previously described nod.2m.hhd mice ( 17 ) are maintained by sibling matings at the jackson laboratory .
some experiments used an n10 backcross generation nod stock congenically expressing the cd45.2 rather than the cd45.1 variant leukocyte marker .
nod.2m.hhd mice in which antigen - presenting cells ( apcs ) specifically express an mhc class ii promoter ( h2-e)driven mouse proinsulin 2 transgene ( nod.2m.hhd-pi ) were generated by crossing nod.2m.hhd with previously described nod.pi mice ( 27 ) .
the institutional animal care and use committee at the jackson laboratory approved all animal experiments .
synthetic peptides ins1/2 a210 ( ivdqcctsi ) , ins1 b515 ( hlcgphlvea ) , igrp228236 ( fgidllwsv ) , igrp265273 ( vlfglgfai ) , and flu16 ( flu mp5866 ) ( gilgfvftl ) were purchased from mimotopes pty , melbourne , australia .
monoclonal antibodies specific for cd11c ( clone n418 ) , cd8 ( clone 536.72 ) , cd45.1 ( clone a201.7 ) , and cd45.2 ( clone 1042.1 ) were purchased from bd biosciences , san jose , ca and ebiosciences , san diego , ca .
ecdi peptide - coupled splenocyte ( peptide - sps ) treatment was carried out as previously described ( 28 ) .
briefly , spleens were removed from syngeneic female mice , collagenase d treated ( roche diagnostics , manheim , germany ) , and the erythrocytes lysed .
the splenocytes were incubated with ecdi ( 150 mg/3.2  10 cells [ calbiochem , la jolla , ca ] ) , and as indicated , a mixture of ins and/or igrp peptide(s ) ( 1 mg / ml each ) on ice for 1 h , hand - shaking every 10 min .
the peptide - sps were washed , centrifuged , filtered to remove cell clumps , and resuspended in pbs .
nod.2m.hhd female mice ( 46 weeks old ) received 50  10 peptide - sps in 200 l pbs by intravenous injection .
controls consisted of nod.2m.hhd female mice treated with splenocytes bearing the ecdi - coupled hla - a2.1 binding but diabetes - irrelevant flu16 peptide .
diabetes onset was defined by a urinary glucose value of 3 as assessed with ames diastix ( bayer , diagnostics division , elkhart , nj ) .
mice remaining free of overt diabetes through 26 weeks posttreatment were killed and their pancreata fixed in bouin s solution and sectioned at three nonoverlapping levels .
islets ( at least 20 per mouse ) were individually scored as follows : 0 , no lesions ; 1 , peri - insular leukocytic aggregates , usually periductal infiltrates ; 2 , < 25% islet destruction ; 3 , > 25% islet destruction ; and 4 , complete islet destruction . an insulitis score for each mouse was obtained by dividing the total score for each pancreas by the number of islets examined .
interferon ( ifn)- enzyme - linked immunospot ( elispot ) assays were performed as previously described ( 17 ) .
spots were counted using an automated elispot reader system ( immunospot ; ctl analyzers , cleveland , oh ) .
numbers of ifn- or il-10 spots were normalized to the indicated numbers of cd8 t - cells .
nod.2m.hhd mice treated every 5 weeks with peptide - sps were primed in a rear footpad 2 days after the third treatment with a cocktail containing 20 g each of the hla - a2.1restricted ins1/2 a210 , ins1 b515 , igrp228236 , and igrp265273 peptides emulsified in 50 l of complete freund s adjuvant .
ten days after priming , the mice were killed , the popliteal lymph nodes were isolated and dispersed by collagenase d treatment , and cd8 t - cell reactions to each individual peptide were determined by ifn- elispot analyses .
splenic cd8 t - cells were enriched from nod.2m.hhd mice by the previously described magnetic bead  based negative selection approach ( 29 ) and incubated in vitro at a concentration of 5  10/ml with 5  10/ml ins- and/or igrp - sps .
the next day , the cd8 t - cells were recovered and assessed by ifn- elispot analyses for reactivity against hla - a2.1restricted ins and igrp peptides .
starting at 3 weeks of age , nod.2 m .hhd mice were treated with repeated intraperitoneal injections of a soluble mixture containing 25 g of each peptide ( ins a210 , ins b515 , igrp228236 , and igrp265273 ) .
the log - rank test was used to compare diabetes incidence curves and the nonparametric unpaired test to compare antigen - reactive cd8 t - cell numbers between different treatment groups .
previously described nod.2m.hhd mice ( 17 ) are maintained by sibling matings at the jackson laboratory .
some experiments used an n10 backcross generation nod stock congenically expressing the cd45.2 rather than the cd45.1 variant leukocyte marker .
nod.2m.hhd mice in which antigen - presenting cells ( apcs ) specifically express an mhc class ii promoter ( h2-e)driven mouse proinsulin 2 transgene ( nod.2m.hhd-pi ) were generated by crossing nod.2m.hhd with previously described nod.pi mice ( 27 ) .
the institutional animal care and use committee at the jackson laboratory approved all animal experiments .
synthetic peptides ins1/2 a210 ( ivdqcctsi ) , ins1 b515 ( hlcgphlvea ) , igrp228236 ( fgidllwsv ) , igrp265273 ( vlfglgfai ) , and flu16 ( flu mp5866 ) ( gilgfvftl ) were purchased from mimotopes pty , melbourne , australia .
monoclonal antibodies specific for cd11c ( clone n418 ) , cd8 ( clone 536.72 ) , cd45.1 ( clone a201.7 ) , and cd45.2 ( clone 1042.1 ) were purchased from bd biosciences , san jose , ca and ebiosciences , san diego , ca .
ecdi peptide - coupled splenocyte ( peptide - sps ) treatment was carried out as previously described ( 28 ) .
briefly , spleens were removed from syngeneic female mice , collagenase d treated ( roche diagnostics , manheim , germany ) , and the erythrocytes lysed .
the splenocytes were incubated with ecdi ( 150 mg/3.2  10 cells [ calbiochem , la jolla , ca ] ) , and as indicated , a mixture of ins and/or igrp peptide(s ) ( 1 mg / ml each ) on ice for 1 h , hand - shaking every 10 min .
the peptide - sps were washed , centrifuged , filtered to remove cell clumps , and resuspended in pbs .
nod.2m.hhd female mice ( 46 weeks old ) received 50  10 peptide - sps in 200 l pbs by intravenous injection .
controls consisted of nod.2m.hhd female mice treated with splenocytes bearing the ecdi - coupled hla - a2.1 binding but diabetes - irrelevant flu16 peptide .
diabetes onset was defined by a urinary glucose value of 3 as assessed with ames diastix ( bayer , diagnostics division , elkhart , nj ) .
mice remaining free of overt diabetes through 26 weeks posttreatment were killed and their pancreata fixed in bouin s solution and sectioned at three nonoverlapping levels .
islets ( at least 20 per mouse ) were individually scored as follows : 0 , no lesions ; 1 , peri - insular leukocytic aggregates , usually periductal infiltrates ; 2 , < 25% islet destruction ; 3 , > 25% islet destruction ; and 4 , complete islet destruction . an insulitis score for each mouse was obtained by dividing the total score for each pancreas by the number of islets examined .
interferon ( ifn)- enzyme - linked immunospot ( elispot ) assays were performed as previously described ( 17 ) .
spots were counted using an automated elispot reader system ( immunospot ; ctl analyzers , cleveland , oh ) .
numbers of ifn- or il-10 spots were normalized to the indicated numbers of cd8 t - cells .
nod.2m.hhd mice treated every 5 weeks with peptide - sps were primed in a rear footpad 2 days after the third treatment with a cocktail containing 20 g each of the hla - a2.1restricted ins1/2 a210 , ins1 b515 , igrp228236 , and igrp265273 peptides emulsified in 50 l of complete freund s adjuvant .
ten days after priming , the mice were killed , the popliteal lymph nodes were isolated and dispersed by collagenase d treatment , and cd8 t - cell reactions to each individual peptide were determined by ifn- elispot analyses .
splenic cd8 t - cells were enriched from nod.2m.hhd mice by the previously described magnetic bead  based negative selection approach ( 29 ) and incubated in vitro at a concentration of 5  10/ml with 5  10/ml ins- and/or igrp - sps .
the next day , the cd8 t - cells were recovered and assessed by ifn- elispot analyses for reactivity against hla - a2.1restricted ins and igrp peptides .
starting at 3 weeks of age , nod.2 m .hhd mice were treated with repeated intraperitoneal injections of a soluble mixture containing 25 g of each peptide ( ins a210 , ins b515 , igrp228236 , and igrp265273 ) .
the log - rank test was used to compare diabetes incidence curves and the nonparametric unpaired test to compare antigen - reactive cd8 t - cell numbers between different treatment groups .
cd8 t - cells were enriched from pooled spleens of 9- to 16-week - old nod.2m.hhd mice and cocultured in vitro with syngeneic splenocytes bearing an ecdi - coupled cocktail of the hla - a2.1restricted ins1/2 a210 , ins1 b515 , igrp228236 , and igrp265273 -cell autoantigens ( ins / igrp - sps ) and assessed the next day for responsiveness to each individual epitope by ifn- elispot analyses . compared with those exposed to the control flu16 peptide ( flu - sps ) , cd8 t - cells from nod.2m.hhd mice cocultured with ins
/ igrp - sps displayed significantly decreased responses to restimulation by igrp epitopes ( fig .
levels of reactivity to the hla - a2.1restricted ins peptides was low among control cd8 t - cells exposed to flu16 and not further influenced by previous coculture with ins / igrp - sps ( fig .
coincubation with syngeneic splenocytes bearing an ecdi cross - linked mixture of the two ins peptides ( ins - sps ) did not diminish cd8 t - cell responsiveness to ins or igrp epitopes ( fig .
however , preincubation with igrp - sps did significantly diminish the ability of cd8 t - cells from nod.2m.hhd mice to respond to restimulation by igrp but not ins epitopes ( fig .
il-10 elispot analyses also indicated that regardless of preincubation conditions , no cd8 t - cells from nod.2m.hhd mice responding to ins or igrp stimulation produced this immunosuppressive cytokine . on the basis of these collective data
, we subsequently assessed syngeneic ins / igrp - sps as a possible diabetes intervention approach in nod.2m.hhd mice .
hla - a2.1restricted -cell antigenic peptides that are ecdi cross - linked to syngeneic splenocytes significantly diminish responsiveness by cognate cd8 t - cells from nod.2m.hhd mice .
a : cd8 t - cells from nod.2m.hhd mice were incubated with syngeneic flu - sps or ins / igrp - sps ( bearing the complete mixture of igrp265273 , igrp228236 , ins1/2 a210 , and ins1 b514 peptides ) . the next day , recovered cd8 t - cells were cocultured for 48 h with 1
mol / l of each of the individual ins or igrp peptides , and antigen reactivity was assessed by elispot analyses of ifn- production .
b : cd8 t - cells from nod.2m.hhd mice were incubated with syngeneic flu - sps , igrp - sps ( mixture of igrp265273 and igrp228236 ) , or ins - sps ( mixture of ins1/2 a210 and ins1 b514 ) .
t - cell reactivity to individual igrp or ins peptides was then determined as described above .
p values are based on comparison with flu - sps . * p < 0.01 , * * p < 0.05 ; p values ins - sps compared with igrp - sps , * * * p < 0.01 , # # p < 0.001 .
initial analyses found that repeated injections of a soluble mixture of the four ins and igrp peptides ( 25 g each ) did not inhibit diabetes development in nod.2 m .hhd mice ( data not shown ) .
thus , given the in vitro results shown in fig . 1 , we assessed whether a single intravenous injection of ins / igrp - sps given at 46 weeks of age could protect nod.2m.hhd female mice from diabetes development .
diabetes development was inhibited in mice treated with the ins / igrp - sps , compared with controls receiving flu - sps ( fig .
nod.2m.hhd mice were injected with syngeneic splenocytes separately bearing either a mixture of the two ecdi cross - linked ins ( ins - sps ) or igrp ( igrp - sps ) peptides . because one injection of ins / igrp - sps initially afforded a partial diabetes protective effect , treatments with ins - sps and igrp - sps
were repeated at 5-week intervals . compared with controls receiving flu - sps , only igrp - sps and not ins - sps exerted a diabetes protective effect in nod.2m.hhd mice ( fig .
treatments with syngeneic splenocytes separately bearing either the ecdi - coupled igrp228236 or igrp265273 peptide failed to significantly inhibit diabetes development in nod.2m.hhd mice ( fig .
thus , splenocytes must bear a combination of the ecdi - coupled igrp228236 and igrp265273 peptides in order to elicit robust diabetes protective effects in nod.2m.hhd mice .
furthermore , although both ins / igrp - sps and igrp - sps treatment could inhibit overt diabetes development in nod.2m.hhd mice , neither intervention significantly suppressed insulitis levels ( fig .
nod.2m.hhd female mice were injected intravenously at 46 weeks of age with ins / igrp - sps ( bearing the ecdi - linked mixture of igrp265273 , igrp228236 , ins1/2 a210 , and ins1 b514 peptides ) .
syngeneic splenocytes bearing a mixture of ecdi - coupled igrp , but not ins peptides , inhibit diabetes but not insulitis development in nod.2m.hhd mice .
beginning at 46 weeks of age , nod.2m.hhd female mice were injected intravenously at 5-week intervals with either igrp - sps , ins - sps , igrp265273-sps , or igrp228236-sps .
igrp - sps developed diabetes at a significantly lower rate ( p = 0.04 ) than flu - sps  treated controls .
b : percentage of nod.2m.hhd mice in each treatment group with the indicated range of insulitis scores .
we assessed whether the diabetes protective effect of ins / igrp - sps treatment was associated with altered in vivo responsiveness of either hla - a2.1restricted ins or igrp autoreactive cd8 t - cells in nod.2m.hhd mice .
two days following a third ins / igrp - sps or flu - sps treatment given at 5-week intervals , nod.2m.hhd mice were primed in the footpad with a cocktail of the four ins and igrp peptides .
ten days postpriming , the mice were killed and cd8 t - cells within the draining popliteal lymph nodes were assessed for reactivity to each individual peptide by ifn- elispot analyses .
all mice were still nondiabetic at the time of analysis . in ins / igrp - sps
 treated nod.2m.hhd mice , cd8 t - cell responses to both the hla - a2.1restricted igrp228236 and igrp265273 peptides but neither ins epitope were significantly decreased ( p = 0.03 and 0.004 ) ( fig .
4 ) . treatment with igrp - sps but not with ins - sps also showed significantly decreased cd8 t - cell responses to both the igrp228236 and igrp265273 peptides ( p = 0.05 and 0.002 ) ( fig .
these results further indicate that in the context of the human hla - a2.1 class i variant , autoreactive cd8 t - cells recognizing the igrp228236 and/or igrp265273 peptides are of significant pathogenic importance during diabetes development in nod.2m.hhd mice .
ins / igrp - sps and igrp - sps inhibit in vivo responses of hla - a2.1restricted igrp but not ins autoreactive cd8 t - cells .
beginning at 46 weeks of age , nod.2m.hhd female mice were injected intravenously with ins / igrp - sps , igrp - sps , ins - sps , or flu - sps at 5-week intervals .
two days after the third treatment , mice were footpad primed with a mixture of the four ins / igrp peptides .
ten days after priming , cd8 t - cells within the draining popliteal lymph nodes were cocultured for 48 h with 1
previous studies have indicated that ( pro)insulin is a key autoantigen for diabetes development in standard nod mice ( 27,3033 ) .
this was partly demonstrated by studies showing that mhc class ii promoter  driven transgenic expression of proinsulin-2 in apcs of nod mice inhibits insulitis and diabetes development ( 27,30 ) .
hence , we used such a transgenic approach as an alternative means to test whether ( pro)insulin may also be an autoantigen of pathogenic importance in nod.2m.hhd mice .
nod.2m.hhd mice expressing the previously described ( 27 ) proinsulin-2 transgene in apcs ( designated nod.2m.hhd-pi ) were generated and assessed for diabetes development .
5a , compared with nontransgenic controls , the rate of type 1 diabetes development was somewhat retarded in nod.2m.hhd-pi female mice but did not quite achieve statistical significance ( p = 0.08 ) by kaplan - meier analyses .
however , as assessed by  analyses , the cumulative frequency of diabetes development by 35 weeks of age was significantly lower ( p < 0.005 ) in nod.2m.hhd-pi mice than in nontransgenic controls .
insulitis levels were also significantly lower in nod.2m.hhd-pi mice than in nontransgenic controls ( fig .
5b ) . baseline and primed levels of cd8 t - cell responses to the hla - a2.1restricted ins1/2 a210 or ins1 b514 epitopes were found not to differ in nod.2m.hhd-pi mice and nontransgenic controls ( data not shown ) .
these findings do not eliminate the possibility that like those targeting igrp epitopes , hla - a2.1restricted insulin autoreactive cd8 t - cells are also important pathogenic contributors to diabetes development in nod.2m.hhd mice .
however , these apc transgenic expression studies indicate that even if they do not represent autoantigens recognized by pathogenic hla - a2.1restricted cd8 t - cells , ( pro)insulin derived epitopes are important targets of at least diabetogenic cd4 t - cells in nod.2m.hhd mice .
insulitis and diabetes development in nod.2m.hhd-pi mice . a : incidence curve of diabetes development in nod.2m.hhd-pi female mice and nontransgenic littermates .
the rate of diabetes development did not quite achieve a statistical difference ( p = 0.08 ) by kaplan - meier analyses .
the cumulative frequency of diabetes development by 35 weeks of age was significantly different ( p < 0.005 ) as assessed by  analyses .
b : histological grading of insulitis in pancreas sections of 35-week - old female mice ( nod.2m.hhd-pi mice , n = 14 ; nod.2m.hhd mice , n = 16 ) .
it has been previously reported that splenocytes bearing ecdi - coupled proteins or peptides do not directly induce tolerogenic responses by cd4 t - cells in an efficient manner , but rather do so indirectly following their uptake and processing by host - type apc ( 21 ) . if this is also the case for inducing cd8 t - cell tolerance , we reasoned it should remain possible to inhibit diabetes development in the hla - a2.1expressing nod.2m.hhd stock by treatments with splenocytes from totally mhc class i 
deficient nod.2 m mice bearing the complete cocktail of ecdi cross - linked ins and igrp epitopes ( designated ins / igrp-2 m sps ) . unlike those receiving flu-2 m sps , diabetes development was completely abrogated in ins / igrp-2 m sps - treated nod.2m.hhd mice ( fig .
ins / igrp - sps do not have to share host - type mhc class i identity to attenuate diabetes development in nod.2m.hhd mice . beginning at 46 weeks of age nod.2m.hhd mice were injected intravenously at 5-week intervals with ins / igrp-2 m sps or flu-2 m sps .
b : ins / igrp-2 m sps inhibit in vivo responses of hla - a2.1restricted igrp but not ins autoreactive cd8 t - cells in nod.2m.hhd mice .
two days after the third treatment , mice were footpad primed with a mixture of the four ins / igrp peptides .
ten days after priming , cd8 t - cells within the draining popliteal lymph nodes of nondiabetic mice were cocultured for 48 h with 1
mol / l of each of the individual ins or igrp peptides , and antigen reactivity was assessed by elispot analyses of ifn- production .
two days after a third ins / igrp-2 m sps or flu-2 m sps treatment given at 5-week intervals , nod.2m.hhd mice were primed in the footpad with a cocktail of the four ins and igrp peptides .
ten days postpriming , cd8 t - cells within the draining popliteal lymph nodes were assessed for reactivity to each individual peptide by ifn- elispot analyses .
cd8 t - cell responses to both the hla - a2.1restricted igrp228236 and igrp265273 peptides but again not the ins epitopes were significantly decreased in ins / igrp-2 m sps - treated nod.2m.hhd mice ( fig .
these collective results indicate that in order to induce cd8 t - cell tolerance and to elicit diabetes protective effects in nod.2m.hhd recipients , donor cells bearing ecdi cross - linked hla - a2.1 restricted igrp autoantigenic peptides do not also have to express the relevant host - type mhc class i variant .
the results described above also indicated that rather than directing inducing tolerogenic responses inhibiting diabetogenic cd8 cell activity in nod.2m.hhd mice , ins / igrp - sps or igrp - sps instead do so in an indirect manner dependent on their uptake by host - type apc . to directly test this possibility , we compared the ability of ecdi - treated or untreated donor cells that were also labeled with the carboxyfluorescein diacetate succinimidyl ester ( cfse ) tracker dye to be taken up by host - type antigen - presenting dendritic cells ( dcs ) .
donor- and recipient - type cells were also distinguished by respective expression of the cd45.2 versus cd45.1 pan - leukocyte marker variant .
there was a significantly greater uptake by host - type splenic cd8 + dcs of ecdi - treated than untreated donor cells that had been intravenously infused 1 day earlier ( fig .
this likely explains why fewer ecdi - treated than untreated donor cells could be detected in the recipients , but with the apoptotic status of the former also allowing them to be more efficiently engulfed by host - type dcs .
these results indicate the ability of donor cells bearing ecdi - coupled igrp peptides to elicit diabetes protective effects likely entails their uptake by host - type dcs that then display the autoantigenic epitopes to cd8 t - cells in a tolerance - inducing manner .
more efficient uptake by host - type splenic cd8 + dcs of ecdi - treated than untreated donor cells .
splenocytes from nod.cd45.2 congenic mice were labeled with the cfse tracker dye and then subsequently treated or not with ecdi before being infused intravenously into standard nod cd45.1-expressing recipients .
the following day , viable host - type splenic dcs were assessed by flow cytometry for comparative uptake of cfse - labeled ecdi - treated or untreated donor cells .
a : depiction of gating strategy to assess uptake by viable ( propidium iodide negative ) host - type cd45.1-expressing splenic dc subsets of cfse - labeled donor - type leukocytes that had or had not been treated with ecdi .
b : mean proportions  sem of host - type splenic cd8 + dcs that had taken up cfse - labeled ecdi - treated or untreated donor - type leukocytes ( n = 3/group ) .
cd8 t - cells were enriched from pooled spleens of 9- to 16-week - old nod.2m.hhd mice and cocultured in vitro with syngeneic splenocytes bearing an ecdi - coupled cocktail of the hla - a2.1restricted ins1/2 a210 , ins1 b515 , igrp228236 , and igrp265273 -cell autoantigens ( ins / igrp - sps ) and assessed the next day for responsiveness to each individual epitope by ifn- elispot analyses . compared with those exposed to the control flu16 peptide ( flu - sps ) , cd8 t - cells from nod.2m.hhd mice cocultured with ins
/ igrp - sps displayed significantly decreased responses to restimulation by igrp epitopes ( fig .
levels of reactivity to the hla - a2.1restricted ins peptides was low among control cd8 t - cells exposed to flu16 and not further influenced by previous coculture with ins / igrp - sps ( fig .
coincubation with syngeneic splenocytes bearing an ecdi cross - linked mixture of the two ins peptides ( ins - sps ) did not diminish cd8 t - cell responsiveness to ins or igrp epitopes ( fig .
however , preincubation with igrp - sps did significantly diminish the ability of cd8 t - cells from nod.2m.hhd mice to respond to restimulation by igrp but not ins epitopes ( fig .
il-10 elispot analyses also indicated that regardless of preincubation conditions , no cd8 t - cells from nod.2m.hhd mice responding to ins or igrp stimulation produced this immunosuppressive cytokine . on the basis of these collective data
, we subsequently assessed syngeneic ins / igrp - sps as a possible diabetes intervention approach in nod.2m.hhd mice .
hla - a2.1restricted -cell antigenic peptides that are ecdi cross - linked to syngeneic splenocytes significantly diminish responsiveness by cognate cd8 t - cells from nod.2m.hhd mice .
a : cd8 t - cells from nod.2m.hhd mice were incubated with syngeneic flu - sps or ins / igrp - sps ( bearing the complete mixture of igrp265273 , igrp228236 , ins1/2 a210 , and ins1 b514 peptides ) . the next day , recovered cd8 t - cells were cocultured for 48 h with 1
mol / l of each of the individual ins or igrp peptides , and antigen reactivity was assessed by elispot analyses of ifn- production .
b : cd8 t - cells from nod.2m.hhd mice were incubated with syngeneic flu - sps , igrp - sps ( mixture of igrp265273 and igrp228236 ) , or ins - sps ( mixture of ins1/2 a210 and ins1 b514 ) .
t - cell reactivity to individual igrp or ins peptides was then determined as described above .
p values are based on comparison with flu - sps . * p < 0.01 , * * p < 0.05 ; p values ins - sps compared with igrp - sps , * * *
initial analyses found that repeated injections of a soluble mixture of the four ins and igrp peptides ( 25 g each ) did not inhibit diabetes development in nod.2 m .hhd mice ( data not shown ) .
thus , given the in vitro results shown in fig . 1 , we assessed whether a single intravenous injection of ins / igrp - sps given at 46 weeks of age could protect nod.2m.hhd female mice from diabetes development .
diabetes development was inhibited in mice treated with the ins / igrp - sps , compared with controls receiving flu - sps ( fig .
nod.2m.hhd mice were injected with syngeneic splenocytes separately bearing either a mixture of the two ecdi cross - linked ins ( ins - sps ) or igrp ( igrp - sps ) peptides . because one injection of ins / igrp - sps initially afforded a partial diabetes protective effect , treatments with ins - sps and igrp - sps
were repeated at 5-week intervals . compared with controls receiving flu - sps , only igrp - sps and not ins - sps exerted a diabetes protective effect in nod.2m.hhd mice ( fig .
treatments with syngeneic splenocytes separately bearing either the ecdi - coupled igrp228236 or igrp265273 peptide failed to significantly inhibit diabetes development in nod.2m.hhd mice ( fig .
thus , splenocytes must bear a combination of the ecdi - coupled igrp228236 and igrp265273 peptides in order to elicit robust diabetes protective effects in nod.2m.hhd mice .
furthermore , although both ins / igrp - sps and igrp - sps treatment could inhibit overt diabetes development in nod.2m.hhd mice , neither intervention significantly suppressed insulitis levels ( fig .
nod.2m.hhd female mice were injected intravenously at 46 weeks of age with ins / igrp - sps ( bearing the ecdi - linked mixture of igrp265273 , igrp228236 , ins1/2 a210 , and ins1 b514 peptides ) .
syngeneic splenocytes bearing a mixture of ecdi - coupled igrp , but not ins peptides , inhibit diabetes but not insulitis development in nod.2m.hhd mice .
beginning at 46 weeks of age , nod.2m.hhd female mice were injected intravenously at 5-week intervals with either igrp - sps , ins - sps , igrp265273-sps , or igrp228236-sps .
a : mice were monitored for diabetes development . only mice receiving igrp - sps developed diabetes at a significantly lower rate ( p = 0.04 ) than flu - sps  treated controls .
b : percentage of nod.2m.hhd mice in each treatment group with the indicated range of insulitis scores .
we assessed whether the diabetes protective effect of ins / igrp - sps treatment was associated with altered in vivo responsiveness of either hla - a2.1restricted ins or igrp autoreactive cd8 t - cells in nod.2m.hhd mice .
two days following a third ins / igrp - sps or flu - sps treatment given at 5-week intervals , nod.2m.hhd mice were primed in the footpad with a cocktail of the four ins and igrp peptides .
ten days postpriming , the mice were killed and cd8 t - cells within the draining popliteal lymph nodes were assessed for reactivity to each individual peptide by ifn- elispot analyses .
in ins / igrp - sps  treated nod.2m.hhd mice , cd8 t - cell responses to both the hla - a2.1restricted igrp228236 and igrp265273 peptides but neither ins epitope were significantly decreased ( p = 0.03 and 0.004 ) ( fig .
4 ) . treatment with igrp - sps but not with ins - sps also showed significantly decreased cd8 t - cell responses to both the igrp228236 and igrp265273 peptides ( p = 0.05 and 0.002 ) ( fig .
these results further indicate that in the context of the human hla - a2.1 class i variant , autoreactive cd8 t - cells recognizing the igrp228236 and/or igrp265273 peptides are of significant pathogenic importance during diabetes development in nod.2m.hhd mice .
ins / igrp - sps and igrp - sps inhibit in vivo responses of hla - a2.1restricted igrp but not ins autoreactive cd8 t - cells .
beginning at 46 weeks of age , nod.2m.hhd female mice were injected intravenously with ins / igrp - sps , igrp - sps , ins - sps , or flu - sps at 5-week intervals .
two days after the third treatment , mice were footpad primed with a mixture of the four ins / igrp peptides .
ten days after priming , cd8 t - cells within the draining popliteal lymph nodes were cocultured for 48 h with 1
previous studies have indicated that ( pro)insulin is a key autoantigen for diabetes development in standard nod mice ( 27,3033 ) .
this was partly demonstrated by studies showing that mhc class ii promoter  driven transgenic expression of proinsulin-2 in apcs of nod mice inhibits insulitis and diabetes development ( 27,30 ) .
hence , we used such a transgenic approach as an alternative means to test whether ( pro)insulin may also be an autoantigen of pathogenic importance in nod.2m.hhd mice .
nod.2m.hhd mice expressing the previously described ( 27 ) proinsulin-2 transgene in apcs ( designated nod.2m.hhd-pi ) were generated and assessed for diabetes development . as shown in fig .
5a , compared with nontransgenic controls , the rate of type 1 diabetes development was somewhat retarded in nod.2m.hhd-pi female mice but did not quite achieve statistical significance ( p = 0.08 ) by kaplan - meier analyses .
however , as assessed by  analyses , the cumulative frequency of diabetes development by 35 weeks of age was significantly lower ( p < 0.005 ) in nod.2m.hhd-pi mice than in nontransgenic controls .
insulitis levels were also significantly lower in nod.2m.hhd-pi mice than in nontransgenic controls ( fig .
5b ) . baseline and primed levels of cd8 t - cell responses to the hla - a2.1restricted ins1/2 a210 or ins1 b514 epitopes were found not to differ in nod.2m.hhd-pi mice and nontransgenic controls ( data not shown ) .
these findings do not eliminate the possibility that like those targeting igrp epitopes , hla - a2.1restricted insulin autoreactive cd8 t - cells are also important pathogenic contributors to diabetes development in nod.2m.hhd mice .
however , these apc transgenic expression studies indicate that even if they do not represent autoantigens recognized by pathogenic hla - a2.1restricted cd8 t - cells , ( pro)insulin derived epitopes are important targets of at least diabetogenic cd4 t - cells in nod.2m.hhd mice .
insulitis and diabetes development in nod.2m.hhd-pi mice . a : incidence curve of diabetes development in nod.2m.hhd-pi female mice and nontransgenic littermates .
the rate of diabetes development did not quite achieve a statistical difference ( p = 0.08 ) by kaplan - meier analyses .
the cumulative frequency of diabetes development by 35 weeks of age was significantly different ( p < 0.005 ) as assessed by  analyses .
b : histological grading of insulitis in pancreas sections of 35-week - old female mice ( nod.2m.hhd-pi mice , n = 14 ; nod.2m.hhd mice , n = 16 ) .
it has been previously reported that splenocytes bearing ecdi - coupled proteins or peptides do not directly induce tolerogenic responses by cd4 t - cells in an efficient manner , but rather do so indirectly following their uptake and processing by host - type apc ( 21 ) . if this is also the case for inducing cd8 t - cell tolerance , we reasoned it should remain possible to inhibit diabetes development in the hla - a2.1expressing nod.2m.hhd stock by treatments with splenocytes from totally mhc class i 
deficient nod.2 m mice bearing the complete cocktail of ecdi cross - linked ins and igrp epitopes ( designated ins / igrp-2 m sps ) . unlike those receiving flu-2 m sps , diabetes development was completely abrogated in ins / igrp-2 m sps - treated nod.2m.hhd mice ( fig .
ins / igrp - sps do not have to share host - type mhc class i identity to attenuate diabetes development in nod.2m.hhd mice . beginning at 46 weeks of age nod.2m.hhd mice were injected intravenously at 5-week intervals with ins / igrp-2 m sps or flu-2 m sps .
b : ins / igrp-2 m sps inhibit in vivo responses of hla - a2.1restricted igrp but not ins autoreactive cd8 t - cells in nod.2m.hhd mice .
two days after the third treatment , mice were footpad primed with a mixture of the four ins / igrp peptides .
ten days after priming , cd8 t - cells within the draining popliteal lymph nodes of nondiabetic mice were cocultured for 48 h with 1 mol / l of each of the individual ins or igrp peptides , and antigen reactivity was assessed by elispot analyses of ifn- production .
two days after a third ins / igrp-2 m sps or flu-2 m sps treatment given at 5-week intervals , nod.2m.hhd mice were primed in the footpad with a cocktail of the four ins and igrp peptides .
ten days postpriming , cd8 t - cells within the draining popliteal lymph nodes were assessed for reactivity to each individual peptide by ifn- elispot analyses .
cd8 t - cell responses to both the hla - a2.1restricted igrp228236 and igrp265273 peptides but again not the ins epitopes were significantly decreased in ins / igrp-2 m sps - treated nod.2m.hhd mice ( fig .
these collective results indicate that in order to induce cd8 t - cell tolerance and to elicit diabetes protective effects in nod.2m.hhd recipients , donor cells bearing ecdi cross - linked hla - a2.1 restricted igrp autoantigenic peptides do not also have to express the relevant host - type mhc class i variant .
the results described above also indicated that rather than directing inducing tolerogenic responses inhibiting diabetogenic cd8 cell activity in nod.2m.hhd mice , ins / igrp - sps or igrp - sps instead do so in an indirect manner dependent on their uptake by host - type apc . to directly test this possibility , we compared the ability of ecdi - treated or untreated donor cells that were also labeled with the carboxyfluorescein diacetate succinimidyl ester ( cfse ) tracker dye to be taken up by host - type antigen - presenting dendritic cells ( dcs ) .
donor- and recipient - type cells were also distinguished by respective expression of the cd45.2 versus cd45.1 pan - leukocyte marker variant .
there was a significantly greater uptake by host - type splenic cd8 + dcs of ecdi - treated than untreated donor cells that had been intravenously infused 1 day earlier ( fig .
this likely explains why fewer ecdi - treated than untreated donor cells could be detected in the recipients , but with the apoptotic status of the former also allowing them to be more efficiently engulfed by host - type dcs .
these results indicate the ability of donor cells bearing ecdi - coupled igrp peptides to elicit diabetes protective effects likely entails their uptake by host - type dcs that then display the autoantigenic epitopes to cd8 t - cells in a tolerance - inducing manner .
more efficient uptake by host - type splenic cd8 + dcs of ecdi - treated than untreated donor cells .
splenocytes from nod.cd45.2 congenic mice were labeled with the cfse tracker dye and then subsequently treated or not with ecdi before being infused intravenously into standard nod cd45.1-expressing recipients .
the following day , viable host - type splenic dcs were assessed by flow cytometry for comparative uptake of cfse - labeled ecdi - treated or untreated donor cells .
a : depiction of gating strategy to assess uptake by viable ( propidium iodide negative ) host - type cd45.1-expressing splenic dc subsets of cfse - labeled donor - type leukocytes that had or had not been treated with ecdi .
b : mean proportions  sem of host - type splenic cd8 + dcs that had taken up cfse - labeled ecdi - treated or untreated donor - type leukocytes ( n = 3/group ) .
our results indicate that treatment with ins / igrp - sps ( bearing an ecdi cross - linked mixture of the ins1/2 a210 , ins1 b515 , igrp228236 , and/or igrp265273 peptides ) inhibits diabetes development in  humanized 
we found igrp - sps to be more effective than ins - sps in inhibiting diabetes development in nod.2m.hhd mice .
furthermore , the inhibition of diabetes development in nod.2m.hhd mice by ins / igrp - sps treatment was associated with an attenuation of igrp but not ins - specific autoreactive cd8 t - cell responses .
treatment with igrp228236 or igrp265273 single peptide - sps did not significantly inhibit diabetes development in nod.2m.hhd mice .
this indicated that cd8 t - cell tolerance must be established to both the hla - a2.1restricted igrp228236 and igrp265273 epitopes in order to elicit diabetes - protective effects in nod.2m.hhd mice . in standard nod mice
, insulin appears to be an earlier target than igrp of cd4 and cd8 t - cells initiating diabetes development ( 3133 ) .
it is unclear why ins / igrp - sps or ins - sps treatment did not alter levels of hla - a2.1restricted cd8 responses against the ins1/2 a210 or ins1 b514 epitopes in nod.2m.hhd mice .
however , nod.2m.hhd mice transgenically expressing proinsulin-2 in apcs were largely protected from insulitis and diabetes development .
these apc transgenic studies also indicated that even if diabetes development in nod.2m.hhd mice does not require hla - a2.1restricted cd8 responses targeting ( pro)insulin epitopes , an important component of their disease susceptibility still entails cd4 t - cell responses against this pancreatic -cell antigen .
it has been previously reported that although peptide - sps can directly induce cd4 t - cell tolerance induction processes in an inefficient manner , they do so more efficiently through an indirect mechanism involving their uptake and processing by host - type apcs ( 25 ) . because of these alternative mechanisms , mhc compatibility between splenotype donor and host is not required in order to induce cd4 t - cell tolerance to ecdi - coupled antigens , although syngeneic donor cells are more efficient at doing so .
we found ins / igrp peptides ecdi coupled to completely mhc class i  deficient donor splenocytes strongly inhibited igrp - specific cd8 t - cell responses and diabetes development in nod.2m.hhd mice .
hence , donor / host mhc class i compatibility is not required to efficiently induce cd8 t - cell tolerance to ecdi - coupled self - antigenic peptides .
these findings indicate that igrp - sps inhibit diabetes development in nod.2m.hhd mice by inducing cd8 t - cell tolerance through an indirect host - type apc - dependent pathway .
indeed , other data indicate that host - type dcs more efficiently take up donor ecdi - treated than untreated leukocytes . a review by luo et al .
( 21 ) discusses efforts by the immune tolerance network to develop a clinical trial using ecdi insulin - coupled peripheral blood lymphocytes as a possible diabetes intervention in humans .
our current results indicate that the use of  humanized  nod.2m.hhd mice and other related strains may facilitate the development of clinically translatable peptide - based therapies for diabetic patients .
humanized  mouse resources make it possible to determine which autoantigenic peptides when ecdi cross - linked to autologous leukocytes are most likely to attenuate hla - a2.1restricted cd8 t - cell responses that recent evidence ( 1016 ) indicates may be important for diabetes development in many human patients .
 objectivetype 1 diabetes can be inhibited in standard nod mice by autoantigen - specific immunotherapy targeting pathogenic cd8 + t - cells . 
 nod.2mnull.hhd mice expressing human hla - a2.1 but lacking murine major histocompatibility complex class i molecules develop diabetes characterized by cd8 t - cells recognizing certain autoantigenic peptides also targeted in human patients . 
 these include peptides derived from the pancreatic -cell proteins insulin ( ins1/2 a210 and ins1 b514 ) and islet - specific glucose-6-phosphatase catalytic subunit  related protein ( igrp265273 and igrp228236 ) . 
 hence , nod.2mnull.hhd mice represent a model system for developing potentially clinically translatable interventions for suppressing diabetogenic hla - a2.1restricted t - cell responses.research design and methodsstarting at 46 weeks of age , nod.2mnull.hhd female mice were injected intravenously with syngeneic splenocytes to which various admixtures of the four above - mentioned peptides were bound by the cross - linking agent ethylene carbodiimide ( ecdi).resultstreatment with such cells bearing the complete cocktail of ins and igrp epitopes ( designated ins / igrp - sps ) significantly inhibited diabetes development in nod.2mnull.hhd recipients compared with controls receiving splenocytes coupled with an irrelevant hla - a2.1restricted flu16 peptide . 
 subsequent analyses found syngeneic splenocytes bearing the combination of the two ecdi - coupled igrps but not ins peptides ( igrp - sps or ins - sps ) effectively inhibited diabetes development in nod.2mnull.hhd mice . 
 this result was supported by enzyme - linked immunospot ( elispot ) analyses indicating combined ins / igrp - sps diminished hla - a2.1restricted igrp but not ins autoreactive cd8 + t - cell responses in nod.2mnull.hhd mice.conclusionsthese data support the potential of a cell therapy approach targeting hla - a2.1restricted igrp autoreactive cd8 t - cells as a diabetes intervention approach in appropriate human patients . 
RESEARCH DESIGN AND METHODS
Mice.
Peptides and antibodies.
ECDI peptide-coupled cell treatment.
Assessment of diabetes and insulitis development.
Enzyme-linked immunospot assays, CD8 T-cell culture, and in vivo T-cell priming.
In vitro assessment of peptide-SP effects on diabetogenic CD8 T-cells.
Soluble peptide treatment.
Statistical analyses.
RESULTS
Splenocytes bearing ECDI-coupled peptides ablate IGRP-specific CD8 T-cell responses in vitro.
Syngeneic splenocytes bearing ECDI-coupled autoantigenic IGRP, but not INS peptides, inhibit diabetes development in NOD.
INS/IGRP-SPs and IGRP-SPs inhibit in vivo responses of HLA-A2.1restricted IGRP but not INS autoreactive CD8 T-cells in NOD.
Proinsulin is a pathogenic autoantigen in NOD.
INS/IGRP-SPs do not have to share host MHC class I identity to tolerize IGRP-specific CD8 T-cells and to attenuate diabetes development in NOD.
DISCUSSION