We previously developed NOD mice transgenically expressing the TCR from the diabetogenic AI4 CD8 T-cell clone and also homozygous for the Rag1null
). Adoptively transferred AI4 T-cells from such donors rapidly induce diabetes in both sublethally irradiated NOD and B6.H2g7
). Thus, we were surprised AI4 T-cells failed to transfer diabetes or significant levels of insulitis to sublethally irradiated (NODxB6.H2g7
)F1 hybrids (). This difference was not due to varying post-transfer stimulation of AI4 T-cells within pancreatic lymph nodes, because the proliferation of such effectors at this site in F1 hybrids was similar to that of NOD mice and even greater than in B6.H2g7
recipients (). Furthermore, no differences in expression of CD44, CD25, and CD62 L activation markers were detected between AI4 T-cells isolated from the parental or F1 mice ( and data not shown). However, AI4 T-cells preactivated in culture rapidly transferred diabetes to F1 recipients (). Hence, the F1 genetic environment allows for less efficient initial pathogenic activation of diabetogenic CD8 T-cells than in either parental strain.
FIG. 1. NOD and B6.H2g7 but not (NODxB6.H2g7)F1 mice succumb to AI4 T-cell–induced diabetes. A: Incidence of diabetes in 6- to 8-week-old female NOD, B6.H2g7, F1, and (F1xB6.H2g7)BC1 recipient mice that were sublethally irradiated (600 R) and injected (more ...)
F1 hybrid resistance to AI4-induced diabetes indicates the NOD and B6.H2g7 genomes harbor separate recessively acting alleles supporting pathogenic CD8 T-cell activation. To map such unrecognized recessive B6 alleles, NOD.Rag1null.AI4 splenocytes were transferred into 91 sublethally irradiated female (NODxB6.H2g7)F1xB6.H2g7 BC1 progeny that were then monitored for diabetes development and also genotyped. Diabetes developed in 40% of BC1 segregants at a rate similar to both NOD and B6.H2g7 mice (). Nondiabetic BC1 mice were examined for insulitis levels. Similar to F1 hybrids, insulitis levels were low in nondiabetic BC1 mice (). This sharp dichotomy among BC1 progeny suggested that rather than contributions from multiple loci, which would result in a broad spectrum of insulitis scores, a limited number of genes rendered B6.H2g7 mice susceptible to AI4 T-cell–induced diabetes. Indeed, a SNP-based genome-wide one-dimensional scan of BC1 progeny revealed only one B6 genomic region on chromosome 11 that was highly linked to diabetes susceptibility and insulitis (logarithm of odds [LOD] score = 13.2; ).
FIG. 2. A single genetic locus primarily contributes to B6.H2g7 susceptibility to AI4 T-cell–induced diabetes. Whole genome (A) and chromosome 11–specific (B) LOD score analysis of SNP markers linked to diabetes susceptibility and insulitis severity (more ...)
On the basis of original typing of SNP markers only, the 95% CI for the region of interest was originally narrowed to a 3.6-Mb segment at the distal end of chromosome 11 (112.6–116.2 Mb), ~40 Mb below the previously identified Idd4
locus (). Susceptibility to AI4 T-cell–induced diabetes/insulitis was associated with homozygosity for B6 markers within this region of chromosome 11, with LOD scores increasing up to and including the most distal NOD/B6 distinguishing SNP (rs3675087 at 116.2 Mb) that was typed. However, it remained possible that a gene(s) controlling susceptibility to AI4 transfer resided distally to the 116.2-Mb position. Thus, BC1 progeny were regenotyped for the polymorphic microsatellite marker D11Mit48
located at 117.76 Mb. Linkage between B6 homozygosity at D11Mit48
and diabetes/insulitis was very similar to that for rs3675087 (Supplementary Table 1
). Sanger sequence analysis (www.sanger.ac.uk
) indicated there are NOD/B6 polymorphisms in protein-encoding regions of two genes distal to D11Mit48
, but these were not covered by the SNP typing panel available to us. For this reason, we redefined the support interval controlling differential sensitivity to type 1 diabetes induced by transferred AI4 T-cells to between 112.6 Mb and the end of chromosome 11 (121.8 Mb; ).
We tested whether genotyping BC1 mice for D11Mit48 alone could predict susceptibility to AI4 T-cell–induced diabetes. An additional cohort of 31 BC1 mice was genotyped for the D11Mit48 polymorphism before receiving NOD.Rag1null.AI4 splenocytes. None of the 15 heterozygous (D11Mit48NOD/B6) animals developed diabetes and usually no more than mild peri-insulitis (). Conversely, 14 of 16 homozygous (D11Mit48B6/B6) mice developed diabetes, and the remaining two mice were severely insulitic (). This simple segregation pattern strongly indicates that a recessively acting B6 origin gene(s) tightly linked to D11Mit48 is a primary contributor to the pathogenic activation of diabetogenic CD8 T-cells, whereas the NOD allelic variant actually dominantly suppresses this process. However, the protective effect of this NOD chromosome 11 allelic variant must normally be masked by the large number of other diabetes susceptibility genes characterizing this strain.
FIG. 3. A polymorphic gene(s) in close linkage with the D11Mit48 microsatellite marker controls susceptibility to AI4 T-cell–induced diabetes through effects on a CD4 T-cell population other than CD25+ Tregs. A: Mice homozygous for the B6 allele (B6/B6) (more ...)
To determine whether the chromosome 11 gene(s) controls diabetes susceptibility through effects on hematopoietic cells, NOD.Rag1null.AI4 splenocytes were transferred into B6.H2g7 mice previously reconstituted with bone marrow from D11Mit48B6/B6 or D11Mit48NOD/B6 BC1 progeny. Only recipients of D11Mit48B6/B6 but not D11Mit48NOD/B6 bone marrow developed diabetes () or significant levels of insulitis (). Therefore, the D11Mit48-linked gene(s) controlling susceptibility to AI4 T-cell–mediated diabetic functions through a hematopoietic cell population(s).
Next, we established this gene(s) controls type 1 diabetes susceptibility through a lymphocyte population(s). This was determined by demonstrating NOD.Rag1null.AI4
splenocytes transferred diabetes with equal efficiency to B6.H2g7
, NOD, and F1 mice all homozygous for the Rag1null
mutation, eliminating all endogenous lymphocytes (). We tested whether regulatory T-cells (Tregs) were the lymphocyte population rendering F1 mice resistant to AI4-mediated diabetes. NOD and F1 mice were treated with a CD25-depleting antibody 1 day before receiving NOD.Rag1null.AI4
splenocytes. This eliminated most CD4+
Tregs for the duration of the 15-day postadoptive transfer period during which AI4 T-cells normally induce diabetes (Supplementary Fig. 1
). Anti-CD25–treated F1 mice remained resistant to AI4-induced diabetes ().
We next tested whether the chromosome 11 gene(s) controls type 1 diabetes susceptibility through effects on a CD4 T-cell population other than Tregs. Total CD4 T-cells purified from D11Mit48B6/B6 or D11Mit48NOD/B6 BC1 progeny were cotransferred with NOD.Rag1null.AI4 splenocytes into B6.H2g7.Rag1null recipients. Only recipients of D11Mit48B6/B6 but not D11Mit48NOD/B6 CD4 T-cells developed AI4 T-cell–induced diabetes or high levels of insulitis (). Therefore, the D11Mit48-linked gene(s) controlling susceptibility to AI4 T-cell–mediated diabetic functions through a non-Treg CD4 T-cell population(s).
We used microarray-based comparisons of mRNA transcript levels to identify candidates for a CD4 T-cell–expressed gene(s) within the chromosome 11 support interval regulating pathogenic activation of diabetogenic AI4 CD8 T-cells. This was accomplished by recovering CD4 T-cells purified from D11Mit48B6/B6
BC1 progeny that were previously cotransferred into B6.H2g7
recipients with NOD.Rag1null.AI4
splenocytes. CD4 T-cells were repurified from spleens once all recipients of D11Mit48B6/B6
CD4 T-cells developed diabetes (all D11Mit48NOD/B6
CD4 T-cell recipients were disease-free). describes 12 genes mapping within the chromosome 11 support interval (112.6–121.8 Mb) that were differentially expressed by these two classes of CD4 T-cells and also characterized by NOD/B6 polymorphisms (www.sanger.ac.uk
). Interestingly, three of these genes (Rab37
, and CD300lf
) map to the PSORS2 psoriasis susceptibility locus in humans (17
List of polymorphic genes within the chromosome 11 interval 112.6 to 121.8 Mb that are differentially expressed in CD4 T-cells purified from D11Mit48B6/B6 vs. D11Mit48NOD/B6 genotyped BC1 progeny