J. Exp. Med.
© The Rockefeller University Press
0022-1007/97/10/1059/17 $2.00
Volume 186, Number 7, October 6, 1997 1059-1075

A Mechanism for the Major Histocompatibility Complex-linked Resistance to Autoimmunity

By Dennis Schmidt, Joan Verdaguer, Nuzhat Averill, and Pere Santamaria

From the Department of Microbiology and Infectious Diseases and Julia McFarlane Diabetes Research Centre, Faculty of Medicine, Health Sciences Centre, The University of Calgary, Calgary, Alberta T2N 4N1, Canada

Summary

MATERIALS AND METHODS

RESULTS

DISCUSSION

Footnotes

Acknowledgements

REFERENCES

Summary

Insulin-dependent diabetes mellitus (IDDM),¹ a prototype of organ-specific autoimmune diseases, results from selective destruction of pancreatic beta cells by a T lymphocyte-dependent autoimmune process in genetically predisposed individuals (1). Genetic susceptibility and/or resistance to most autoimmune disorders, including IDDM, is associated with highly polymorphic genes of the MHC complex and, to a lesser extent, with polygenic modifiers on other chromosomes (2).

Population and animal studies have suggested that the MHC class II-linked susceptibility and resistance to IDDM are inherited as dominant traits with incomplete penetrance (2, 3). In humans, the MHC-associated IDDM susceptibility and resistance are predominantly, but not exclusively, determined by polymorphisms at the human leukocyte antigen (HLA) DQB1 locus. Alleles encoding DQ chains with serine, alanine, or valine at position 57 provide susceptibility, whereas those encoding DQ chains with aspartic acid at position 57 provide resistance with differing degrees of dominance (1, 2). In mice, susceptibility and resistance to spontaneous IDDM are also linked to the MHC (H-2). The diabetes-prone nonobese diabetic (NOD) mouse, which spontaneously develops a form of diabetes closely resembling human IDDM, is homozygous for a unique H-2 haplotype (H-2^g7). This haplotype carries a nonproductive I-E gene and encodes an I-A^d/I-A^g7 heterodimer in which the histidine and aspartic acid found at positions 56 and 57 in most murine I-A chains (the counterpart of human DQ chains) are replaced by proline and serine, respectively (4, 5). Studies of congenic NOD mice expressing non-NOD MHC haplotypes, and of NOD mice expressing I-E^d, modified I-A^g7, I-A^k/I-A^k, or I-A^d transgenes, have demonstrated that MHC class II molecules encoded by H-2 haplotypes derived from NOD or IDDM-resistant mice play a direct role in providing either susceptibility or resistance to spontaneous IDDM, respectively (6).

The precise mechanisms through which MHC genes provide autoimmune disease susceptibility and resistance, however, remain mysterious. MHC molecules are cell-surface receptors that present short fragments of self and foreign proteins to T lymphocytes and play a pivotal role in instructing T lymphocytes maturing in the thymus how to discriminate between self- and nonself-antigens (19, 20). Thymocytes bearing TCRs capable of recognizing self-peptide-MHC complexes with high affinity/avidity die or are rendered unresponsive to antigenic stimulation (21). In contrast, thymocytes bearing TCRs capable of engaging self-peptide-MHC complexes with intermediate affinity/ avidity survive and are exported to the peripheral lymphoid organs as cells capable of recognizing foreign antigens bound to self-MHC molecules (25). On the basis of some of this knowledge, a number of authors hypothesized that MHC molecules providing resistance to autoimmune diseases, including IDDM, might do so by mediating the clonal deletion or anergy of autoreactive T cells (8, 11, 31). Studies in MHC-congenic NOD mice and in I-A-, I-E-, and TCR-transgenic NOD mice, however, did not find evidence of T cell tolerance induction, and suggested that the diabetes resistance provided by protective MHC genes likely involved the induction of immunoregulatory functions (10, 13, 16, 18, 32, 33).

The studies presented here were initiated to test the hypothesis that diabetes-resistant genetic backgrounds express non-MHC-linked genetic elements other than endogenous superantigens that are tolerogenic for diabetogenic T cells. To that end, we followed the fate of an NOD islet-derived, beta cell-specific, I-A^g7-restricted transgenic TCR in diabetes-prone NOD mice, and in diabetes-resistant F1 hybrid mice lacking endogenous superantigens binding to the transgenic TCR. In NOD mice, T cells expressing the transgenic TCR underwent positive thymic selection and triggered a dramatic acceleration of the onset of IDDM. In certain F1 hybrid strains, however, the same cells underwent negative selection and the mice did not develop IDDM. Contrary to what we expected, the thymocyte deletion and IDDM resistance observed in these mice cosegregated as a single locus trait with MHC haplotypes known to provide dominant resistance to IDDM in MHC-transgenic and/or -congenic NOD mice (H-2^b and H-2^k). Thymocyte deletion in these mice was not mediated by endogenous superantigens, since it was absent in single-chain TCR--transgenic H-2^g7/b and H-2^g7/k F1 mice. Studies of bone marrow chimeras, I-A^b-deficient H-2^g7/b mice and I-A^k-transgenic NOD mice demonstrated that the thymocyte deletion and IDDM resistance of these mice resulted from the ability of the transgenic TCR to engage I-A^b, I-A^k, and possibly, I-E^k molecules on thymic bone marrow-derived cells. Additional experiments with H-2^g7/q- and H-2^g7/nb1-congenic NOD mice revealed that this highly pathogenic TCR was also deleted in the presence of I-A^q and I-A/I-E^nb1 molecules, which also provide dominant resistance to autoimmune diabetes in non-TCR-transgenic NOD mice. Our unexpected results provide an explanation as to how protective MHC class II molecules may provide resistance to spontaneous T cell-mediated autoimmune disorders, i.e., by removing certain highly pathogenic autoreactive T cells.

MATERIALS AND METHODS

Generation of TCR Transgenes. The TCR- and - cDNAs of NY4.1 were cloned by anchored PCR and multiple recombinants sequenced as described (34). The cDNAs were then amplified by PCR using primers containing L-V and J-C intron sequences, the corresponding splice donor and acceptor sites and convenient restriction sites, cloned into pBS-SK⁺ (Stratagene, La Jolla, CA), and sequenced. Inserts with the expected sequences were released from the vector by digestion with ClaI and NotI (TCR-) or XhoI and NotI (TCR-). The 4.1-VDJ sequence was subcloned into a TCR- shuttle vector carrying the endogenous TCR- enhancer and 5 regulatory sequences (3A9; gift from M. Davis, Stanford University, Stanford, CA). The 4.1-VJ cDNA was subcloned into PRE53 (from M. Davis). A 2.4-kb ClaI-KpnI fragment from 4.1-VJ/PRE53, containing 5 regulatory sequences and the 4.1-VJ sequence, was then coligated into pBS-SK⁺ with a genomic 11.9-kb KpnI-NotI fragment from AN6.2 (provided by S. Hedrick, University of California San Diego, San Diego, CA), containing the four C exons and the downstream TCR- enhancer.

Production of Transgenic Mice. After removing prokaryotic sequences by digestion with PvuI and SalI (TCR-) or ClaI and SacII (TCR-), the constructs (21.5- and 14.5-kb, respectively) were injected into fertilized (SJL × C57BL/6) F2 eggs (DNX, Princeton, NJ). Offspring were screened for integration of the transgenes by Southern blotting using VDJ and VJ cDNA probes. Transgenic founder mice (4.1-AN6A3-TCR-/, 4.1-AN6B3-TCR- and 4.1-AN6B7-TCR-) were backcrossed with NOD/Lt mice (I-A^g7, I-E; Jackson Laboratory, Bar Harbor, ME) for 3-5 generations to generate TCR-/-, TCR--, and TCR--transgenic NOD mice. TCR--, TCR--, and TCR-/-transgenic NOD mice (of the N5 backcross) were crossed with SJL/J (S; I-A^s, I-E), C57BL/6 (B; I-A^b, I-E), C58/J (C; I-A^k, I-E^k), NOD.H-2^g7/q (I-A^g7/q, I-E), or NOD.H-2^g7/nb1 (I-A^g7/nb1, I-E^nb1) mice (Jackson Laboratory), to generate TCR-transgenic F1 mice or H-2 heterozygous TCR-transgenic NOD mice. TCR-/-transgenic F1 (4.1-F1) mice were also backcrossed with NOD, C57BL/6, or C58/J mice, to generate H-2^g7, H-2^g7/b, H-2^g7/k 4.1 mice with 75% NOD genotype, and H-2^b or H-2^k 4.1 mice with 75% C57BL/6 or C58/J genotypes, respectively. I-A^b-deficient 4.1-(N × B) F1 mice were generated by crossing I-A^b C57BL-6 mice (Taconic, Germantown, NY) with 4.1-NOD mice. 2 microglobulin (2m) 4.1-(N × B) F1 mice were obtained by breeding the 2m mutation of 2m NOD/Lt mice (Jackson Laboratory) into 4.1-NOD mice, to obtain 2m 4.1-NOD mice, and then by crossing these mice with 2m C57BL/6 mice (Taconic). CD8- H-2^g7/b mice were obtained by crossing CD8- mice (I-A^b, I-E) (gift from T. Mak, University of Toronto, Toronto, Canada) with 4.1-NOD mice, followed by crossing CD8-^/+ 4.1-H-2^g7/b mice with CD8- H-2^b mice. I-A^k-transgenic 4.1-NOD mice were obtained by crossing I-A^k/I-A^k-transgenic NOD mice (from G. Morahan and J.F.A.P. Miller, The Walter and Eliza Hall Institute, Victoria, Australia) with 4.1-NOD mice. Mice were screened for inheritance of the transgenes, mutated alleles, and MHC haplotypes by PCR of tail DNA (4.1, 4.1, 2m, I-A^g7, I-A^k) and/ or by flow cytometry (4.1, CD8-, I-A^b, I-A^s, K^d, K^k). All mice were housed in a specific pathogen-free facility.

Antibodies and Flow Cytometry. Hybridomas secreting mAbs H57-597 (anti-TCR-), 53-6.7 (anti-CD8-), B220 (anti-B cells), and M1/70 (anti-Mac-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Anti- Lyt-2 (CD8-)-PE (53-6.7), anti-L3T4-FITC (IM7), or anti- L3T4-biotin (CD4; H129.19), anti-V11-FITC (RR3-15), anti-H-2K^d-FITC (SF1-1.1), and anti-I-A^b-biotin (25-9-3) were purchased from PharMingen (San Diego, CA). Purified mouse anti-H-2K^k (11-4.1) was from Becton Dickinson (San Jose, CA). Mouse IgG-absorbed FITC-conjugated goat anti-rat IgG and FITC-conjugated goat anti-mouse IgG were from CALTAG Labs. (South San Francisco, CA) and Becton Dickinson, respectively. Streptavidin-PerCP was from Becton Dickinson. Thymi, spleens, and islet-derived T cells were analyzed by three-color flow cytometry using a FACScan^® as described (35).

Cloning and Sequencing of TCR- cDNAs. The TCR- cDNA molecules of splenic CD4⁺ T cells were cloned and sequenced by anchored PCR as described previously (35).

Preparation of CD8⁺ T Cell-depleted Splenic and Islet-derived T Cells. Spleens were disrupted into single cell suspensions and red blood cells lysed in 0.87% ammonium chloride. Remaining cells were washed with complete medium (CM: RPMI 1640 media containing 10% heat-inactivated fetal bovine serum [GIBCO BRL, Gaithersburg, MD], 50 U/ml penicillin, 50 µg/ml streptomycin [Flow Labs., McLean, VA], and 50 µM 2-ME [Sigma Chemical Co., St. Louis, MO]) and then depleted of CD8⁺ T cells using anti-CD8 mAb (53-6.7)-coated magnetic beads as described (35). Islet-infiltrating CD4⁺ T cells were isolated from acutely diabetic 4.1-NOD mice essentially as described previously (35), analyzed by flow cytometry, depleted of CD8⁺ T cells by negative selection with anti-CD8 mAb-coated immunobeads, expanded in rIL-2-containing CM for 1-2 wk, and used for in vitro and in vivo studies.

Proliferation Assays. Pancreatic islets from 5-8-wk-old nontransgenic male NOD mice were dispersed into single cells by incubation in Ca²⁺- and Mg²⁺-free PBS containing 0.125% trypsin and 3 mM EGTA at 37°C for 3 min. 2 × 10⁴ splenic or islet-derived CD4⁺ cells were incubated, in triplicate, with -irradiated (3,000 rad) islet cells (3-100 × 10³/well) and unfractionated NOD splenocytes (10⁵/well), as a source of antigen and APCs, respectively, in 96-well round-bottomed tissue culture plates for 3 d at 37°C in 5% CO₂ in rIL-2-free CM. Cultures were pulsed with 1 µCi of [³H]thymidine during the last 18 h of culture and harvested. The incorporated thymidine was measured by scintillation counting. Specific proliferation was calculated by substracting background proliferation (cpm of cultures containing islet cells plus APCs alone and cpm of cultures of T cells alone) from islet cell-induced proliferation (cpm of cultures containing T cells, APCs, and islet cells).

Histology and Immunopathology. The body-tail of each pancreas was divided into two pieces. One piece was fixed in formalin, embedded in paraffin, sectioned at 4.5 µM, and stained with hematoxylin and eosin. The degree of insulitis was evaluated by scoring 15-30 islets/mouse in a blinded fashion using the following criteria: 0, normal islet; 1, peri-insulitis; 2, mononuclear cell infiltration in <25% of the islet; 3, mononuclear cell infiltration in 25-50% of the islet; 4, >50% of the islet infiltrated. A second piece of tissue was snap frozen, immersed in OCT, sectioned at 6-7 µM, and stored at 80°C for immunopathology. Sections were fixed in cold acetone for 10 min and stained with anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-Mac-1, and anti-B220 mAbs, followed by anti-rat IgG-FITC, or with anti-rat IgG-FITC alone, as described (35).

Adoptive T cell Transfer. CD8⁺ T cell-depleted islet-derived CD4⁺ T cells from diabetic 4.1-NOD mice (5 × 10⁶ cells/ mouse) were transfused into the tail veins of scid-NOD/Lt (Jackson Laboratory) in 200 µl of PBS, pH 7.2. Transfused mice were followed for development of IDDM by monitoring blood glucose levels with Glucostix and a glucometer (Miles Canada, Etobicoke, Ontario). Mice were killed at IDDM onset for flow cytometry and immunopathological studies.

Bone Marrow Chimeras. Bone marrow chimeras were generated following standard protocols (36). In brief, bone marrow suspensions (5-10 × 10⁶ cells) from donor mice (transgenic NOD or [N × B] F1 mice) were injected into the tail vein of recipient mice (nontransgenic NOD, [N × B] F1 or [N × S] F1 mice) treated with two doses of 500 rads 3 h apart from a ¹³⁷Cs source (Gammacell; Atomic Energy of Canada, Ottawa, Ontario). Chimeric mice were killed 5-6 wk after bone marrow transplantation.

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

RESULTS

Generation of Beta Cell-specific, I-A^g7-restricted TCR-/- transgenic NOD Mice.

A CD4⁺ T cell clone (NY4.1) that was derived from pancreatic islets of a diabetic NOD mouse and that recognized a putative beta cell autoantigen in the context of I-A^g7 (37) was chosen as donor of the TCR transgenes. This T cell clone transcribed one functional TCR- rearrangement, carrying V11 and J2.4 sequences and one functional TCR- rearrangement, carrying a novel V gene (Vx4.1) and the J33 element (These sequence data are available from EMBL/GenBank/DDBJ under accession numbers U80816 and U80817). These TCR rearrangements were subcloned into genomic TCR- and - shuttle vectors carrying endogenous TCR enhancers and 5-regulatory sequences. The resulting genomic constructs were then used to produce transgenic mice. Founders expressing the transgenes (4.1-AN63A-TCR-/, 4.1-AN6B3- TCR-, and 4.1-AN6B7-TCR) were crossed with NOD mice for several generations to generate 4.1-TCR-/-, 4.1-TCR--, and 4.1-TCR--transgenic NOD mice, respectively.

Expression of the 4.1-TCR in 4.1-TCR-/-transgenic NOD (4.1-NOD) Mice.

Three-color cytofluorometric studies showed that >90% CD4⁺CD8 thymocytes (Fig. 1 A) and splenic CD4⁺ T cells (Fig. 1 B) from 4.1-NOD mice expressed V11⁺ TCRs, compared to ~6% of the CD4⁺CD8 thymocytes and splenic CD4⁺ T cells from nontransgenic littermates, thus indicating TCR- transgene expression. Although we do not have a transgenic TCR- chain-specific antibody and thus cannot directly quantitate TCR- transgene expression, five different lines of evidence suggest that the transgenic TCR- chain is expressed on a sizeable fraction of thymocytes and peripheral T cells of 4.1-NOD mice. First, CD4⁺CD8⁺ thymocytes from 4.1-NOD mice expressed higher levels of total TCR-/ (as determined by staining with an anti-C mAb) than CD4⁺CD8⁺ thymocytes from TCR--transgenic NOD mice (mean fluorescence intensities: 59 ± 12 versus 31 ± 3, respectively; P <0.001), suggesting early TCR- chain expression (23, 38, 39). Second, thymocyte development in 4.1-NOD mice, but not TCR--transgenic NOD mice, was skewed towards the CD4⁺CD8 subset (Fig. 1 A), compatible with TCR- transgene-dependent positive selection of 4.1-CD4⁺ thymocytes. Third, skewing of thymocytes into the CD4⁺CD8 subset occurred in transgenic mice expressing the selecting I-A^g7 molecule, but not in transgenic mice expressing only nonselecting I-A molecules (i.e., I-A^s, see below), as seen with other MHC class II-restricted TCR-/-transgenic models (38, 40). Fourth, 37 out of 37 4.1-NOD TCR- cDNA sequences, generated from splenic CD4⁺ T cell-derived RNA by anchored PCR, were TCR- transgene-derived. Finally, splenic CD4⁺ T cells from 4.1-NOD, but not TCR--transgenic or nontransgenic, NOD mice proliferated in a dose-dependent manner in response to irradiated NOD islet cells (Fig. 1 C). The islet cell-induced proliferation of 4.1- and control CD4⁺ T cells was quite variable between experiments, perhaps due to variability in the quality of the islet cell preparations; however, the differences within individual experiments were reproducible. Taken together, these results provide strong evidence that in 4.1-NOD mice, the 4.1-TCR specificity is expressed appropriately, and that, in the presence of the selecting I-A^g7 molecule, 4.1-TCR-/ transgene expression fosters the positive selection of beta cell-reactive CD4⁺ T cells.

To determine whether positive selection of the beta cell-specific TCR in 4.1-NOD mice had any pathogenic significance, we followed 4.1-NOD mice of the N3-N5 backcrosses of the founder mouse onto the NOD background, and nontransgenic NOD mice for development of IDDM. As shown in Fig. 2 A, 4.1-NOD mice developed IDDM much earlier than nontransgenic NOD mice (IDDM onset at 43.6 ± 13 versus 119 ± 26 d in females, and at 56 ± 27 versus 157 ± 28 d in males; P <0.0001) (Fig. 2 A). In males, transgene expression also increased the incidence of IDDM (73.3 versus 45.7%; P <0.05). The kinetics of disease penetrance in the transgenic and nontransgenic populations were, however, remarkably similar (Fig. 2 A). Disease acceleration in 4.1-NOD mice required coexpression of the TCR- and - transgenes, since the few 4.1-TCR--NOD mice that became diabetic (3/7, 43%) did so significantly later than 4.1-NOD mice (103 ± 20 versus 46 ± 19 d; P <0.01). These results contrast with those obtained with the only other existing beta cell-specific TCR-transgenic NOD mouse model (32), which, when housed under specific pathogen-free conditions, develops IDDM less frequently and significantly later (10-15% at 6 mo) than 4.1-NOD mice (41). Taken together, our results indicate that the 4.1-TCR-/ is highly diabetogenic in the NOD background, and suggest that the events that trigger accelerated diabetogenesis in 4.1-NOD mice are similar to those that trigger IDDM in nontransgenic NOD mice.

To investigate the mechanisms underlying disease acceleration in 4.1-NOD mice, we then followed the progression of insulitis in prediabetic and diabetic mice. Histopathological studies of pancreata from 3- and 6-wk-old prediabetic 4.1-NOD mice showed that acceleration of diabetes in these mice was a result of faster progression, but not earlier onset, of islet inflammation (Fig. 2 B). As expected, the insulitis lesions of diabetic 4.1-NOD mice contained more CD4⁺ and fewer CD8⁺ T cells (but similar numbers of B cells and macrophages [not shown]), than those of diabetic nontransgenic NOD mice (Fig. 2 C). Islet-derived CD4⁺ T cells from 4.1-NOD mice expressed high levels of the transgene-encoded V11⁺ chain (Fig. 2 D), proliferated in response to NOD islet cells in vitro (Fig. 2 E), and transcribed messenger RNA for IL-2 and IFN-, but not IL-4 (data not shown). These data indicate that these cells were transgenic, beta cell reactive, and of the Th1 type, as expected. Moreover, purified islet-derived CD4⁺ T cells from three different diabetic 4.1-NOD mice were able to transfer IDDM into three different scid-NOD mice shortly after transfusion (36 ± 12 d) in the absence of CD8⁺ T cells in the inflamed islets (Fig. 2 F). We thus conclude that expression of the 4.1-TCR in the NOD background promotes the selection of highly diabetogenic CD4⁺ Th1 cells and their accelerated recruitment into pancreatic islets, leading to massive beta cell destruction and IDDM within the first few weeks of life.

The exquisite pathogenicity of the 4.1-TCR provided us with a powerful tool with which to test our initial hypothesis that diabetes-resistant backgrounds encode non-MHC-linked elements other than endogenous mouse mammary tumor virus superantigens (vSAgs) that are tolerogenic for diabetogenic T cells. To investigate this, we crossed 4.1-NOD mice (H-2^g7) with SJL/J (H-2^s), C57BL/6 (H-2^b), and C58/J mice (H-2^k); F1 mice resulting from crosses of nontransgenic NOD mice with these strains express the diabetogenic I-A^g7 molecule, but are diabetes-resistant, and do not delete V11⁺ T cells (42). The lack of vSAg-mediated deletion of V11⁺ or Vx4.1⁺ cells in these backgrounds was confirmed by the fact that the thymocyte profiles of single-chain TCR-- or TCR--transgenic F1 mice and those of TCR-- or TCR--transgenic NOD mice, respectively, were indistinguishable; as shown in Fig. 3, the percentages of thymic and splenic CD4⁺CD8V11⁺ cells in TCR--transgenic (Fig. 3 A) or nontransgenic F1 mice (Fig. 3 B) were virtually identical to (if not greater than) those seen in TCR--transgenic or nontransgenic NOD mice, respectively.

The flow cytometric profiles of thymocytes (Fig. 4 A) and splenocytes (Fig. 4 B) from 4.1-(N × S) F1 mice (n = 8) were comparable to those seen in 4.1-NOD mice (Fig. 1), indicating that the 4.1-TCR specificity also undergoes positive selection in H-2^g7/s mice. In contrast, all 4.1-(N × B) F1 mice (n = 22) and most 4.1-(N × C) F1 mice (n = 16/19) had only one-third to one-half the number of thymocytes seen in 4.1-NOD mice (and 4.1-[N × S] F1 mice), and displayed flow cytometric profiles of thymocytes and splenocytes compatible with negative selection of the 4.1-TCR, as compared with other TCR-/-transgenic models (23, 24, 39, 43) (Fig. 4). When compared to 4.1-NOD mice, 4.1-(N × B) F1 and 4.1-(N × C) F1 mice (also 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 V11 element, and an increase in the percentage of CD4CD8 thymocytes (Fig. 4 A). In the spleen, 4.1-NOD mice had significantly more CD4⁺ T cells and more CD4⁺ T cells expressing V11⁺ TCRs than deleting mice (Fig. 4 B). The few V11⁺CD4⁺ T cells that matured in deleting mice expressed half as many transgenic TCR- chains on the cell surface (but comparable numbers of total TCR-/ complexes) as V11⁺CD4⁺ T cells of 4.1-NOD mice, both in the thymus and in the spleen (P <0.003; data not shown), suggesting that in deleting mice these cells were selected on endogenous TCR chains that had bypassed allelic exclusion, as seen in other models (23, 24, 39, 43).

Proliferation assays using splenic CD4⁺ T cells as responders and irradiated NOD islet cells and splenocytes as antigen and APCs, respectively, revealed the absence of beta cell-reactive CD4⁺ T cells in the periphery of deleting, but not nondeleting, 4.1-F1 mice (Fig. 5 A). Lack of proliferation in these assays was the result of deletion, rather than anergy, since peripheral CD4⁺ T cells from deleting and nondeleting 4.1-F1 mice proliferated similarly in response to plate bound anti-TCR-/ mAb (Fig. 5 B). The absence of diabetogenic 4.1 T cells in the peripheral lymphoid organs of deleting mice was confirmed by the observation that, like their nontransgenic littermates, 4.1-F1 hybrid mice developed neither diabetes nor insulitis (Table 1).

Table 1. Insulitis and Diabetes in NOD versus F1 Hybrid Mice* n IDDM Age at Onset¶ Insulitis Score (n)¶ d   x ± SD NOD-TG  40  30/40a  48 ± 20g 3.74 ± 0.15i(4) NOD-non-TG 173 123/173b 138 ± 27h 1.59 ± 0.58j(13) (NOD × B6) F1-TG  10   0/10c - 0.42 ± 0.81§k(10) (NOD × B6) F1-non-TG  12   0/12d - 0§l(12) (NOD × C58) F1-TG  15   0/15e - 0.42 ± 0.70§m(15) (NOD × C58) F1-non-TG   4   0/4f - 0§n(4) *  Groups include both males and females.  6-wk-old mice; §  15-wk-old mice. Insulitis (15-30 islets/mouse) was scored as described in Materials and Methods. x ± SD, mean ± standard deviation; TG, transgenic. a versus c,e, b versus d,f, g versus h, j versus l: P <0.0001; i versus j: P <0.003; i versus k: P <0.0005; i versus m, j versus n: P <0.0032. Data was compared by 2 ( ) and Mann-Whitney U test( ¶ ).

Taken together, these data indicate that: (a) C57BL/6 and C58/J mice carry genes encoding elements capable of mediating the deletion of the diabetogenic 4.1-TCR; (b) the deleting element(s) expressed by these strains has complete or incomplete penetrance, respectively; and (c) these elements are not encoded by vSAgs and target T cells coexpressing both chains of the diabetogenic TCR.

Previous studies have suggested that the IDDM resistance provided by certain non-NOD MHC class II genes, including I-A^b and I-A^k, involves the induction of immunoregulatory functions rather than the deletion or anergy of autoreactive T cells (10, 16, 18, 32, 33, 44). Accordingly, we hypothesized that thymocyte deletion in 4.1-F1 hybrid mice would be mediated by elements encoded by non-MHC-linked genes. To test this hypothesis, we backcrossed 4.1-F1 mice with NOD mice and investigated whether thymocyte deletion and IDDM resistance in the transgenic offspring (4.1-F2 mice) segregated away from the H-2 haplotypes of the deleting backgrounds (H-2^b and H-2^k). All 4.1-F2 mice were killed at IDDM onset, or at 10 wk if nondiabetic, to determine: (a) their H-2 phenotypes, (b) the occurrence of thymocyte deletion; and (c) the degree of insulitis. Unexpectedly, we found that deletion of 4.1 thymocytes segregated as a single-locus trait linked to the MHC; it occurred in H-2^g7/b (16/16 mice, 100%) or H-2^g7/k mice (9/15 mice, 60%; incidence of deletion comparable to that seen in [N × C] F1 mice), but never in H-2^g7/g7 mice (0/24 mice, 0%) (Table 2). Like deleting 4.1-F1 mice, deleting 4.1-F2 mice did not harbor detectable beta cell-reactive CD4⁺ T cells in the spleen (data not shown). Furthermore, deleting H-2^g7/b or H-2^g7/k 4.1-F2 mice developed neither IDDM nor insulitis, whereas H-2^g7/g7 4.1-F2 mice developed moderate to severe insulitis (100%) and diabetes (~50%) within 10 wk (Table 2). These data thus indicate that the deleting elements of C57BL/6 and C58/J mice are encoded by genes tightly linked to their H-2^b and H-2^k complexes, and demonstrate that insulitis and diabetes resistance in 4.1-F2 mice carrying these haplotypes results from deletion of diabetogenic thymocytes.

Table 2. Cosegregation of Thymocyte Deletion and Resistance to Insulitis and IDDM with H-2b and H-2k Haplotypes in 4.1-transgenic Mice* H-2 Deleting status Thymocyte profile Age at onset Insulitis score , (n) n CD4+CD8 (V11+) CD4+CD8+ CD4CD8 CD4CD8+ IDDM %, x ± SD n§ d   x ± SD (NOD × B6) F1   × NOD H-2g7 15   31 ± 7a     44 ± 12   22 ± 7f 3 ± 1 7/15k 53 ± 17 2.4 ± 1.4 (8)p (92 ± 2)a H-2g7/b 16 + 15 ± 3b     49 ± 12  33 ± 10g 3 ± 1 0/16l - 0.4 ± 0.6 (16)q (66 ± 5)b (NOD × C58) F1   × NOD H-2g7  9   35 ± 5c       43 ± 7 19 ± 4h 3 ± 2 5/9m 47 ± 16 3.4 ± 0.7 (4)r (93 ± 1)c H-2g7/k  6   35 ± 4d    45 ± 8   16 ± 6i 4 ± 2 0/6n - 1.7 ± 1.2(6)s (89 ± 9)d H-2g7/k  9 + 17 ± 3e    45 ± 6   33 ± 6j 4 ± 3 0/9o - 0.2 ± 0.2 (8)t (58 ± 9)e *  All mice were killed at IDDM onset or at 10 wk if nondiabetic. Flow cytometry was done as described in the legend to Fig. 1.  Nondiabetic mice only. Groups of mice include both male and female mice (~ 50% each). No differences in the incidence of IDDM nor in the degree of insulitis were noted between female and male mice within groups. Insulitis (15-30 islets/mouse) was scored as described in Materials and Methods. a versus b,c versus e,d versus e,f versus g,h versus j,m versus j: P <0.0002; k versus l: P <0.0001; m versus n, m versus o, s versus t: P <0.002; p versus q: P <0.0006; r versus s: P <0.0007; r versus t: P <0.01 (compared by 2[ § ] and Mann-Whitney U test [ ]).

The data presented above suggested that the deletion of transgenic thymocytes and the IDDM resistance observed in 4.1-F1 and -F2 mice might be mediated by MHC class I and/or class II molecules encoded by the protective H-2 haplotypes. To determine whether 4.1 thymocyte deletion required the engagement of MHC class I molecules, we followed the maturation of 4.1 thymocytes in CD8-- or 2m-deficient 4.1-(N × B) F1 mice (H-2^g7/b), which either do not express the MHC class I-binding CD8 coreceptor on thymocytes, or lack MHC class I molecules, respectively. These mice deleted transgenic thymocytes as efficiently as wild-type 4.1-F1 mice (data not shown), thus indicating that deletion of transgenic thymocytes was not mediated by MHC class I molecules.

Since H-2^g7/b mice do not express I-E molecules, we reasoned that deletion in these mice might be mediated by I-A^b. To test this notion, we followed the development of 4.1 thymocytes in I-A^b-deficient 4.1-(N × B) F1 mice; except for the I-A^b mutation, I-A^b-deficient 4.1-(N × B) F1 and 4.1-(N × B) F1 mice are genetically identical. Selective abrogation of I-A^b expression in 4.1-(N × B) F1 mice restored, at least in part, the positive selection of the transgenic TCR, as evidenced by (a) significant increases in the percentage of V11⁺ thymocytes (Table 3), (b) significant increases in the ratio of CD4⁺CD8 to CD4CD8 T cells in the thymus and in the ratio of CD4⁺ to CD8⁺ T cells in the spleen (Table 3), (c) the reappearance of beta cell-reactive CD4⁺ T cells in the spleen (Fig. 6, left), and (d) the reemergence of insulitis (Table 3). The overall positive selection of the 4.1-TCR in I-A^b- deficient 4.1-(N × B) F1 mice, however, was less efficient than in 4.1-NOD mice; I-A^b-deficient 4.1-(N × B) F1 mice had more CD4CD8 thymocytes than, and half the splenic CD4⁺ T cells of, age-matched (3-5-wk-old) 4.1-NOD mice (Table 3). Furthermore, the peripheral CD4⁺ T cells of I-A^b-deficient 4.1-(N × B) F1 mice proliferated less efficiently in response to NOD islet cells than the peripheral CD4⁺ T cells of 4.1-NOD mice (Fig. 6, left). Finally, the insulitis lesions of I-A^b-deficient 4.1-(N × B) F1 mice were milder than those seen in 4.1-NOD mice and did not lead to IDDM in any of the 15 mice that were followed (Table 3). Therefore, the I-A^b gene is not the only protective element present in (N × B) F1 mice, but its expression is sufficient in and of itself to induce deletion of 4.1 thymocytes.

Table 3. Influence of I-Ab,I-Ak, I-Aq, and H2nb1 MHC Class II Molecules on 4.1 Thymocyte Development Percentage of cells* Ratios* IDDM (n) Insulitis score (n) n Organ CD4+CD8(V11+) CD4+CD8+ CD4CD8 CD4CD8+ CD4+/ DN CD4+/CD8+ x ± SD x ± SD (NOD × B6) F1 29 T 15 ± 5a (67 ± 7)    53 ± 10  27 ± 9 4 ± 3     0.6 ± 0.2s -  0 (10)ae 0.42 ± 0.81 (10)ak S 12 ± 6 (78 ± 12)      -  84 ± 8 4 ± 2 -  2.9 ± 1.5y (NOD × I-AbB6) F1 9 T 36 ± 5b (93 ± 3)    37 ± 9  23 ± 4o 4 ± 1 1.6 ± 0.2t -  0 (15)af 1.59 ± 0.45 (10)al S 12 ± 6c (86 ± 11)      -  86 ± 7 2 ± 1 -  4.6 ± 1.4z NOD 9 T 30 ± 7d (94 ± 2)e    53 ± 9  13 ± 4p 4 ± 2 2.2 ± 0.6u - 30 (40)ag 3.74 ± 0.15 (4)am S 24 ± 4f (88 ± 4)g      -    72 ± 5 4 ± 1 - 8.7 ± 2.9aa NOD.H-2g7/I-Ak 7 T 25 ± 3 (93 ± 1)    49 ± 5  21 ± 3q 5 ± 1  1.2 ± 0.2v -  0 (9)ah  2.2 ± 0.8 (7)an S  6 ± 1h (90 ± 1)      -  92 ± 1 2 ± 1 -  2.9 ± 0.3ab NOD.H-2g7/q 7 T 19 ± 3i (88 ± 3)    59 ± 5  19 ± 3 3 ± 1    1.0 ± 0.2w -  0 (7)ai  0.7 ± 0.4 (7)ao S 11 ± 8j (87 ± 4)      - 86 ± 10 3 ± 2 - 2.9 ± 1.4ac NOD.H-2g7/nb1 11 T 11 ± 3k (37 ± 9)l    52 ± 7  34 ± 5r 3 ± 1 0.3 ± 0.1x -  0 (11)aj  0.1 ± 0.1 (7)ap S  6 ± 2m (51 ± 16)n      -  91 ± 4 2 ± 1 -  2.9&nb