Polymorphism of MHC and MHC-linked genes is tightly associated with susceptibility to type 1 diabetes (T1D) in human and animal models. Despite the extensive studies, however, the role of MHC and MHC-linked genes expressed by T cells on T1D susceptibility remains unclear. Because T cells develop from TCR− thymic precursor (pre-T) cells that undergo MHC restriction mediated by thymic stroma cells, we reconstituted the T cell compartment of NOD.scid-RIP-B7.1 mice using pre-T cells isolated from NOD, NOR, AKR, and C57BL/6 (B6) mice. T1D developed rapidly in the mice reconstituted with pre-T cells derived from NOD or NOR donors. In contrast, most of the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from AKR or B6 donors were free of T1D. Further analysis revealed that genes within MHC locus of AKR or B6 origin reduced incidence of T1D in the reconstituted NOD.scid-RIP-B7.1 mice. The expression of MHC class I genes of k, but not b haplotype, in T cells conferred T1D resistance. Replacement of an interval near the distal end of the D region in T cells of B6 origin with an identical allele of 129.S6 origin resulted in T1D development in the reconstituted mice. These results provide evidence that the expression of MHC class I and MHC-linked genes in T cells of NOD mice indeed contributes to T1D susceptibility, while expression of specific resistance alleles of MHC or MHC-linked genes in T cells alone would effectively reduce or even prevent T1D.
The development of type 1 diabetes (T1D)3 in humans and NOD mice is controlled by multiple susceptibility genes. In NOD mice, T1D susceptibility genes have been mapped into >20 chromosomal loci (Idd loci) (1, 2, 3, 4). Although most of the susceptibility genes remain to be identified, it is known that T cells and APCs in NOD mice express different sets of T1D susceptibility genes (5, 6). Among Idd loci, Idd-1 locus contains the MHC complex and contributes significantly to disease susceptibility. The critical role of MHC genes in T1D pathogenesis has been established using congenic, transgenic, and gene-targeted NOD mice (1, 4, 7). Expression of T1D resistance MHC genes, including both class I and class II genes, in APCs of either hemopoietic or nonhemopoietic origin, enhances central and peripheral tolerance or regulatory mechanisms that inhibit pathogenic progression of T1D in NOD mice (7, 8, 9, 10, 11, 12). However, whether the expression of susceptibility MHC class I genes in T cells of NOD mice has any effect on T1D pathogenesis is poorly understood.
In addition to MHC genes, non-MHC genes within or in close proximity to MHC complex in Idd-1 locus also play a role in T1D pathogenesis. It was shown that T1D incidence in NOD mice can be significantly reduced by resistance alleles of MHC-linked genes derived from CTS mice, a NOD-related strain. The region that contains MHC-linked T1D susceptibility genes in NOD mice was then designated as Idd-16 (13). Further genetic analysis indicated that NOD and CTS mice share identical alleles at the class I K end of the H2 complex, suggesting that resistance genes in CTS mice are located near the distal end of D region in MHC complex (14, 15). Using NOD mice congenic for different sections of MHC complex, an interval proximal to the K end of H2 complex in B10.A (r209) and B6, as well as in MHC complex of k haplotype, was found to contain T1D resistance genes in subsequent studies (16, 17). However, the specific cell populations that express MHC-linked T1D resistance genes were not identified in these studies, because every cell contained the identical MHC and MHC-linked genes in the congenic NOD mice.
To determine whether expression of resistant alleles of MHC and MHC-linked genes by T cells alone is sufficient to alter T1D pathogenesis in NOD mice, we reconstituted the T cell compartment in NOD.scid-RIP-B7.1 mice (18) using thymic pre-T (CD3−CD25+CD44−) cells from T1D-resistant strains. Because pre-T cells committed to the T lineage have yet to express TCR genes (19, 20), T cell development in the thymus of the recipient mice would be restricted by the MHC genes of NOD haplotype. Furthermore, in the pre-T cell reconstituted mice, the donor-derived MHC and non-MHC genes are exclusively expressed in T cells. In contrast to T cells, APCs and other cells express genes of NOD origin (6). NOD.scid-RIP-B7.1 mice were used in this study because the RIP-B7.1 transgene is a potent enhancer for T1D pathogenesis. RIP-B7.1 can trigger and accelerate development of T1D in NOD and other strains of mice when islet-specific autoreactive T cells are present (18, 21, 22, 23, 24). Therefore, accelerated T1D is anticipated in NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells, unless T cells of the donor origin express T1D resistance genes. We found that the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from AKR or B6 mice were almost free of insulitis and T1D, whereas the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from NOD or NOR mice developed accelerated T1D. Our results further show that MHC class I genes of k haplotype and the genes located between MHC class II and the distal end of D region of B6 origin contribute to T1D resistance when expressed in T cells.
Materials and Methods
Mice and Abs
NOD, NOD.H2b, NOR, AKR, il4 gene-deficient C57BL/6 (B6-il4), β2-microglobulin gene-deficient AKR (AKR-β2m), and C57BL/6 (B6-β2m) mice were purchased from The Jackson Laboratory. NOD.scid-RIP-B7.1 mice were a kind gift of D. Serreze (The Jackson Laboratory, Bar Harbor, ME). C57BL/6 (B6), I-A gene-deficient C57BL/6 (B6-Abb), wild-type control (B6-Abb-N6), and 129.S6 (parental strain for B6-Abb) mice were purchased from Taconic Farms. The mice were maintained in specific pathogen-free units in the Animal Resource Centre at the University of Calgary, and Guidelines of Animal Care Committee of University of Calgary were followed. All Abs used in this study were purchased from BD Pharmingen.
Reconstitution of NOD.scid-RIP-B7.1 mice with purified thymic pre-T cells
The CD3−CD4−CD8−CD25+CD44− pre-T cells were isolated from the thymus of 4-wk-old donor mice using anti-CD25 Ab-conjugated microbeads (Miltenyi Biotec) and FACS. The purified pre-T cells (>95% pure) were suspended in PBS and injected into both lobes of the thymus (0.5 × 106 cells/recipient) of 3-day-old NOD.scid-RIP-B7.1 mice, as previously described (6). Littermates of the recipients were injected with PBS alone and used as control. Reconstitution of the T cell compartment by donor-derived T cell populations in the recipient mice was monitored biweekly by the examination of CD4+/CD8+TCR+ cells in blood with strain-specific markers until the end of experiment. When reconstituted recipient mice were sacrificed, lymph node cells and splenocytes were analyzed by FACS, and cell counting was performed to determine the proportion and numbers of donor-derived T cells in each organ.
FACS analysis of T cell populations in recipients and donor mice
Peripheral monocytes, thymocytes, or lymph node cells isolated from reconstituted NOD.scid-RIP-B7.1 and donor mice were preincubated with Ab against CD16/32 (2.4G2) in PBS containing 2% FCS. The cells were then incubated with biotinylated anti-TCR β-chain Ab (H57-597), washed, and further incubated with a mixture of streptavidin-PerCP and anti-Kd PE, anti-Kb PE, anti-Thy-1.1 PE, or anti-Thy-1.2 PE for 20 min. The cells were then washed and analyzed using FACScan.
Assay for cytokine production
CD4+ T cells were isolated from splenocytes using magnetic cell sorting (Miltenyi Biotec). The purified (>95%) CD4+ cells were incubated in 96-well plates (2 × 105 cells/well) with immobilized anti-CD3 Ab (5 μg/ml) in the presence of anti-CD28 Ab (2 μg/ml). The culture supernatant was collected 72 h later, and IL-2, IFN-γ, and IL-4 were determined by ELISA (R&D Systems).
Tail DNA was prepared from NOD, NOD.H2b, 129.S6, B6, B6-Abb, and B6-Abb-N6 mice. Microsatellite analysis was performed using genomic markers drawn from the Massachusetts Institute of Technology database. For the Idd-2 locus, D9Mit91; Idd-3 locus, il2; Idd-4 locus, acrb; Idd-5 locus, D1Mit3, D1Mit 144, and D1Mit 216; Idd-6 locus, D6Mit14; Idd-7 locus, D7Mit263; Idd-8 locus, D14Mit40; Idd-9 locus, D4Mit28, D4Mit251, D4Mit33, and D4Mit180; Idd-10 locus, D3Mit57; Idd-11 locus, Slc9a1; Idd-12 locus, D14Mit45; Idd-13 locus, II1b; Idd-14 locus, D13Mit61; Idd-15 locus, D5Mit48; Idd-17 locus, D3Mit230; Idd-18 locus, D3Mit109; Idd-19 locus, D6Mit194; the markers used for the Idd-1 and Idd-16 loci are shown in Table I. Genetic distance was derived from the Mouse Genome Database.
|Position .||Marker .||Mouse Strain .||.||.||.||.||.|
|.||.||AKR .||B6-Abb .||B6 .||NOD .||129.S6 .||NOD.H2b .|
|Position .||Marker .||Mouse Strain .||.||.||.||.||.|
|.||.||AKR .||B6-Abb .||B6 .||NOD .||129.S6 .||NOD.H2b .|
Data are expressed as the mean + SD. Statistical analyses were performed using the Welch t test (for ELISA data).
The origin of pre-T cells determined development of T1D in the reconstituted NOD.scid-RIP-B7.1 mice
Because MHC-linked T1D resistance genes were identified in the H-2 complex of both k and b haplotypes (17), we reconstituted NOD.scid-RIP-B7.1 mice with thymic pre-T cells isolated from young (3–4 wk) AKR (H2k) or B6 (H2b) mice (Table I) to determine whether MHC-linked T1D resistance genes expressed specifically in T cells alter T1D pathogenesis. NOD.scid-RIP-B7.1 mice were also reconstituted with pre-T cells from young NOD and NOR mice, because these cells express the H2g7 MHC susceptibility haplotype. Pre-T cells were TCR−CD3−CD4−CD8− (Fig. 1,A); however, both CD4+TCR+ and CD8+TCR+ populations developed in the periphery of all recipient mice 3 wk after intrathymic injection of the pre-T cells. In contrast, T cells were not detected in the control NOD.scid-RIP-B7.1 mice. Strain-specific markers were analyzed to ascertain the origins of different cell populations in the recipient mice. FACS analysis of blood monocytes, lymph node cells, and splenocytes from the recipient mice showed that T cells in the mice reconstituted with pre-T cells of AKR origin expressed the Thy-1.1 marker, while T cells in the mice reconstituted with pre-T cells of NOD origin expressed the Thy-1.2 marker. T cells in the mice reconstituted with pre-T cells of B6 origin expressed MHC class I Kb, but non-T cells were Kb negative in these mice. Both T and other cells expressed Kd in the NOD.scid.RIP-B7.1 mice reconstituted with pre-T cells of NOD or NOR origin (Fig. 1 B). Thus, T cells were all of donor origin, while other cells were from the recipient NOD.scid.RIP-B7.1 mice. The levels of reconstitution detected in the circulation were consistent from early to late stages, and the total number of T cells in these reconstituted mice at the end of the experiment varied from 5 to 10 × 106/recipient; however, the variation in absolute number of T cells was not correlated with different donor origins. These results show that pre-T cells, regardless of their origins, developed into mature T cells in the thymus and reconstituted both CD4+ and CD8+ T cell compartments in the periphery of NOD.scid-RIP-B7.1 mice.
None of control NOD.scid.RIP-B7.1 mice developed T1D (0 of 6), while T1D developed rapidly in the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells of NOD origin. Nine of 11 recipient mice developed diabetes by 14 wk after pre-T cell transfer (Fig. 2,A), even though the number of T cells in the hosts was low (5–8 × 106/recipient) due to a limited number of pre-T cells injected. Thus, the T cell compartment of NOD.scid-RIP-B7.1 recipients was sufficiently reconstituted by the intrathymic injection of pre-T cells. The recipients of pre-T cells of NOR origin also developed T1D with a high incidence within 20 wk (7 of 8), although NOR mice are resistant to both insulitis and T1D. It is clear that T cells of NOR origin can become diabetogenic when they develop in NOD.scid-RIP-B7.1 mice. In contrast, only 1 of 8 NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells of AKR origin developed T1D, and the recipients of B6 pre-T cells were free of T1D (0 of 13) for over 30 wk (Fig. 2,A). The lack of islet Ag-specific autoreactive T cells may prevent T1D in NOD.scid-RIP-B7.1 mice reconstituted by pre-T cells of AKR or B6 origin. To test this possibility, pancreata were removed at the end of experiments to determine the islet-specific lymphocyte infiltration. Residual islets were severely infiltrated in diabetic NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells of AKR origin, and peri-insulitis was detected in nondiabetic mice receiving either AKR or B6 pre-T cells (Fig. 2 B). These data show that islet-specific autoreactive T cells developed in the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells of AKR or B6 origins, most likely due to thymic education mediated by H2g7 MHC genes of host origin (25, 26). However, most of the autoreactive T cells of AKR and B6 origins did not become diabetogenic in the reconstituted mice, possibly because the resistance genes expressed in T cells of AKR and B6 origins abrogated the diabetic potential of these cells.
T1D resistance genes in T cells of AKR origin prevent T1D development in a dose-dependent manner
To directly test the effects of the genes expressed in T cells of AKR origin on T1D pathogenesis, we crossed NOD and AKR mice and used pre-T cells from the offspring, F1(AKR × NOD), to reconstitute NOD.scid-RIP-B7.1 mice. F1(AKR × NOD) mice develop neither insulitis nor T1D (data not shown), most likely because of the expression of resistance MHC class II I-Ak genes in thymic and peripheral APCs (9). When NOD.scid-RIP-B7.1 mice were reconstituted with pre-T cells of F1(AKR × NOD) origin, T cells were positively stained by both anti-Kd and anti-Kk, as well as both anti-Thy-1.1 and anti-Thy-1.2 Abs (Fig. 3,A), showing that all T cells express genes of both NOD and AKR alleles. T1D developed in 50% of the recipients (four of eight), and the onset of the disease in these mice was delayed compared with the recipients of pre-T cells of NOD origin (Fig. 3 B). The genes of AKR origin expressed in T cells are therefore inhibitory to T1D pathogenesis, even in the T cells that are heterozygous for the resistant allele.
To determine whether T cells of AKR origin can suppress the diabetogenic T cells of NOD origin, we reconstituted NOD.scid-RIP-B7.1 mice with mixed thymic pre-T cells (1:1 ratio) from AKR and NOD (AKR + NOD) donors. In these mice, T cell populations of both AKR and NOD origins were present 3–4 wk after intrathymic injection of pre-T cells (data not shown). Surprisingly, in some recipients of mixed pre-T cells, the number of T cells of AKR origin (Thy-1.1+) gradually declined, resulting in predominant T cell populations of NOD origin in the blood. These mice developed T1D when T cells of AKR origin were barely detectable (Fig. 3 B). In contrast, none of the reconstituted mice containing a significant T cell population of AKR origin developed diabetes. It is not clear how T cells of NOD origin became an overwhelming population in some, but not all, of the reconstituted mice. However, the presence of T cells, or subsets of T cells of AKR origin had an inhibitory effect on the diabetogenic T cells of NOD origin, and inhibitory effects were dependent on the number of cells expressing T1D resistance genes.
One of the major functions of T cells that affects development of autoimmune diseases is cytokine production, and IL-4-producing Th2 cells display regulatory functions against islet-specific pathogenic T cells in NOD mice. If polymorphic genes in T cells of AKR and F1(AKR × NOD) origins prevent T1D in the reconstituted mice through increased IL-4 production, then the levels of IL-4 production may correlate with dosage effects of resistance genes in these T cells. To test this possibility, CD4+ T cells from AKR, F1(AKR × NOD), and NOD mice were isolated and activated, and levels of cytokine production were determined. We found that all of these T cells produced similar levels of IL-2 and IFN-γ. CD4+ T cells from NOD mice produced IL-4 at a level much lower than that produced by T cells of other origins. However, IL-4 production by T cells of AKR origin was not significantly different from that of F1(AKR × NOD) origin (Fig. 3 C). Therefore, the dosage effect of resistance genes in T cells of AKR origin is not associated with levels of IL-4 production, suggesting that genes other than cytokine or cytokine-regulating genes expressed in T cells of AKR origin play a role in T1D resistance.
T1D resistance by T cells of AKR origin is β2m gene dependent
T cells of both NOD and NOR origins share a T1D-susceptible MHC haplotype; in contrast, T cells of AKR and B6 mice express class I genes from T1D-resistant MHC. To test the potential role of T1D resistance class I MHC genes expressed on T cells in T1D prevention, we reconstituted a group of NOD.scid-RIP-B7.1 mice with thymic pre-T cells from β2m gene-deficient AKR (AKR-β2m) donors. In these recipient mice, all T cells were Thy-1.1+, but did not express MHC class I molecules. Nevertheless, both CD4+ and CD8+ T cells developed normally in these reconstituted mice (Fig. 3,A). It is apparent that the development of CD8+ T cells was due to positive selection by MHC class I molecules on the thymic stroma of the host. In contrast to the recipients of pre-T cells of AKR origin, NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells of AKR-β2m origin developed T1D rapidly with an incidence almost identical with that in the recipients of pre-T cells of NOD origin (Fig. 3 B). These results demonstrate the requirement of β2m gene for T1D prevention, and indicate that the formation of MHC class I complex of AKR origin on the surface of T cells was inhibitory for T1D pathogenesis.
Partial T1D protection by genes in H2b complex expressed in T cells
To test the effects of T1D resistance genes expressed in T cells of B6 origin, we crossed NOD and B6 mice and used pre-T cells from the offspring, F1(B6 × NOD), to reconstitute NOD.scid-RIP-B7.1 mice. It has been known that F1(B6 × NOD) mice do not develop insulitis and T1D (1). In the reconstituted NOD.scid-RIP-B7.1 mice, T cells expressed both MHC class I Kb and Kd molecules, as expected (Fig. 4,A), and ∼30% of reconstituted mice developed T1D (Fig. 4,B). It is clear that the reconstituted mice heterozygous for T1D resistance genes in T cells of F1(B6 × NOD) origin were not fully protected from T1D. T cells of B6 origin produce less IFN-γ, but more IL-4 than T cells of NOD mice, and the MHC-independent, T cell-intrinsic differences in cytokine production were suggested as a part of T1D susceptibility/resistance (27, 28, 29). To determine whether resistance genes in T cells of B6 origin protect the reconstituted mice from T1D through increased IL-4 production, we reconstituted NOD.scid-RIP-B7.1 mice with pre-T cells from il4 gene-deficient B6 mice (B6-il4). None of these mice developed diabetes (Fig. 4 B), showing that high levels of IL-4 produced by T cells of B6 origin did not play a role in protection of the reconstituted mice from T1D.
To examine the effects of MHC class I genes expressed in T cells of B6 origin, NOD.scid-RIP-B7.1 mice were reconstituted with pre-T cells from B6 mice with a deficient β2m gene (B6-β2m). In these reconstituted mice, MHC class I of NOD haplotype was expressed normally in non-T cells, but T cells expressed neither NOD nor B6 MHC class I genes (Fig. 5,A). None of the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from donor mice developed T1D in 30 wk (Fig. 5,B), suggesting that in contrast to T cells of AKR origin, expression of β2m and MHC class I genes in T cells of B6 origin is not required for T1D prevention. To further test the potential role of MHC complex in T cells of B6 origin in T1D prevention, we reconstituted a group of NOD.scid-RIP-B7.1 mice with pre-T cells from NOD.H2b donors. NOD and NOD.H2b mice are identical except for the MHC complex. NOD.H2b mice possess the MHC complex of B6 origin and are free of T1D. However, T1D developed in the recipient mice reconstituted with pre-T cells from NOD.H2b donors, although incidence was significantly lower in these recipient mice than the mice reconstituted with pre-T cells of NOD origin (Fig. 5 B). Therefore, genes, within MHC locus of B6 origin, but not class I genes, contribute to T1D protection mediated by T cells.
MHC-linked genes within H2b complex confer partial T1D protection
Because the MHC complex of B6 origin contributes to T1D resistance conferred by T cells in the absence of β2m gene and thus MHC class I expression, we next reconstituted NOD.scid-RIP-B7.1 recipient mice with pre-T cells from B6-Abb mice. B6-Abb mice share an identical genome with B6 mice, except for a disrupted MHC class II I-A gene. T cells of B6-Abb origin expressed class I genes of b haplotype at levels similar to that in T cells of NOD.H2b or B6 origin (Fig. 5,A). Genotyping of Idd loci, from Idd2 to Idd19 loci, confirmed that B6-Abb and B6 mice are identical on these loci. However, >30% of the NOD.scid-RIP-B7.1 mice reconstituted by T cells of B6-Abb origin developed T1D (Fig. 5 B). This result was unexpected, because murine T cells do not express MHC class II genes, and thus a disrupted class II gene should have no direct effect on the functions of T cells. Nevertheless, the development of T1D in the reconstituted mice clearly indicates a genetic polymorphism in MHC complex between B6 and B6-Abb origins. Because the I-A gene was first disrupted in 129.S6 mice and then introduced into B6 mice to generate B6-Abb mice, it is possible that polymorphic alleles in the MHC complex of 129.S6 origin linked with the disrupted I-A gene were introduced into B6-Abb mice, and that the polymorphic alleles of 129.S6 origin reduce T1D resistance that is encoded in the alleles of B6 origin and expressed in T cells.
To identify the regions within MHC complex of B6 and B6-Abb origin that contain polymorphic alleles, the MHC loci of B6-Abb, B6, and NOD.H2b mice were genotyped with 11 microsatellite markers across an interval of 7 cM on chromosome 17. The MHC loci of NOD and 129.S6 mice were also analyzed. Within the MHC locus, B6-Abb, B6, and NOD.H2b mice were identical from K (D17Mit28) through D regions. FACS analysis confirmed that T cells of B6 and B6-Abb origin expressed MHC class I Kb and Db molecules at similar levels (Fig. 4,A, Table I, and data not shown). However, microsatellite analysis revealed different alleles between B6 and B6-Abb mice in an interval from D17Mit47 to D17Mit50 (Fig. 6 and Table I). This interval spans ∼4 cM at the distal end of the D region. Because B6-Abb and 129.S6 mice are identical for this interval, it is likely that a small piece of chromosome 17 of 129.S6 origin was introduced into the B6 genome along with the targeted I-A gene. In contrast to B6-Abb mice, NOD.H2b mice possess both the proximal region of K and distal end of the D region of B6 origin. We also analyzed and compared the distal end of D region in AKR, BALB/c, and NOD mice, because MHC-linked resistance was not detected in T cells of AKR origin, whereas a strong T1D resistance was reported at the distal D end of MHC complex of BALB/c origin (30). The results show a similar pattern between AKR and NOD mice; however, BALB/c mice are different from all the other strains at D17mit47 (Fig. 5). Thus, this locus is diverse among strains, even in mice sharing an identical MHC complex, such as B6 and 129.S6 mice.
Genetic susceptibility of T1D has been extensively studied, and a number of candidate genes, including MHC and non-MHC genes, have been identified (7, 31, 32, 33, 34, 35). Although the identity of the susceptibility genes that are specifically active in T cells is not clear, the expression of susceptibility genes in T cells may help autoreactive T cells escape from negative selection or enhance responses in the periphery (36, 37). In contrast, selective expression of T1D resistance genes in T cells of NOD mice may be inhibitory for the activation and expansion of pathogenic T cell populations. In the present study, NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from different donors allowed us to test the effects of T1D susceptibility/resistance genes expressed specifically in T cells on T1D pathogenesis, because these mice can develop accelerated T1D or remain T1D free, depending solely on the origin of T cells. Although islet-specific autoreactive T cells developed in all of the reconstituted mice, the mice reconstituted with T cells of AKR or B6 origins displayed strong T1D resistance. Furthermore, the results from the recipient mice reconstituted with T cells of F1(AKR × NOD) or F1(B6 × NOD) revealed that the effects of T1D resistance genes in T cells were dose dependent. Because anti-inflammatory cytokines, such as IL-4, produced by T cells display suppressive effects on T1D pathogenesis, we examined the levels of IL-4 production by T cells of different origins to determine whether IL-4 played a role in T1D prevention in the reconstituted mice. However, the levels of IL-4 production by T cells of AKR and F1(AKR × NOD) were not correlated with the degree of T1D protection, and T cells of B6 origin fully protected the reconstituted mice from T1D even when they failed to produce IL-4. These results indicate no or only a minor role of IL-4 production for T1D resistance in T cell-reconstituted NOD mice.
It is striking that T1D developed in the NOD.scid-RIP-B7.1 mice reconstituted with T cells expressing T1D susceptibility MHC genes, but not in the mice reconstituted with T cells expressing T1D resistance MHC genes. The heterozygous MHC allele in T cells of F1(AKR × NOD) and F1(B6 × NOD) correlated with reduced T1D incidence in the reconstituted NOD.scid-RIP-B7.1 mice. These observations suggest potential roles of MHC and MHC-linked genes expressed in T cells of NOD mice in T1D pathogenesis. The results of the present study further reveal that MHC class I genes of k haplotype expressed in T cells provide T1D resistance, because T1D developed with a high incidence in the recipient mice of pre-T cells derived from β2m gene-deficient AKR donors, but not in the recipients of pre-T cells derived from β2m-sufficient AKR donors. The difference in T1D development in these two groups of reconstituted mice was not due to β2m gene itself, although polymorphism of β2m gene is associated with T1D susceptibility (11, 38). The T1D susceptibility encoded by β2m gene of the NOD allele is manifested in nonhemopoietic cells, possibly by shaping the structure of MHC class I complex and thus the presentation of autoantigens (11). Therefore, β2m gene deficiency alone was unlikely to be responsible for T1D development in the reconstituted mice, because APC populations, including cells of nonhemopoietic lineage, in the reconstituted mice all expressed the susceptible form of β2m and MHC genes of NOD origin. In addition, T cells of NOR origin express resistant allele of β2m gene derived from B6 origin (11, 38), but they conferred no T1D resistance in the reconstituted mice. Furthermore, the decreased incidence of T1D detected in the mice reconstituted with T cells of F1(AKR × NOD) origin correlated with the expression of a single dose of class I genes of k haplotype in T cells. Therefore, the failed expression of MHC class I complex on the surface of T cells because of the deficient β2m gene results in total loss of T1D resistance conferred by T cells of AKR origin. We cannot rule out the possibility that other T1D resistance genes flanking β2m gene in T cells of AKR origin may have been replaced by susceptibility alleles in T cells of AKR-β2m origin during the development of these mice. However, those resistance genes may not be expressed in T cells, as T cells of NOR origin also contain resistance alleles flanking β2m genes, but did not confer resistance to T1D.
A recent study showed that due to allelic variations, the MHC class I K gene in CTS mice is homologous to K gene of k haplotype, and may be responsible for decreased incidence of T1D in NOD.CTS-H2 congenic mice (39). It is possible that expression of Kk gene in T cells provided T1D resistance, although Dk gene in T cells may also play a role. However, we could not discriminate the role of Kk and Dk expressed in T cells of AKR origin in the reconstituted NOD.scid-RIP-B7.1 mice. The mechanisms of T1D prevention by MHC class I genes of k haplotype expressed in T cells remain unclear, although a previous study suggested regulatory mechanisms induced by the expression of a T1D-resistant class I gene (40). Indeed, the development of regulatory NKT cells requires expression of MHC-I-like CD1d/β2m complex on the surface of double-positive thymocytes (41) that were mostly donor origin in the reconstituted mice. The observation that the T cells of AKR origin inhibited diabetogenic T cells of NOD origin in the reconstituted mice supports the induction of regulatory mechanisms by resistance class I genes; however, the specific pathway remains to be identified.
In contrast to T cells of AKR origin, T cells of B6 origin do not require the expression of β2m and MHC class I genes to provide T1D resistance in the reconstituted mice. T cells of B6 and NOD origins share an identical class I Db gene that displays pathogenic contribution to T1D in NOD mice (12). We could not detect any protective effect of the class I Kb gene in T cells, because Kb gene has no known functions in the absence of β2m gene, and the mice reconstituted with the T cells of B6-β2m origin were fully protected from T1D. In addition, the mice reconstituted with pre-T cells from B6-Abb donors developed T1D, although T cells in these mice expressed the Kb gene at normal levels. These results suggest that T1D resistance by MHC class I genes in T cells is k haplotype specific, whereas class I genes of H2g7 and H2b haplotypes in T cells do not encode T1D resistance.
Even though class I genes of b haplotype in T cells are not T1D protective, a markedly reduced T1D incidence in the NOD.scid-RIP-B7.1 reconstituted with pre-T cells of NOD.H2b origin demonstrated that the genes within MHC locus of B6 origin contributed to T1D resistance. The effects of MHC-linked genes on T1D pathogenesis were detected in NOD.scid-RIP-B7.1 mice reconstituted with T cells of B6-Abb origin. The development of T1D in these mice, even with a low incidence, reflected significantly reduced T1D resistance by T cells of B6-Abb origin compared with that by T cells of B6 origin. Furthermore, the results of genetic analysis showed that T1D developed in the reconstituted mice when T cells of B6 origin had an interval from class II to the distal end of D region replaced by an identical allele of 129.S6 origin. Although 129.S6 mice do not develop T1D, they may possess T1D susceptibility alleles in this interval. The survival or death of T cells bearing islet-specific TCR of NOD and B6 origins during thymic selection is determined by surface levels of TCR expression (37); it remains to be determined whether 129.S6 allele lowered levels of TCR expression in thymocytes of B6-Abb origin, allowing more autoreactive T cells to escape from negative selection. It is also noted that the interval replaced in B6-Abb mice includes MHC class III region between class II and D region. Many class III genes are also expressed by T cells and affect T cell functions.
In this study, we show that T cells can confer T1D resistance in NOD mice if they express MHC class I genes of k haplotype or MHC-linked genes of B6 origin. Although we are not able to identify a candidate T1D resistance gene, the results provide the first evidence for resistance alleles near or around the D region in B6 origin. We did not detect a similar resistance allele in MHC complex of AKR origin. A strong T1D resistance was reported near D region of BALB/c origin (30); it would be interesting to know whether MHC-linked T1D resistance genes of BALB/c origin are also expressed by T cells. Taken together, genetic polymorphism of MHC-linked T1D susceptibility/resistance genes may be associated with specific strains, but not MHC haplotypes, because mice sharing an identical MHC haplotype, such as B6, B6-Abb, and 129.S6 mice, possess different alleles of MHC-linked genes. Regardless of genetic polymorphism among different strains, at least one of MHC-linked susceptibility genes is expressed in T cells or a subset of T cells in NOD mice and displays its functional association with T1D pathogenesis.
We are grateful to Laurie Robertson for assistance with flow cytometry.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by Juvenile Diabetes Foundation International (to Y.Y.).
Abbreviations used in this paper: T1D, type 1 diabetes; β2, β2-microglobulin.