nod mice transgenically expressing the tcr from the diabetogenic cd8 t - cell clone ai4 plus a functionally inactivated rag1 gene ( nod.rag1.ai4 ) have been described ( 8) . indicated female mice were sublethally irradiated ( 600 r ) and injected intravenously with 1  10 nod.rag1.ai4 splenocytes to induce diabetes . in one experiment , recipients were injected intraperitoneally with 250 g / mouse of the cd25-depleting pc61.5 antibody 1 day before ai4 t - cell transfer . in another experiment , donor cells were prelabeled with 2.5 mol / l carboxy fluorescein succinimidyl ester ( cfse ) . after 4 days , viable ai4 t - cells from collagenase d - digested spleen and pancreatic lymph nodes were identified by flow cytometry using cfse and a cd8-specific antibody ( 536.7 ) . expression of various t - cell surface markers was assessed using antibodies specific for cd44 ( im7.8.1 ) , cd25 ( pc61.5 ) , and cd62 l ( mel-14 ) . another experiment used female mice injected with ai4 t - cells preactivated in culture for 3 days with 100 nmol / l antigenic mimotope peptide yfienylel and 50 units / ml interleukin 2 . in a separate experiment , female b6.h2 mice were lethally irradiated ( 1,200 r ) and injected intravenously with 5  10 bone marrow cells from bc1 progeny heterozygous or homozygous for the microsatellite marker d11mit48 . fourteen weeks later , recipient mice were sublethally irradiated and injected intravenously with 1  10 nod.rag1.ai4 splenocytes to induce diabetes . in another study , b6.h2 , nod and f1 mice all homozygous for the rag1 mutation were injected with 1  10 nod.rag1.ai4 splenocytes . finally , magnetic bead purified cd4 t - cells from d11mit48 or d11mit48 bc1 progeny were cotransferred with 1  10 nod.rag1.ai4 splenocytes into b6.h2.rag1 recipients . diabetes was assessed by daily monitoring of glycosuria with ames diastix ( bayer , diagnostics division , elkhart , in ) , with disease onset defined by two consecutive values of 3 . previously described criteria ( 9 ) were used to establish insulitis scores ranging from 0 ( individual islet with no leukocytic infiltration , normal -cell mass ) to 4 ( complete destruction ) for the indicated mice . bc1 progeny were genotyped as previously described ( 10 ) for 131 single nucleotide polymorphisms ( snp ) at approximately 20-mb intervals across the genome ( national center for biotechnology information [ ncbi ] build 37 marker positions ) . linkage markers for genes controlling insulitis severity and diabetes development in response to ai4 t - cell transfer were identified as previously described ( 11 ) . cd4 t - cells were purified from bc1 progeny either heterozygous or homozygous for d11mit48 and cotransferred into female b6.h2.rag1 recipients with 1  10 nod.rag1.ai4 splenocytes . rna was isolated from cd4 t - cells sorted from spleens of these recipients by flow cytometry 3 weeks after adoptive transfer . three biologic replicates were generated for both genotypes , with each sample emanating from purified cd4 t - cells pooled from two donor mice . microarray analysis of comparative cd4 t - cell gene expression was conducted using the affymetrix 430v2 genechip array ( santa clara , ca ) . r / bioconductor software summarized the probe intensities for each gene using the robust multiaverage method ( 12,13 ) . r / maanova software ( churchill group , bar harbor , me ) was used to generate lists of differentially expressed genes between the tested samples ( 14 ) . differentially expressed genes were identified by using ts , a modified t statistic incorporating shrinkage estimates of variance components from within the r / maanova ( 15 ) . nod mice transgenically expressing the tcr from the diabetogenic cd8 t - cell clone ai4 plus a functionally inactivated rag1 gene ( nod.rag1.ai4 ) have been described ( 8) . indicated female mice were sublethally irradiated ( 600 r ) and injected intravenously with 1  10 nod.rag1.ai4 splenocytes to induce diabetes . in one experiment , recipients were injected intraperitoneally with 250 g / mouse of the cd25-depleting pc61.5 antibody 1 day before ai4 t - cell transfer . in another experiment , donor cells were prelabeled with 2.5 mol / l carboxy fluorescein succinimidyl ester ( cfse ) . after 4 days , viable ai4 t - cells from collagenase d - digested spleen and pancreatic lymph nodes were identified by flow cytometry using cfse and a cd8-specific antibody ( 536.7 ) . expression of various t - cell surface markers was assessed using antibodies specific for cd44 ( im7.8.1 ) , cd25 ( pc61.5 ) , and cd62 l ( mel-14 ) . another experiment used female mice injected with ai4 t - cells preactivated in culture for 3 days with 100 nmol / l antigenic mimotope peptide yfienylel and 50 units / ml interleukin 2 . in a separate experiment , female b6.h2 mice were lethally irradiated ( 1,200 r ) and injected intravenously with 5  10 bone marrow cells from bc1 progeny heterozygous or homozygous for the microsatellite marker d11mit48 . fourteen weeks later , recipient mice were sublethally irradiated and injected intravenously with 1  10 nod.rag1.ai4 splenocytes to induce diabetes . in another study , b6.h2 , nod and f1 mice all homozygous for the rag1 mutation were injected with 1  10 nod.rag1.ai4 splenocytes . finally , magnetic bead purified cd4 t - cells from d11mit48 or d11mit48 bc1 progeny were cotransferred with 1  10 nod.rag1.ai4 splenocytes into b6.h2.rag1 recipients . diabetes was assessed by daily monitoring of glycosuria with ames diastix ( bayer , diagnostics division , elkhart , in ) , with disease onset defined by two consecutive values of 3 . previously described criteria ( 9 ) were used to establish insulitis scores ranging from 0 ( individual islet with no leukocytic infiltration , normal -cell mass ) to 4 ( complete destruction ) for the indicated mice . bc1 progeny were genotyped as previously described ( 10 ) for 131 single nucleotide polymorphisms ( snp ) at approximately 20-mb intervals across the genome ( national center for biotechnology information [ ncbi ] build 37 marker positions ) . linkage markers for genes controlling insulitis severity and diabetes development in response to ai4 t - cell transfer were identified as previously described ( 11 ) . cd4 t - cells were purified from bc1 progeny either heterozygous or homozygous for d11mit48 and cotransferred into female b6.h2.rag1 recipients with 1  10 nod.rag1.ai4 splenocytes . rna was isolated from cd4 t - cells sorted from spleens of these recipients by flow cytometry 3 weeks after adoptive transfer . three biologic replicates were generated for both genotypes , with each sample emanating from purified cd4 t - cells pooled from two donor mice . microarray analysis of comparative cd4 t - cell gene expression was conducted using the affymetrix 430v2 genechip array ( santa clara , ca ) . r / bioconductor software summarized the probe intensities for each gene using the robust multiaverage method ( 12,13 ) . r / maanova software ( churchill group , bar harbor , me ) was used to generate lists of differentially expressed genes between the tested samples ( 14 ) . differentially expressed genes were identified by using ts , a modified t statistic incorporating shrinkage estimates of variance components from within the r / maanova ( 15 ) . we previously developed nod mice transgenically expressing the tcr from the diabetogenic ai4 cd8 t - cell clone and also homozygous for the rag1 mutation ( nod.rag1.ai4 ) . adoptively transferred ai4 t - cells from such donors rapidly induce diabetes in both sublethally irradiated nod and b6.h2 mice ( 16 ) . thus , we were surprised ai4 t - cells failed to transfer diabetes or significant levels of insulitis to sublethally irradiated ( nodxb6.h2)f1 hybrids ( fig . 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.h2 recipients ( fig . 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 ( fig . however , ai4 t - cells preactivated in culture rapidly transferred diabetes to f1 recipients ( fig . hence , the f1 genetic environment allows for less efficient initial pathogenic activation of diabetogenic cd8 t - cells than in either parental strain . nod and b6.h2 but not ( nodxb6.h2)f1 mice succumb to ai4 t - cell  induced diabetes . a : incidence of diabetes in 6- to 8-week - old female nod , b6.h2 , f1 , and ( f1xb6.h2)bc1 recipient mice that were sublethally irradiated ( 600 r ) and injected intravenously with 1  10 nod.rag1.ai4 splenocytes . incidence of diabetes in bc1 recipients was significantly different ( p < 0.0001 ) from nod , b6.h2 , and f1 mice . b : insulitis scores ( 0 = no insulitis to 4 = no remaining islet cell mass ) for surviving nondiabetic nod.rag1.ai4 splenocyte recipients . insulitis severity was significantly greater in surviving nod mice compared with f1 and bc1 recipients according to the mann - whitney test . c : in vivo proliferation and activation of cfse - labeled nod.rag1.ai4 t - cells at 4 days after transfer in pancreatic lymph nodes of nod , b6.h2 , and f1 mice . d : incidence of diabetes in 6- to 8-week - old sublethally irradiated female nod and f1 recipients of in vitro activated ai4 t - cells . f1 hybrid resistance to ai4-induced diabetes indicates the nod and b6.h2 genomes harbor separate recessively acting alleles supporting pathogenic cd8 t - cell activation . to map such unrecognized recessive b6 alleles , nod.rag1.ai4 splenocytes were transferred into 91 sublethally irradiated female ( nodxb6.h2)f1xb6.h2 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.h2 mice ( fig . similar to f1 hybrids , insulitis levels were low in nondiabetic bc1 mice ( fig . 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.h2 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 . a single genetic locus primarily contributes to b6.h2 susceptibility to ai4 t - cell  induced diabetes . whole genome ( a ) and chromosome 11specific ( b ) lod score analysis of snp markers linked to diabetes susceptibility and insulitis severity in sublethally irradiated bc1 recipients of 1  10 nod.rag1.ai4 splenocytes . horizontal lines depict lod scores indicative of 1 , 5 , 10 , and 63% linkage support thresholds . 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 ( 112.6116.2 mb ) , 40 mb below the previously identified idd4 locus ( fig . 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 ; fig . 2c ) . 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.rag1.ai4 splenocytes . none of the 15 heterozygous ( d11mit48 ) animals developed diabetes and usually no more than mild peri - insulitis ( fig . 3a and b ) . conversely , 14 of 16 homozygous ( d11mit48 ) mice developed diabetes , and the remaining two mice were severely insulitic ( fig . 3a and b ) . 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 . 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 ) vs. heterozygous ( nod / b6 ) for d11mit48 were , respectively , highly susceptible and resistant to ai4 t - cell  induced diabetes . b : insulitis scores are shown for nondiabetic heterozygous and homozygous bc1 nod.rag1.ai4 splenocyte recipients . c and d : b6.h2 mice previously reconstituted with bone marrow from d11mit48 or d11mit48 bc1 segregants are , respectively , susceptible or resistant to diabetes and insulitis induced by subsequently infused ai4 t - cells . e : incidence of diabetes in 6- to 8-week - old female b6.h2.rag1 , nod.rag1 , and f1.rag1 recipients of 1  10 nod.rag1.ai4 splenocytes . f : incidence of diabetes in 6- to 8-week - old sublethally irradiated nod and f1 recipients of 1  10 nod.rag1.ai4 splenocytes . recipients were also injected intraperitoneally with a cd25-depleting antibody ( pc61 250 g / mouse ) 1 day before ai4 t - cell transfer . g and h : b6.h2.rag1 mice infused with purified cd4 t - cells from d11mit48 or d11mit48 bc1 segregants are , respectively , susceptible or resistant to diabetes and insulitis induced by subsequently infused ai4 t - cells . to determine whether the chromosome 11 gene(s ) controls diabetes susceptibility through effects on hematopoietic cells , nod.rag1.ai4 splenocytes were transferred into b6.h2 mice previously reconstituted with bone marrow from d11mit48 or d11mit48 bc1 progeny . 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.rag1.ai4 splenocytes transferred diabetes with equal efficiency to b6.h2 , nod , and f1 mice all homozygous for the rag1 mutation , eliminating all endogenous lymphocytes ( fig . 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.rag1.ai4 splenocytes . this eliminated most cd4cd25 tregs for the duration of the 15-day postadoptive transfer period during which ai4 t - cells normally induce diabetes ( supplementary fig . 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 d11mit48 or d11mit48 bc1 progeny were cotransferred with nod.rag1.ai4 splenocytes into b6.h2.rag1 recipients . only recipients of d11mit48 but not d11mit48 cd4 t - cells developed ai4 t - cell  induced diabetes or high levels of insulitis ( fig . 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 d11mit48 and d11mit48 bc1 progeny that were previously cotransferred into b6.h2.rag1 recipients with nod.rag1.ai4 splenocytes . cd4 t - cells were repurified from spleens once all recipients of d11mit48 cd4 t - cells developed diabetes ( all d11mit48 cd4 t - cell recipients were disease - free ) . table 1 describes 12 genes mapping within the chromosome 11 support interval ( 112.6121.8 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 , slc9a3r1 , and cd300lf ) map to the psors2 psoriasis susceptibility locus in humans ( 1719 ) . 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 d11mit48 vs. d11mit48 genotyped bc1 progeny * marker positions were taken from ncbi build 37.1 ( www.ncbi.nlm.nih.gov ) . although the nod genome contains a collection of genes supporting development of spontaneous diabetes , additional susceptibility alleles remain masked within normally disease - resistant mouse strains . indeed , we found the distal region of chromosome 11 in b6.h2 mice harbors what is likely a single recessive gene acting through cd4 t - cells that more strongly promotes pathogenic activation of diabetogenic cd8 t - cells than the nod allelic variant . presumably , the normal efficient thymic deletion of pathogenic cd8 t - cells in b6.h2 mice ( 4,11 ) masks the activity of their prodiabetogenic gene(s ) on chromosome 11 . determination of diabetes susceptibility genes hidden in normally resistant mouse strains might ultimately aid the identification of individuals among heterogeneous humans who are at high risk of future disease .
objectivenominally resistant mouse strains such as c57bl/6 ( b6 ) harbor latent type 1 diabetes susceptibility genes uncovered in outcross to disease - susceptible nod mice . however , 
 identification of possible recessively acting b6-derived susceptibility genes is limited because very few f2 progeny derived from outcrossing this strain with nod develop spontaneous autoimmune diabetes . 
 thus , we assessed whether a transgenic t - cell receptor ( tcr ) disease transfer model allowed the mapping of recessively acting b6 genetic loci that in the proper context contribute to diabetes.research design and methodscd8 t - cells transgenically expressing the diabetogenic ai4 tcr were transferred into 91 ( nodxb6.h2g7)f1xb6.h2g7 first - backcross ( bc1 ) females . 
 a genome - wide scan was performed for loci affecting clinical diabetes and insulitis severity.resultsa major locus on chromosome 11 in tight linkage with the marker d11mit48 ( logarithm of odds score = 13.2 ) strongly determined whether bc1 progeny were susceptible to ai4 t - cell  mediated diabetes . 
 mice homozygous versus heterozygous for b6 markers of this chromosome 11 genetic locus were , respectively , highly susceptible or resistant to ai4-induced insulitis and diabetes . 
 the genetic effect is manifest by host cd4 t - cells . 
 microarray analyses of mrna transcript expression identified a limited number of candidate genes.conclusionsthe distal region of chromosome 11 in b6 mice harbors a previously unrecognized recessively acting gene(s ) that can promote autoreactive diabetogenic cd8 t - cell responses . 
 future identification of this gene(s ) may further aid the screening of heterogeneous humans at future risk for diabetes , and might also provide a target for possible disease interventions .