cells).

Radiation bone marrow chimeras

Bone marrow chimeras were generated following standard protocols (45). Briefly, recipient mice (I-A^g7/g7, I-A^g7/b, or I-A^b/b mice) were first treated with two doses of 500 rad 3 h apart from a ¹³⁷Cs source (Gammacell, Atomic Energy of Canada, Ottawa, Canada) and then transfused with bone marrow cell suspensions (8–10 x 10⁶ cells/mouse) from donor mice (4.1-A^bE_pli^0/0, 4.1-H-2 M^-/-, 4.1-I-A^g7/g7, or 4.1-I-A^g7/b). Chimeric mice were sacrificed 6–8 wk after bone marrow transplantation.

Statistical analyses

Statistical analyses were performed using Mann-Whitney U and ² tests.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Positive selection of 4.1-thymocytes in I-A^g7+ mice expressing deleting I-A^b molecules exclusively on cortical thymic epithelial cells

Our first set of experiments focused on confirming that 4.1-thymocytes can undergo positive selection in I-A^g7 hemizygous mice, and that I-A^b molecules (capable of inducing complete 4.1-thymocyte deletion in H-2^g7/b mice (28)) can only trigger the deletion of 4.1-thymocytes when expressed on bone marrow-derived APCs, but not when expressed on cortical thymic epithelial cells. Our first approach involved following the fate of 4.1-thymocytes in I-A^b+/I-Aß^b- (I-A⁰) or I-A^b/d/I-Aß^b-/g7 mice expressing an I-Aß^b transgene under the control of the K14 (keratin) promoter (referred to as K14 mice). K14 mice express deleting I-A^b molecules (transgenic I-Aß^b chains complexed with endogenous I-A^b chains) exclusively on cortical thymic epithelial cells (22) (Table 1).

The thymocyte cell number and the thymic and splenic cytofluorometric profiles of either I-A^g7 hemizygous or I-A^g7 homozygous 4.1-TCR-positive/K14-negative offspring from 4.1/K14-I-A^g7/0 x 4.1/K14-I-A^g7/0 intercrosses (designated 4.1-I-A^g7/0 and 4.1-I-A^g7/g7 mice, respectively) were virtually identical (Table 1 and Fig. 1). This indicated that positive selection of 4.1-thymocytes does not require expression of two copies of the I-A^d and I-Aß^g7 genes. Subsequent cytofluorometric studies of thymi from 4.1-I-A^g7/0 mice expressing the K14-I-Aß^b transgene (4.1/K14-I-A^g7/0 mice) revealed that, unlike I-A^b molecules expressed on bone marrow-derived APCs (28), I-A^b molecules expressed on cortical thymic epithelial cells cannot trigger the deletion of 4.1-thymocytes (Table 1 and Fig. 2): the thymi of 4.1-I-A^g7/0 and 4.1/K14-I-A^g7/0 mice contained significantly more thymocytes (p < 0.0001), greater percentages of CD4⁺CD8^-Vß11⁺ thymocytes (p < 0.0009), and lower percentages of CD4^-CD8^- thymocytes (p < 0.002 and p < 0.009, respectively) than the thymi of 4.1-I-A^g7/b mice (Figs. 1A and 2A). As expected, the spleens of 4.1-I-A^g7/0 and 4.1/K14-I-A^g7/0 (but not control K14-I-A^g7/0) mice also had significantly more CD4⁺Vß11⁺ T cells than the spleens of deleting 4.1-I-A^g7/b mice (p < 0.003 and p < 0.017, respectively; Fig. 2B).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. 4.1-thymocyte selection in I-Ag7 hemizygous mice. A and B, CD4, CD8, and Vß11 cytofluorometric profiles of thymocytes (A) and splenocytes (B) from 4.1-I-Ag7/g7 and 4.1-I-Ag7/0 mice. A and B(top panels), CD4 vs CD8 contour plots of single cell suspensions stained with anti-CD4-biotin plus streptavidin-PerCP, anti-CD8-phycoerythrin, and anti-Vß11-FITC. A and B(bottom panels), Vß11 fluorescence histograms of each T cell subset after electronic gating. Numbers indicate the average percentage of cells (A and B, top panels) or the average number of Vß11+ cells (A andB, bottom panels) in each subset. Data correspond to 7–12 mice/group (6–8 wk old). DP, double-positive cells; DN, double-negative cells. The numbers of thymocytes in 4.1-I-Ag7/0 and 4.1-I-Ag7/g7 mice were 24 ± 4 x 106 and 19 ± 4 x 106, respectively.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Absence of 4.1-thymocyte deletion in 4.1-TCR transgenic mice selectively expressing deleting MHC class II molecules (I-Ab) on cortical thymic epithelial cells. A and B, CD4, CD8, and Vß11 cytofluorometric profiles of thymocytes (A) and splenocytes (B) from 4.1-I-Ag7/b, 4.1/K14-I-Ag7/0, and K14-I-Ag7/0 mice. See Fig. 1 for details. The numbers of thymocytes in 4.1/K14-I-Ag7/0, 4.1-I-Ag7/b, and K14-I-Ag7/0 mice were 23 ± 6 x 106, 6 ± 1 x 106, and 60 ± 7 x 106, respectively. Data shown are average values of 4–12 mice/group (6–8 wk old). See text for statistical values.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Splenic CD4+ T cells from 4.1-I-Ag7/0 and 4.1/K14-I-Ag7/0, but not 4.1-I-Ag7/b or K14-I-Ag7/0, mice are islet reactive. Proliferation of splenic CD4+ T cells from 4.1-I-Ag7/g7, 4.1-I-Ag7/0, 4.1/K14-I-Ag7/0, K14-I-Ag7/0, and 4.1-I-Ag7/b mice in response to NOD islet cells. Cultures of 2 x 104 splenic CD4+ T cells (CD8+ T cell depleted) were incubated with irradiated NOD islet cells and splenocytes (105 cells/well) for 3 days, pulsed with [3H]thymidine, and harvested. Bars show the SEMs.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. 4.1-thymocyte development in radiation bone marrow chimeras expressing I-Ab molecules on thymic epithelial cells. Bone marrow cells (7–12 x 106) from 4.1-I-Ag7/g7 mice were transfused into the tail veins of lethally irradiated I-Ag7/g7 (A), I-Ag7/b (B), or I-Ab/b (C) mice. The thymocyte cytofluorometric profiles of the radiation bone marrow chimeras were analyzed 6–8 wk after transplantation (see Fig. 1). The cells lying in the center of the right panel correspond to dead thymocytes displaying nuclear autofluorescence. The numbers of thymocytes in I-Ag7/g7, I-Ag7/b, and I-Ab/b hosts were 6 ± 1 x 106, 10 ± 4 x 106, and 1 ± 1 x 106, respectively. Results shown are average values from two to eight mice per group.

I-A^b molecules on cortical thymic epithelial cells cannot induce the positive selection of 4.1-thymocytes

Having established that I-A^b molecules on cortical thymic epithelial cells cannot delete 4.1-thymocytes, we proceeded to investigate whether cortical I-A^b molecules could actually induce their positive selection. This was first investigated by comparing the fate of 4.1-thymocytes in 4.1-TCR-transgenic I-A^b+/I-Aß^b- mice expressing or lacking the K14 transgene (4.1/K14-I-A^0/0 and 4.1-I-A^0/0 mice, respectively; Table 1). Surprisingly, these studies revealed that cortical I-A^b molecules could not positively select 4.1-thymocytes; thymi from both 4.1/K14-I-A^0/0 and 4.1-I-A^0/0 mice had very few CD4⁺CD8^- cells and numerous CD4^-CD8^- cells (Fig. 5A, two left panels), probably reflecting the massive developmental arrest of 4.1-CD4⁺CD8⁺ thymocytes in the absence of selecting MHC class II molecules. The absence of 4.1-thymocyte positive selection in these mice was not the result of a general inability of K14 transgene-encoded I-A^b molecules to drive the positive selection of thymocytes; as shown in Fig. 5 (two right panels), non-TCR transgenic K14-I-A^0/0 mice were efficient positive selectors of CD4⁺CD8^- T cells: these mice had more thymocytes (p < 0.0001) and greater percentages of CD4⁺CD8^- thymocytes (Fig. 5A) and splenic CD4⁺ T cells (Fig. 5B) than I-A^0/0 mice (p < 0.0012), which do not express MHC class II molecules (Table 1). Furthermore, 4.1/K14-I-A^0/0 mice, which express I-A^b molecules in the thymic cortex and thus can select non-TCR transgenic thymocytes, had more thymocytes than 4.1-I-A^0/0 mice (p < 0.0001; Fig. 5A). Non-TCR transgenic K14-I-A^g7/0 mice also had more thymocytes than I-A^g7/g7 mice (61 ± 13 vs 32 ± 5 x 10⁶; p < 0.001).

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Developmental arrest of 4.1-thymocytes in I-Ag7-negative 4.1-TCR transgenic mice expressing deleting MHC class II molecules only on cortical thymic epithelial cells. A andB, CD4, CD8, and Vß11 cytofluorometric profiles of thymocytes (A) and splenocytes (B) from 4.1-I-A0/0, 4.1/K14-I-A0/0, I-A0/0, and K14-I-A0/0 mice. See Fig. 1 for details. The numbers of thymocytes in 4.1-I-A0/0, 4.1/K14-I-A0/0, I-A0/0, and K14-I-A0/0 mice were 6 ± 2 x 106, 22 ± 3 x 106, 55 ± 14 x 106, and 108 ± 13 x 106, respectively. Data are average values from 7–13 mice/group. See text for statistical comparisons.

A small percentage of CD4⁺CD8^-Vß11⁺ cells, however, did appear to mature in 4.1/K14-I-A^0/0 mice (Fig. 5B). Proliferation assays employing splenic CD4⁺ T cells from 4.1/K14-I-A^g7/0, 4.1/K14-I-A^0/0, or K14-I-A^g7/0 mice as responders and irradiated NOD islet cells and splenocytes (as a source of antigen and APCs, respectively) revealed that, unlike the CD4⁺ T cells from K14-I-A^g7/0 mice, the CD4⁺ T cells from 4.1/K14-I-A^0/0 mice contained some ß-cell-reactive cells (Fig. 6A). These cells, however, displayed significantly lower islet reactivity (Fig. 6A), contained fewer Vß11⁺ T cells (p < 0.006), and expressed lower levels of the transgenic TCR ß-chain (Vß11 mean fluorescence intensities, 61 ± 13 vs 78 ± 14, respectively; p < 0.006) than the CD4⁺ T cells from 4.1/K14-I-A^g7/0 mice (compare middle panel of Fig. 2B with the second panel of Fig. 5B). This suggested that the few islet-reactive CD4⁺ T cells that matured in 4.1/K14-I-A^0/0 mice were actually selected on endogenous TCRs (i.e., the I-A^b-autoreactive TCRs that are found in K14-I-A^0/0 mice (22)) rather than on the transgenic 4.1-TCR itself.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Islet and MHC auto/alloreactivity in 4.1-TCR-transgenic mice expressing deleting or selecting MHC class II molecules on cortical thymic epithelial cells. In vitro proliferation of purified splenic CD4+ T cells from 4.1/K14-I-A0/0, K14-I-Ag7/0, K14-I-A0/0, 4.1-I-Ag7/0, and/or 4.1/K14-I-Ag7/0 mice in response to RAG-2-/- NOD (A andB), C57BL/6 (C), and C57BL/6.I-Aßb- islet cells (D; all insulitis free) in the presence (A) or the absence (B–D) of exogenous APCs. E, Proliferation of purified splenic CD4+ T cells from 4.1-I-Ag7/0 mice against C57BL/6 or C57BL/10.H-2g7 islet cells (also insulitis free) in the absence of exogenous APCs. Proliferation assays were performed as described in Fig. 3, except that CD4+ T cells were purified using the MACS purification system (>94% purity).

Our next set of experiments was aimed at determining whether the I-A^b-induced deletion of 4.1-thymocytes in H-2^g7/b mice might be triggered by the target autoantigen of the 4.1-TCR. Since purified splenic CD4⁺ T cells from 4.1-NOD mice engage their target autoantigen on a resident, highly stimulatory intraislet APC population in the context of I-A^g7 (28), we first asked whether I-A^b molecules expressed on intraislet APCs from I-A^b/b mice could restrict the recognition of this autoantigen by 4.1-CD4⁺ T cells from 4.1-I-A^g7/0 mice. We also asked whether I-A^b molecules on such intraislet APCs could trigger the proliferation of the I-A^b-autoreactive CD4⁺ T cells that are selected in K14-I-A^0/0 mice (22) and, possibly, 4.1/K14-I-A^0/0 mice. This was done by determining whether purified splenic CD4⁺ T cells from each of these three types of mice (4.1-I-A^g7/0, 4.1/K14-I-A^0/0, and K14-I-A^0/0) could proliferate in response to I-A^g7/g7 (Fig. 6B), I-A^b/b (Fig. 6C), or I-A⁰ islet cells (Fig. 6D; from insulitis-free RAG-2^-/- NOD, C57BL/6, and C57BL/6.I-Aß^b-/- mice, respectively). As shown in Figs. 6 B–D, the CD4⁺ T cells of 4.1-I-A^g7/0 mice contained cells capable of recognizing islet Ag in the context of I-A^g7, but not in the context of I-A^b (proliferation in response to I-A⁰ islet cells was similar to proliferation in response to I-A^b/b islet cells). Lack of proliferation in response to I-A^b/b islet cells was due to an inability of I-A^b molecules to present ß-cell autoantigen, since the CD4⁺ T cells of 4.1-I-A^g7/0 mice proliferated well in assays employing islet cells from H-2^g7-congenic C57BL/10.H-2^g7 mice (Fig. 6E), which are also insulitis free and diabetes resistant. These I-A^g7-dependent responses were not the result of I-A^g7 autoreactivity, since 4.1-CD4⁺ T cells from 4.1-NOD mice did not proliferate against I-A^g7/g7 splenocytes in the absence of islet Ag (data not shown). In contrast, the CD4⁺ T cells from K14-I-A^0/0 and, to a lesser extent, 4.1/K14-I-A^0/0 mice proliferated vigorously in response to I-A^b/b islet cells (Fig. 6, B and C) and only slightly in response to I-A^g7/g7 islet cells, confirming the presence of I-A^b-expressing APCs in I-A^b/b islets. These responses were the result of I-A^b autoreactivity, since they were not observed in assays employing islet cells, where I-A molecules are absent (I-A⁰ islet cells; Fig. 6D).

Taken together, these results demonstrated that 1) I-A^b molecules on intraislet APCs cannot present the putative ß-cell autoantigen recognized by the 4.1-TCR, but are highly stimulatory for I-A^b-autoreactive CD4⁺ T cells; 2) I-A^b molecules on intraislet APCs cannot stimulate 4.1-CD4⁺ T cells; and 3) the few ß-cell-reactive CD4⁺Vß11⁺ T cells that mature in 4.1/K14-I-A^0/0 mice are actually selected on endogenous, I-A^b-autoreactive, TCRs.

Deletion of the 4.1-TCR by I-A^b molecules on hemopoietic cells is peptide dependent

The inability of cortical I-A^b molecules to trigger the positive selection of 4.1-thymocytes and the inability of the 4.1-TCR to recognize its target Ag in the context of I-A^b, or I-A^b molecules as allogeneic indicated that the MHC class II promiscuity of the diabetogenic 4.1-TCR is a phenomenon that is restricted to the thymic medulla. In turn, these results suggested that the ability of different antidiabetogenic MHC class II molecules (i.e., I-A^b) to trigger 4.1-thymocyte deletion cannot be accounted for by their ability to present the target ß-cell autoantigen of the 4.1-TCR in the thymic medulla. How, then, can a single, nonalloreactive TCR engage so many different MHC class II molecules selectively on thymic (but not peripheral) hemopoietic cells? We entertained two alternative possibilities to explain this phenomenon: 1) the 4.1-TCR undergoes thymocyte deletion by reacting with residues shared by cross-reactive MHC class II molecules on the surface of a specialized thymic APC regardless of the nature of the peptides bound to these molecules (i.e., deletion is peptide independent); or 2) the 4.1-TCR undergoes thymocyte deletion by engaging a peptide that binds efficiently to protective, but not pathogenic, MHC class II molecules and that is exclusively presented in sufficiently high quantities by APCs of the thymic medulla, but not by peripheral or intraislet APCs (i.e., deletion is peptide dependent).

To distinguish between these two alternative possibilities, we followed the fate of the 4.1-TCR in radiation bone marrow chimeras bearing thymic epithelial cells expressing the selecting I-A^g7 molecule and bone marrow-derived APCs expressing single peptide/I-A^b complexes. Our first experiments were performed with irradiated NOD mice (H-2^g7) that had been transfused with marrow from 4.1-A^bE_pli^0/0 mice (invariant chain-deficient 4.1-TCR transgenic mice expressing I-A^b molecules covalently linked to the I-E_52–68 peptide) (32). NOD mice transfused with marrow from 4.1-H-2^g7 or 4.1-H-2^g7/b mice were used as controls for positive and negative selection, respectively. Cytofluorometric studies of thymocytes (Fig. 7A) and proliferation assays employing splenic CD4⁺ T cells from these chimeras (Fig. 7B) indicated that the 4.1-TCR underwent positive selection in 4.1-H-2^g7NOD and 4.1-A^bE_pli^0/0NOD chimeras, but not in 4.1-H-2^g7/bNOD chimeras. As shown in Fig. 7A, 4.1-H-2^g7/bNOD chimeras had significantly fewer thymocytes and greater percentages of CD4^-CD8^- thymocytes than 4.1-H-2^g7NOD and 4.1-A^bE_pli^0/0NOD chimeras (p < 0.01). Since the expression levels of I-A^b molecules in A^bE_pli^0/0 mice are significantly lower than those in wild-type H-2^b mice (32), there was still the possibility that 4.1-thymocytes did engage the transgenic I-A^b/I-E_52–68 complex on hemopoietic cells of the 4.1-A^bE_pli^0/0NOD chimeras, but with an avidity that was too low to result in their negative selection. To address this possibility, we repeated these experiments in irradiated NOD mice (H-2^g7) that had been transfused with marrow from 4.1-H-2 M^0/0 mice (4.1-TCR transgenic H-2^b/b mice almost exclusively expressing I-A^b molecules bound to the invariant chain-derived CLIP peptide) (43, 46, 47). Unlike 4.1-A^bE_pli^0/0 mice, 4.1-H-2M^0/0 mice express even greater levels of total I-A^b molecules than 4.1-H-2^g7/b mice (Fig. 7B), which are efficient deleters of 4.1-thymocytes. As shown in Fig. 7A, 4.1-thymocytes underwent unopposed positive selection in these chimeras. The absence of 4.1-thymocyte negative selection in I-A^g7+ mice expressing single-peptide/I-A^b complexes on hemopoietic cells therefore demonstrated that the I-A^b-induced negative selection of 4.1-thymocytes in 4.1-H-2^g7/b mice is a peptide-dependent process.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Bone marrow-derived APCs expressing deleting MHC class II molecules bound to single peptides cannot trigger the deletion of 4.1-thymocytes. A, Thymocyte cytofluorometric profiles of radiation bone marrow chimeras. Bone marrow cells (7–12 x 106) from 4.1-I-Ag7/b, 4.1-I-Ag7/g7, 4.1-H-2M0/0, or 4.1-AbEpli0/0 were transfused into the tail vein of lethally irradiated NOD mice (H-2g7). The chimeras were analyzed 6–8 wk after transplantation. The numbers of thymocytes in 4.1-H-2g7/bNOD, 4.1-H-2g7NOD, 4.1-H-2M0/0NOD, and 4.1-AbEpli0/0NOD chimeras were 2 ± 1 x 106, 5 ± 1 x 106, 5 ± 1 x 106, and 6 ± 2 x 106, respectively. Results shown are average values from six to eight mice per group. B, Levels of I-Ab molecules on splenocytes from 4.1-I-Ag7/g7, 4.1-I-Ag7/b, and 4.1-H-2M0/0 mice, as determined by staining with an anti-I-Ab mAb (AF6–120.1). C, Islet reactivity of splenic CD4+ T cells from bone marrow chimeras. Proliferation assays were performed as described in Fig. 6.

	Discussion

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View larger version (20K): [in this window] [in a new window]   FIGURE 8. The MHC class II promiscuity of the 4.1-TCR is geographically restricted to the thymic medulla and is peptide specific. This figure shows a model of 4.1-thymocyte selection based on the results of this study. 4.1-CD4+CD8+ thymocytes can only undergo positive selection by engaging diabetogenic (I-Ag7), but not anti-diabetogenic (I-Ab and, possibly, I-Aq, I-Ek and/or I-Ak, I-Anb1 and/or I-Enb1) (28), MHC class II molecules on cortical thymic epithelial cells. The positively selecting peptide(s) presented by I-Ag7 molecules in the thymic cortex may also be able to bind to deleting MHC molecules, but the 4.1-TCR cannot recognize these peptides in the context of these MHC molecules. In the thymic medulla, the positively selected 4.1-CD4+CD8- thymocytes cross-react with high affinity/avidity with deleting MHC class II molecules bound to a peptide that is selectively expressed on a specialized bone marrow-derived APC. The deleting peptide(s) may not be able to bind to I-Ag7 molecules or may do so without being able to trigger the negative selection of 4.1-thymocytes. In the periphery, 4.1-CD4+ T cells recognize a putative peptide/I-Ag7 complex on intraislet APCs, but cannot engage deleting MHC class II molecules on these cells, either because these molecules cannot present this peptide or because the resulting complexes cannot be recognized by the 4.1-TCR.

Whatever the nature of the peptide ligand(s) involved, our data demonstrate that the ability of a given MHC molecule to restrict the negative selection of a TCR in the thymic medulla does not imply that the same MHC molecule should also be able to restrict this TCR’s prior positive selection in the thymic cortex. Assuming that positive selection of 4.1-thymocytes in 4.1-NOD mice is a peptide-dependent process, our data suggest that there are no peptides available in the thymic cortex to positively select 4.1-thymocytes in the context of I-A^b. In turn, this implies that this TCR cannot undergo positive selection via low affinity interactions with multiple I-A^b/peptide complexes totalling a positively selecting avidity threshold. Whether this unique ability of the 4.1-TCR to undergo sequential positive and negative selection on different MHC class II molecules is a peculiarity of certain autoreactive TCRs (i.e., those that are highly pathogenic and MHC promiscuous) or is also shared by some MHC-alloreactive TCRs and foreign Ag-specific TCRs that can cross-react with allo-MHC molecules remains to be determined. It appears, however, that most MHC-promiscuous TCRs do not behave like the 4.1-TCR; the ability of a given MHC molecule to restrict the deletion of a TCR in the thymic medulla is usually associated with the ability of this MHC molecule to restrict this TCR’s positive selection in the thymic cortex and its activation in the periphery. The K^b-restricted and L^d-alloreactive 2C TCR, for example, can undergo positive (but not negative) selection on K^b molecules and both positive and negative selection on L^d molecules (36). The conalbumin-specific D10-TCR undergoes weak positive selection in H-2^k mice and negative selection in H-2^d mice, but, unlike the 4.1-TCR, can also engage deleting MHC molecules (H-2^d) on peripheral APCs (35, 53). The foreign Ag-specific AND and DO11.10 TCRs (specific for pigeon cytochrome c/I-A^k and OVA/I-A^d complexes, respectively) can undergo positive selection on more than one MHC class II molecule (I-E^k and I-A^b, or I-A^d and I-A^b, respectively), but these MHC molecules do not trigger the negative selection of these TCRs in the thymic medulla (21, 22, 30, 31). On the basis of these differences, it is tempting to speculate that the selective promiscuity of the 4.1-TCR for antidiabetogenic MHC molecules in the thymic medulla is somehow associated with its exquisite diabetogenic potential, and that selection of this type of TCRs is promoted by the unique MHC class II molecule of the NOD mouse (I-A^g7).

Our findings also provide some insights into our understanding of the mechanisms underlying the MHC-associated susceptibility to autoimmunity. Previous studies have proposed that diabetogenic MHC class II molecules (which are poor peptide binders) provide susceptibility to diabetes by being able to reach expression levels on thymic APCs that are sufficient to induce the positive, but not the negative, selection of autoreactive thymocytes (5, 27). This hypothesis implies that differences in the putative ability of diabetogenic and protective MHC class II molecules to delete pathogenic T cells is the result of quantitative differences in the abilities of these molecules to form stable peptide/MHC complexes on thymic APCs and thus to present peptides at deleting doses on hemopoietic cells. Yet, pathogenic MHC molecules can positively select autoreactive T cells in the thymus, and can efficiently present autoantigenic peptides to mature T cells in the periphery. Two of our observations challenge the pure quantitative aspects of this theory, and provide one solution to this paradox. First, I-A^g7-hemizygous 4.1-TCR transgenic mice selected 4.1-thymocytes as efficiently as I-A^g7 homozygous mice, which are thought to express more I-A^g7 molecules than the former. Second, protective I-A^b molecules could efficiently delete, but could not positively select, 4.1-thymocytes, yet these molecules could not present the target ß-cell autoantigen to 4.1-CD4⁺ T cells. These two observations favor the view that the proposed differences in the abilities of diabetogenic and protective MHC class II molecules to delete pathogenic T cells may be both quantitative and qualitative in nature; diabetogenic MHC class II molecules would be able to present the selecting and the autoantigenic peptides, but not the deleting ones. Finally, it is worth emphasizing that our results should not be taken to imply that deletion of pathogenic thymocytes is the only mechanism through which protective MHC molecules afford autoimmune disease protection. As suggested recently, these MHC class II molecules may also afford diabetes protection by selecting regulatory (protective) T cells or islet Ag-specific Th2 cells (54, 55).

In summary, our study on the mechanisms that control the selection of the highly diabetogenic, MHC-promiscuous, 4.1-TCR has demonstrated that the MHC class II promiscuity of the 4.1-TCR is a phenomenon that is peptide dependent but autoantigen-independent and apparently restricted to the thymic medulla. The observation that this pathogenic TCR undergoes sequential positive and negative selection in diabetes-resistant H-2^g7/x mice by engaging different peptide/MHC complexes on two geographically distinct regions of the thymus not only has important implications for our understanding of the mechanisms through which protective MHC class II molecules afford autoimmune disease resistance, but also provides one solution of the so-called thymic paradox, i.e., how apparently similar interactions between individual TCRs and self peptide/MHC complexes on different thymic cell types can result in the formation of a T cell repertoire that can recognize foreign Ag in the context of self MHC yet be tolerant to self.

	Acknowledgments

 
We thank Drs. L. Glimcher, T. Laufer, P. Marrack, A. Rudensky, L. Van Kaer, L. Wicker, and L. Peterson for their generosity in providing mice or reagents. We also thank Y. Yang for helpful comments on the manuscript, L. Bryant for flow cytometry, the Animal Resources Center’s technicians for excellent animal care, Judy Patterson for secretarial help, and H. Kominek for editorial assistance.

	Footnotes

 
¹ This work was supported by a grant from the Medical Research Council of Canada, a Canadian Diabetes Association postdoctoral fellowship (to J.V.), and a Senior Scholarship from the Alberta Heritage Foundation for Medical Research (to P.S.).

² Address correspondence and reprint requests to Dr. Pere Santamaria, Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. N.W., Calgary, Alberta, Canada T2N 4N1. E-mail address:

³ Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; NOD, nonobese diabetic; RAG, recombination-activating gene; CLIP, class II-associated invariant chain peptide.

Received for publication November 3, 1998. Accepted for publication January 20, 1999.

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