type 1 diabetes ( t1d ) is an autoimmune disease characterized by t cell - mediated destruction of the pancreatic islet beta cells .
multiple genetic loci contribute to t1d susceptibility in humans , with the most responsible locus being the major histocompatibility complex ( mhc ) .
the ability of certain class ii mhc genes to influence disease risk has long been appreciated [ 2 , 3 ] .
multiple studies have also revealed an association with certain class i mhc alleles , including the common hla - a02:01 [ 413 ] .
these findings are not surprising , given that cd4 and cd8 t cell responses to a variety of beta cell antigens , including insulin , are observed in t1d patients .
after the mhc , the locus that confers the strongest susceptibility to t1d in humans is the variable number of tandem repeats ( vntr ) regulatory region of the insulin gene [ 1 , 15 ] .
vntr alleles with the smallest number of repeats , designated as class i , predispose to t1d [ 16 , 17 ] , while the longer class iii alleles have a dominant protective effect [ 15 , 18 ] .
class iii vntr alleles are associated with thymic insulin rna levels that are increased two- to threefold compared to class i alleles , leading to the hypothesis that impaired negative selection of insulin - specific t cells in individuals with class i vntr alleles explains their predisposition to t1d [ 19 , 20 ] . while findings from a single human study are consistent with this idea , the development of a mouse model for t1d that incorporates the reduced , but not abolished , thymic insulin expression observed in patients would allow for more rigorous future testing of this hypothesis .
the nod mouse is the primary rodent model used for studying t1d . unlike humans ,
while both genes are expressed in beta cells , ins2 expression predominates in the thymus [ 2427 ] , with little   to no [ 2527 ] detectable thymic ins1 expression .
ins2-deficient ( ins2 ) nod mice develop diabetes at an accelerated rate [ 2830 ] , as do hla - a02:01-transgenic ins2 nod mice , and both ins2-deficient strains have increased insulin - specific islet - infiltrating cd8 t cells compared to their wild - type ( wt ) counterparts .
while these ins2 mouse strains highlight the importance of thymic insulin expression , they do not accurately represent a human patient , where thymic insulin expression is diminished but still present [ 19 , 20 ] .
here we have developed an hla - a02:01-transgenic nod - based t1d model that is heterozygous ( het ) for the ins2 allele , resulting in thymic insulin expression that is decreased but not eliminated .
the mice develop accelerated disease compared to ins2 mice , and this is true regardless of gender .
immune cell populations are not grossly altered , and the mice exhibit typical signs of islet autoimmunity , including cd8 t cell responses to beta cell peptides also targeted in hla - a02:01-positive t1d patients .
this model should find utility as a tool to uncover the mechanisms underlying the association between class i vntr alleles and t1d in humans .
it should also aid in preclinical studies to evaluate insulin - targeted immunotherapies for the disease .
nod.2m.hhd mice   transgenically express a single - chain chimeric hla - a02:01 molecule in which human 2-microglobulin ( 2 m ) is covalently linked to the 1 and 2 domains of hla - a02:01 . the 3 , transmembrane , and cytoplasmic portions of the molecule are derived from h-2d .
mouse class i mhc molecules are not expressed in these mice due to the murine 2 m deficiency .
the resulting progeny were bred as appropriate to obtain ins2 and ins2 nod.2m.hhd mice for our studies .
female 2 m mice breed poorly in our hands and so were rarely used for this purpose .
similarly , nod and nod.ins2 mice were intercrossed and the resulting progeny bred as appropriate to obtain ins2 and ins2 nod mice .
the hhd transgene and the wt and ko 2 m and ins2 alleles were identified by pcr using the following primer pairs : hhd , 5-cttcatcgcagtgggctac-3 and 5-cggtgagtctgtgagtggg-3 ; 2 m , 5-gaaacccctcaaattcaagtatactca-3 and 5-gacggtcttgggctcggccatact-3 ; 2 m , 5-gaaacccctcaaattcaagtatactca-3 and 5-tcgaattcgccaatgacaagacgct-3 ; ins2 , 5-ggcagagaggaggtgctttg-3 and 5-agaaaaccaccagggtagttagc-3 ; ins2 , 5-ggcagagaggaggtgctttg-3 and 5-attgaccgtaatgggatagg-3. all animal experiments were approved by the institutional animal care and use committee of albert einstein college of medicine .
female ins2 and ins2 nod.2m.hhd mice ( four each ) were sacrificed and thymus was harvested .
total thymic rna was isolated using the rneasy midi kit ( qiagen , valencia , ca ) and treated with dnase i ( qiagen ) to eliminate dna contamination .
1.52.3  g of rna was reverse - transcribed to cdna using random hexadeoxynucleotides and oligo dt primers ( invitrogen ) .
equal amounts of cdna were mixed with sybr green pcr master mix ( qiagen ) and each ins2 primer ( 5-cttcttctacacacccatgtcc-3 and 5-tctacaatgccacgcttctg-3 ) or primers for the u6 normalization control ( 5-ctcgcttcggcagcacatatacta-3 and 5-acgaatttgcgtgtcatccttgcg-3 ) and brought to a final volume of 25  l .
real - time quantitative rt - pcr was performed in triplicate using an iq5 optical system ( bio - rad , hercules , ca ) .
amplification was carried out as follows : a single denaturing step at 95c for 10  min followed by 40 cycles of 95c for 15  sec , 59c for 30  sec , and 72c for 30  sec , followed by a final extension step of 72c for 3  min .
results were analyzed using the relative expression software tool ( rest ) [ 32 , 33 ] .
glucosuria was monitored weekly using diastix reagent strips ( bayer , elkhart , in ) .
mice were considered diabetic after two consecutive positive tests , and the date of the first positive test was recorded as the time of onset of disease .
cells were stained with anti - b220 , anti - cd11c , anti - cd4 , and anti - cd8 ( all from bd biosciences , san jose , ca ) . in some samples ,
cells were stained with anti - cd25 ( bd biosciences ) , fixed and permeabilized with fixation / permeabilization buffer ( ebioscience , san diego , ca ) , and stained with anti - foxp3 ( ebioscience ) . to assess insulitis in female nod.2m.hhd and nod.2m.hhd.ins2 mice at 4 and 8 weeks of age ,
pancreata were fixed in bouin 's solution , embedded in paraffin , and sectioned at nonoverlapping levels .
sections were stained with aldehyde fuchsine to readily visualize granulated beta cells and counterstained with hematoxylin and eosin for detection of leukocytes .
islets were scored as previously described : 0 , no insulitis ; 1 , local insulitis without infiltration of islet itself ; 2 , less than 25% infiltration ; 3 , 2575% infiltration ; or 4 , greater than 75% infiltration .
an insulitis index was calculated by adding the scores of all islets and dividing by four times the number of islets scored .
islets were isolated from female nod.2m.hhd.ins2 mice at 8 weeks of age by collagenase p perfusion of the common bile duct as previously described .
islets were handpicked using a micromanipulator and a dissecting microscope and up to 50 islets were transferred per well to 24-well plates in 500  l r-10 medium ( rpmi 1640 ( invitrogen , carlsbad , ca ) containing 10% fbs , 1  mm sodium pyruvate , 28  m -mercaptoethanol , 1x nonessential amino acids ( invitrogen ) ) with 50  u / ml recombinant human il-2 ( peprotech , rocky hill , nj ) .
cells were cultured for 7 days at 37c in 5% co2 , at which point the majority of the cells are expected to be cd8 t cells .
human hla - a02:01-positive t2 cells , deficient for the transporter associated with antigen processing , were cultured at 26c overnight prior to use .
elispot plates ( millipore maha s4510 , billerica , ma ) were coated with anti - mouse ifn antibody ( bd biosciences ) and blocked with 1% bovine serum albumin ( sigma - aldrich , st .
t2 cells were plated at 2  10 cells / well and pulsed with 10  m of the indicated peptides for 1 hour at 26c .
cultured islet - infiltrating t cells from nod.2m.hhd.ins2 mice were added at 2  10 cells / well in 50  l r-10 .
wells were then washed with 0.05% tween 20/pbs and biotinylated anti - mouse ifn detection antibody ( bd biosciences ) was added for 2 hours at 37c .
after washing , streptavidin - alkaline phosphatase ( zymed laboratories , carlsbad , ca ) was added and incubated for 1 hour at 37c .
wells were washed and spots were developed using 5-bromo-4-chloro-3-indolyl - phosphate / nitro - blue tetrazolium substrate ( sigma - aldrich ) .
spots were counted using an automated elispot reader system ( autoimmun diagnostika , strassberg , germany ) .
responses are reported as a stimulation index , which is defined as spot number in response to the test peptide divided by spot number in response to an irrelevant hiv - derived hla - a02:01-binding peptide ( slyntvatl ) .
the cutoff for positivity is a stimulation index greater than 2 and a test peptide spot number greater than 5 per 1  10 t cells .
a previous study had demonstrated that ins2 mice of mixed , but primarily c57bl/6 , background experience a reduction in thymic insulin expression of approximately 40% . to develop a mouse model of t1d
having reduced thymic insulin quantity , and also expressing the human class i mhc molecule hla - a02:01 , we generated nod.2m.hhd.ins2 mice .
using quantitative rt - pcr , we similarly found a reduction in thymic insulin expression of 35% in female ins2 compared to ins2 mice ( n = 4 mice of each genotype ) .
both female ( figure 1(a ) ) and male nod.2m.hhd.ins2 mice ( figure 1(b ) ) demonstrated accelerated diabetes development compared to their ins2 counterparts .
female nod.2m.hhd.ins2 mice developed diabetes as early as 9 weeks of age and all were diabetic by 27 weeks ( figure 1(a ) ) .
the first onset of diabetes in ins2 female mice was at 11 weeks , and only 47% developed diabetes by 30 weeks .
as also seen in standard nod males [ 39 , 40 ] , diabetes development was slowed and overall incidence was reduced in nod.2m.hhd males ( figure 1(b ) ) compared to females . however
, ins2 males exhibited an earlier onset of disease compared to ins2 males ( 10 weeks versus 17 weeks ) , and a larger percentage ( 56% versus 24% ) had developed diabetes by 30 weeks of age ( figure 1(b ) ) .
thus , both genders of nod.2m.hhd.ins2 mice faithfully model the circumstance in humans where reduced thymic insulin expression is predisposing to t1d [ 16 , 17 ] .
note that this is not what we observed in the case of nod.ins2 mice , where both female ( figure 2(a ) ) and male ins2 mice ( figure 2(b ) ) exhibit a diabetes profile that is statistically indistinguishable from that of nod mice . to verify that the accelerated diabetes development observed in nod.2m.hhd.ins2 mice could not be attributed to a gross alteration in immune cell populations
, we examined the splenocyte composition of 8-week - old female nondiabetic nod.2m.hhd and nod.2m.hhd.ins2 mice ( figure 3(a ) ) .
it was previously shown that nod.2m.hhd mice have a reduced cd8 t cell population and elevated b and cd4 t cells compared to standard nod mice .
this was also true for nod.2m.hhd.ins2 mice , and no differences were observed in any of the cell types analyzed as a percentage of total cells . to investigate whether a reduction in regulatory t cells ( treg ) might contribute to disease pathogenesis in the ins2 mice ,
nod.2m.hhd and nod.2m.hhd.ins2 splenocytes were monitored for expression of the characteristic treg cell phenotype , cd4cd25foxp3 .
no difference was observed in treg cells as a percentage of cd4 t cells ( figure 3(b ) ) .
these results indicate that the accelerated diabetes development seen in nod.2m.hhd.ins2 mice is the result of neither an altered immune cell composition nor reduced treg cells , at least at the level investigated here , that is , without regard to antigenic specificity . in mixed background mice carrying zero , one , or two copies of the ins2 gene ,
furthermore , ins2 mice perform identically to their ins2 counterparts in intraperitoneal glucose tolerance tests .
thus , we hypothesized that the diabetes observed in nod.2m.hhd.ins2 mice was of an autoimmune nature , as is the case for the nod.2m.hhd parent strain , and not a deficiency in pancreatic insulin production due to the presence of only one functional copy of the ins2 gene . to verify this ,
histological sections of pancreata from female mice at 4 and 8 weeks of age were examined .
all mice studied exhibited some degree of insulitis , which progressed significantly with age ( figure 3(c ) ) , and islets showing a wide range of immune cell infiltration and beta cell destruction were observed ( figure 3(c ) ) .
we previously identified several hla - a02:01-restricted beta cell epitopes , derived from the autoantigens insulin and islet - specific glucose-6-phosphatase catalytic subunit - related protein ( igrp ) that are recognized by islet - infiltrating t cells from nod.2m.hhd mice [ 31 , 42 ] . to further confirm the autoimmune nature of the diabetes observed in nod.2m.hhd.ins2 mice , islets from 8-week - old females were cultured for 7 days and t cell reactivity to the previously identified beta cell epitopes
was monitored by ifn elispot . all mice harbored autoreactive t cells specific for at least two epitopes ( figure 3(d ) ) , further confirming the autoimmune nature of their disease .
a subset of these epitopes ( ins b514 , igrp 228236 , and igrp 265273 ) have previously been shown to be recognized by cd8 t cells in hla - a02:01-positive t1d patients [ 4346 ] , supporting the clinical relevance of the model .
insulin is an important autoantigen recognized by t cells in both human t1d and the nod mouse model of the disease .
reduced thymic insulin expression is associated with susceptibility to t1d in patients [ 16 , 17 , 19 , 20 ] , suggesting that impaired negative selection of t cells specific for insulin is responsible for this predisposition .
here we have developed and characterized nod.2m.hhd.ins2 mice as a model of t1d that incorporates reduced thymic insulin .
we find that , as in patients , disease is accelerated ( figure 1 ) , and we suggest these mice as a new diabetes model that can be used to better understand this phenomenon .
the nod.2m.hhd.ins2 mice present advantages over other disease models that have been described for this purpose .
for example , thymic insulin expression is abolished in nod.ins2 and nod.2m.hhd.ins2 mice , and both exhibit accelerated t1d [ 2830 ] and increased insulin - specific islet - infiltrating cd8 t cells   when compared to their ins2 counterparts . while these findings suggest the importance of thymic insulin expression , ins2 models do not accurately represent patients , where thymic insulin expression is reduced , but not eliminated [ 19 , 20 ] . as for nod.ins2 mice , in our hands
two earlier studies of nod.ins2 mice also showed no effect on disease in males [ 29 , 30 ] , and only one of the two showed acceleration in females .
in contrast , both male and female nod.2m.hhd.ins2 mice show enhanced disease ( figure 1 ) .
indeed , the female and male incidence curves are nearly overlapping until 15 weeks of age ( cf .
these studies should include the quantification of insulin - specific effector t cells and treg and analysis of their phenotype and function . the recently described ability to isolate insulin - specific cd4 t cells from nod mouse strains using enrichment with peptide / mhc tetramer reagents will facilitate this work . in nod mice , establishment of immunological tolerance to insulin can lead to prevention of t1d [ 4951 ] and remission of established disease . because of these findings , there is great interest in immunological interventions for human t1d that seek to manipulate the t cell response to insulin .
the nod.2m.hhd.ins2 mouse strain should be considered as an additional preclinical model to be used to evaluate such therapies , as it incorporates aspects of the human disease that are not represented in standard nod mice , including reduced thymic insulin expression . in humans , vntr alleles associated with diminished thymic insulin have been shown to alter the frequency and avidity of insulin - specific t cells , both of which could reasonably influence the outcome of therapies designed to manipulate the immune response to insulin .
given that human insulin - specific cd8 t cells have been shown to have cytotoxic activity against islets , an additional advantage of the nod.2m.hhd.ins2 mouse model is the expression of the t1d - predisposing human class i mhc allele hla - a02:01 [ 4 , 6 , 8 , 12 ] , which we have shown as supporting the development of t cells specific for hla - a02:01-restricted insulin epitopes in these mice ( figure 3(d ) ) . in terms of insulin - specific cd4 t cells , the class ii mhc allele expressed in the nod.2m.hhd.ins2 mice is i - a , which is structurally similar to the human t1d - predisposing hla - dq8 [ 55 , 56 ] .
indeed , i - a and hla - dq8 are capable of presenting similar peptides [ 5759 ] .
the nod.2m.hhd.ins2 mouse therefore has a variety of potential uses as a humanized model of t1d , including cd8 and cd4 t cell epitope identification , analysis of the relationship between thymic insulin expression and tolerance , and the evaluation of antigen - specific immunotherapies , particularly those targeting the immune response to insulin .
nod.2m.hhd.ins2 mice represent a model for t1d that incorporates the reduced , but not abolished , thymic insulin expression observed in patients .
this model should find utility in investigations to probe the mechanisms underlying the association between reduced thymic insulin expression and t1d in humans .
it will also be an important tool for t cell epitope discovery and for the preclinical evaluation of insulin - targeted immunotherapies for the disease .
 type 1 diabetes ( t1d ) is an autoimmune disease characterized by t cell - mediated destruction of the pancreatic islet beta cells . 
 multiple genetic loci contribute to disease susceptibility in humans , with the most responsible locus being the major histocompatibility complex ( mhc ) . 
 certain mhc alleles are predisposing , including the common hla - a02:01 . 
 after the mhc , the locus conferring the strongest susceptibility to t1d is the regulatory region of the insulin gene , and alleles associated with reduced thymic insulin expression are predisposing . 
 mice express two insulin genes , ins1 and ins2 . 
 while both are expressed in beta cells , only ins2 is expressed in the thymus . 
 we have developed an hla - a02:01-transgenic nod - based t1d model that is heterozygous for a functional ins2 gene . 
 these mice exhibit reduced thymic insulin expression and accelerated disease in both genders . 
 immune cell populations are not grossly altered , and the mice exhibit typical signs of islet autoimmunity , including cd8 t cell responses to beta cell peptides also targeted in hla - a02:01-positive type 1 diabetes patients . 
 this model should find utility as a tool to uncover the mechanisms underlying the association between reduced thymic insulin expression and t1d in humans and aid in preclinical studies to evaluate insulin - targeted immunotherapies for the disease . 