nod mice were purchased from taconic farms ( germantown , ny ) or bred in the university of toledo animal care facility . nod.bdc2.5 t - cell receptor ( tcr ) transgenic ( tg ) mice were purchased from jackson laboratories ( bar harbor , me ) . the el4 cell line ( atcc , manassas , va ) was cultured in complete medium ( rpmi-1640 supplemented with 10% horse serum ) . gad65 peptides 524 - 543 , 524 - 538 ( p524 ) , and 530 - 543 ( p530 ) , and hen egg white lysozyme peptide hel 1125 were synthesized as described previously ( 9 ) . the p524-reactive t - cell clone gad35za was isolated from peptide - immunized mice ( 9 ) . briefly , spleen and lymph node cells from nod mice immunized with p524 - 538 ( 20 g of peptide p524 in complete freund 's adjuvant ) were challenged in vitro with cognate peptide , and expanded with recombinant human il-2 ( ril-2 ) . t - cell clones were isolated by limiting dilution , and maintained by cycles of restimulation with p524-pulsed irradiated syngeneic spleen cells and expansion with ril-2 . to enumerate endogenously primed p530-specific t cells via enzyme - linked immunospot ( elispot ) , spleen cells from nave female nod mice were cultured in 24-well plates with gad65 peptide 524 - 543 as previously described ( 15 ) . alternatively , to detect responses by enzyme - linked immunosorbent assay ( elisa ) , spleen cells collected from the 24-well plates were resuspended in complete medium and added to 96-well round - bottom plates , with or without gad65 peptide p530 . twenty - four to 72 h later , the culture supernatants were collected and tested for the presence of cytokines ( 9 ) . to evaluate p524 responses in vivo , 2- to 3-week - old nod mice were immunized , once or twice , in the peritoneum with 100 g of p524 , hel 1125 , or pbs , emulsified in incomplete freund 's adjuvant ( ifa ) . four or 9 weeks later , lymphoid tissues were collected and analyzed by elispot or elisa , or flow cytometry , respectively . for transwell experiments , gad35za clonotypic cells ( 5  10 cells / ml ) were coplated with irradiated syngeneic spleen cells ( 4  10 cells / ml ) in the upper chamber of 24-transwell plates , and spleen cells from nave 6- to 8-week - old nod mice were plated at 1  10cells / ml in the lower chamber . monoclonal antibody ( 10 g / ml ) to -interferon ( ifn- ) ( r46a2 ) , il-5 ( trfk5 ) , or il-13 ( mab 413 ) was added to the upper wells ; pbs or rat igg ( 10 g / ml ) was added to control wells . gad65 peptide 524 - 543 was then added to all upper chambers at a final concentration of 10 g / ml . three days later , the spleen cells in the lower chamber were recovered and ifn- spot - forming cells enumerated in duplicate wells by elispot . nod or nod.bdc2.5.tcr.tg spleen cells were labeled with anti - cd4conjugated magnetic beads ( miltenyi biotech , auburn , ca ) and purified according to the manufacturer s instructions . cd4 t - cell enrichment ( > 95% ) was verified by flow cytometry . to assess the effect of ril-13 on cd4 t - cell proliferation , purified nod or bdc2.5.tcr.tg cd4 t cells ( 6  10 cells / well ) were cultured on anti - cd3coated 96-well plates for 72 h in the presence or absence of ril-13 ( r&d systems , minneapolis , mn ) . pbs - coated wells served as negative controls . for the last 18 h of culture , wells were pulse - labeled with 1ci / well h - thymidine ( mp biomedicals , solon , oh ) , harvested , and counted as previously described ( 16 ) . the expression of il-4 , il-10 , transforming growth factor ( tgf)- , and -actin was evaluated in gad35za t cells via rt - pcr . kits containing primers for il-10 , tgf- , and -actin were purchased from r&d systems . primers for il-4 ( forward catcggcattttgaacgaggtca , and reverse cttatcgatgaatccaggcatcg ) were purchased from operon ( alameda , ca ) . briefly , gad35za t cells were stimulated with irradiated nod spleen cells in the presence of p524 ( 10 g / ml ) for 24 to 48 h. mrna was harvested with an rneasy kit ( invitrogen , carlsbad , ca ) and used as a template for complimentary dna ( cdna ) synthesis . rt - pcr was performed according to the manufacturer s instructions for the primer kits , using platinum taq polymerase ( invitrogen ) . for analysis of il-13r1 expression , cd4 t cells purified from nod or nod.bdc2.5.tcr.tg splenocytes were stimulated on anti - cd3coated plates for 0 , 24 , 48 , or 72 h. the cells were harvested , and mrna was extracted with trizol ( invitrogen ) and converted to cdna . cdna was used for quantitative real - time rt - pcr using iq sybr green supermix ( bio - rad , hercules , ca ) . normalized results using the  ct method of quantification are expressed as % gapdh . the pcr primer sequences were as follows : gapdh forward ccaggttgtctcctgcgact , reverse ataccaggaaatgagcttgacaaagt ; il-13r1 forward gaatttgagcgtctctgtcgaa , reverse ggttatgccaaatgcacttgag ; il-4r forward agtgagtggagtcctagcatc , reverse gctgaagtaacagaacaggc . for direct ex vivo analysis of il-13r1 expression on t - cell populations , spleens , inguinal lymph nodes ( ilns ) , and plns were harvested from age - matched mice and stained with anti  tcr - phycoerythrin ( pe)/cy5 ( biolegend ) , anti  cd4-pe ( biolegend ) , and purified goat anti il-13r1 ( santa cruz biotechnology , santa cruz , ca ) or purified goat igg . goat igg - fluoroisothiocyanate ( southern biotech , birmingham , al ) , washed , and analyzed by flow cytometry . for analysis of il-13 signaling , plns from 4-week - old female nod mice were harvested and stained with anti  tcr - fluoroisothiocyanate ( biolegend ) , serum - starved in rpmi for 1 h , and then exposed to ril-13 for 30 min at 37c . the cells were washed , fixed , permeabilized , and then stained with an antibody to phosphorylated signal transducer and activator of transcription ( stat ) 6 ( santa cruz biotechnology ) or goat isotype control , followed by biotinylated donkey anti the pancreas was collected at the same time as lymphoid tissue harvest for il-13r1 evaluation . the pancreas was fixed in 10% formalin , sectioned ( 5 micron sections , 20 microns apart ) , and stained with hematoxylin and eosin . islets were graded as normal or as having peri - insulitis ( inflammation only around the periphery of the islet ) or invasive insulitis ( inflammation was greater than 33% of the islet and disrupted the typical round / oval shape ) . nod mice were purchased from taconic farms ( germantown , ny ) or bred in the university of toledo animal care facility . nod.bdc2.5 t - cell receptor ( tcr ) transgenic ( tg ) mice were purchased from jackson laboratories ( bar harbor , me ) . the el4 cell line ( atcc , manassas , va ) was cultured in complete medium ( rpmi-1640 supplemented with 10% horse serum ) . gad65 peptides 524 - 543 , 524 - 538 ( p524 ) , and 530 - 543 ( p530 ) , and hen egg white lysozyme peptide hel 1125 were synthesized as described previously ( 9 ) . the p524-reactive t - cell clone gad35za was isolated from peptide - immunized mice ( 9 ) . briefly , spleen and lymph node cells from nod mice immunized with p524 - 538 ( 20 g of peptide p524 in complete freund 's adjuvant ) were challenged in vitro with cognate peptide , and expanded with recombinant human il-2 ( ril-2 ) . t - cell clones were isolated by limiting dilution , and maintained by cycles of restimulation with p524-pulsed irradiated syngeneic spleen cells and expansion with ril-2 . to enumerate endogenously primed p530-specific t cells via enzyme - linked immunospot ( elispot ) , spleen cells from nave female nod mice were cultured in 24-well plates with gad65 peptide 524 - 543 as previously described ( 15 ) . alternatively , to detect responses by enzyme - linked immunosorbent assay ( elisa ) , spleen cells collected from the 24-well plates were resuspended in complete medium and added to 96-well round - bottom plates , with or without gad65 peptide p530 . twenty - four to 72 h later , the culture supernatants were collected and tested for the presence of cytokines ( 9 ) . to evaluate p524 responses in vivo , 2- to 3-week - old nod mice were immunized , once or twice , in the peritoneum with 100 g of p524 , hel 1125 , or pbs , emulsified in incomplete freund 's adjuvant ( ifa ) . four or 9 weeks later , lymphoid tissues were collected and analyzed by elispot or elisa , or flow cytometry , respectively . for transwell experiments , gad35za clonotypic cells ( 5  10 cells / ml ) were coplated with irradiated syngeneic spleen cells ( 4  10 cells / ml ) in the upper chamber of 24-transwell plates , and spleen cells from nave 6- to 8-week - old nod mice were plated at 1  10cells / ml in the lower chamber . monoclonal antibody ( 10 g / ml ) to -interferon ( ifn- ) ( r46a2 ) , il-5 ( trfk5 ) , or il-13 ( mab 413 ) was added to the upper wells ; pbs or rat igg ( 10 g / ml ) was added to control wells . gad65 peptide 524 - 543 was then added to all upper chambers at a final concentration of 10 g / ml . three days later , the spleen cells in the lower chamber were recovered and ifn- spot - forming cells enumerated in duplicate wells by elispot . nod or nod.bdc2.5.tcr.tg spleen cells were labeled with anti - cd4conjugated magnetic beads ( miltenyi biotech , auburn , ca ) and purified according to the manufacturer s instructions . cd4 t - cell enrichment ( > 95% ) to assess the effect of ril-13 on cd4 t - cell proliferation , purified nod or bdc2.5.tcr.tg cd4 t cells ( 6  10 cells / well ) were cultured on anti - cd3coated 96-well plates for 72 h in the presence or absence of ril-13 ( r&d systems , minneapolis , mn ) . pbs - coated wells served as negative controls . for the last 18 h of culture , wells were pulse - labeled with 1ci / well h - thymidine ( mp biomedicals , solon , oh ) , harvested , and counted as previously described ( 16 ) . the expression of il-4 , il-10 , transforming growth factor ( tgf)- , and -actin was evaluated in gad35za t cells via rt - pcr . kits containing primers for il-10 , tgf- , and -actin were purchased from r&d systems . primers for il-4 ( forward catcggcattttgaacgaggtca , and reverse cttatcgatgaatccaggcatcg ) were purchased from operon ( alameda , ca ) . briefly , gad35za t cells were stimulated with irradiated nod spleen cells in the presence of p524 ( 10 g / ml ) for 24 to 48 h. mrna was harvested with an rneasy kit ( invitrogen , carlsbad , ca ) and used as a template for complimentary dna ( cdna ) synthesis . rt - pcr was performed according to the manufacturer s instructions for the primer kits , using platinum taq polymerase ( invitrogen ) . for analysis of il-13r1 expression , cd4 t cells purified from nod or nod.bdc2.5.tcr.tg splenocytes were stimulated on anti - cd3coated plates for 0 , 24 , 48 , or 72 h. the cells were harvested , and mrna was extracted with trizol ( invitrogen ) and converted to cdna . cdna was used for quantitative real - time rt - pcr using iq sybr green supermix ( bio - rad , hercules , ca ) . normalized results using the  ct method of quantification are expressed as % gapdh . the pcr primer sequences were as follows : gapdh forward ccaggttgtctcctgcgact , reverse ataccaggaaatgagcttgacaaagt ; il-13r1 forward gaatttgagcgtctctgtcgaa , reverse ggttatgccaaatgcacttgag ; il-4r forward agtgagtggagtcctagcatc , reverse gctgaagtaacagaacaggc . for direct ex vivo analysis of il-13r1 expression on t - cell populations , spleens , inguinal lymph nodes ( ilns ) , and plns were harvested from age - matched mice and stained with anti  tcr - phycoerythrin ( pe)/cy5 ( biolegend ) , anti  cd4-pe ( biolegend ) , and purified goat anti  il-13r1 ( santa cruz biotechnology , santa cruz , ca ) or purified goat igg . goat igg - fluoroisothiocyanate ( southern biotech , birmingham , al ) , washed , and analyzed by flow cytometry . for analysis of il-13 signaling , plns from 4-week - old female nod mice were harvested and stained with anti  tcr - fluoroisothiocyanate ( biolegend ) , serum - starved in rpmi for 1 h , and then exposed to ril-13 for 30 min at 37c . the cells were washed , fixed , permeabilized , and then stained with an antibody to phosphorylated signal transducer and activator of transcription ( stat ) 6 ( santa cruz biotechnology ) or goat isotype control , followed by biotinylated donkey anti the pancreas was collected at the same time as lymphoid tissue harvest for il-13r1 evaluation . the pancreas was fixed in 10% formalin , sectioned ( 5 micron sections , 20 microns apart ) , and stained with hematoxylin and eosin . islets were graded as normal or as having peri - insulitis ( inflammation only around the periphery of the islet ) or invasive insulitis ( inflammation was greater than 33% of the islet and disrupted the typical round / oval shape ) . p524 - 543 and p530-specific ifn-producing cells were prevalent in the spleens of nave prediabetic nod mice ( fig . 1 ) , but were undetectable in healthy balb / c or c57bl/6 mice ( data not shown ) . however , treatment of young 2-week - old nod mice with p524 - 538/ifa led to a significant reduction in the endogenous p530-specific ifn- response , regardless of whether p524 - 543 or p530 was used in the challenge ( fig . in addition to blocking hyperglycemia ( 10 ) , treatment with p524 enhanced or primed an il-5 response to p524 - 543 , which was restricted to the p524 epitope and could not be recalled by the p530 peptide ( fig . these observations indicate that p524/ifa primed a p524-specific th2 response while concomitantly antagonizing p530-specific th1 cells ; this correlated well with prior observations of lost proliferative responses to p530 in p524-treated mice ( 9 ) . furthermore , these findings support the hypothesis that the active induction of p524-reactive t cells directly regulates the activities of endogenously primed gad65-specific t cells . spleen cells pooled from three 6-week - old female nod mice were stimulated in vitro with peptide p524 - 543 ( 10 g / ml ) in 24-well plates . five days later , the cells were resuspended in ifn- elispot plates ( 1  10 cells / well ) with the indicated concentration of peptide gad65 530 - 543 ( p530 ) . the number of spot - forming cells was determined using a stereomicroscope , and the results are representative of four experiments . a and b : six 2-week - old nod mice were immunized once ( 1 ) or twice ( 2 ) with 100 g of p524538 or pbs emulsified in ifa . four weeks later , the spleen cells were collected and analyzed ( as in fig . 1 ) to enumerate ( a ) ifn- spot - forming cells or ( b ) il-5 spot - forming cells , in response to challenge with p524 - 543 ( black bar ) , p530 - 543 ( checkered ) , p524 - 538 ( vertical stripes ) , or concanavalin a ( cona ) ( diagonal stripes ) . background responses to control peptide hel 1125 have been subtracted . the results are representative of two experiments that yielded similar findings . in all experiments , the mean response in peptide - treated mice was compared with control mice ( ifa alone ) using student t test ( * * p < 0.005 ; * * * p < 0.0005 ) . the percentages noted in the chart indicate the reduction in spot - forming cells compared with ifa - immunized control animals . c : three 4-week - old nod mice were immunized once with 100 g of p524 - 538/ifa . the individual spleens ( p524 ) were collected 12 days later and challenged in vitro with p524 - 538 ( 10 g / ml , black bar ) . forty - eight hours later , il-4 , il-5 , il-13 , il-10 , and ifn- levels in the supernatants were measured by elisa . a th0 cell line stimulated with anti - cd3 ( 1.0 g / ml , diagonal stripes ) served as a positive control . the bars represent the mean concentration detected in cultures from the three mice ( minus medium controls ) . the data are representative of four experiments . because it was possible that the observed suppression of the p530 th1 responses was mediated through a regulatory cytokine , we wanted to determine whether p524 preferentially induced a cytokine(s ) known to regulate diabetes in nod mice . although il-13 and il-5 were consistently detected in recall responses of 4-week - old nod mice immunized with p524/ifa , il-4 , il-10 , and ifn- , specific responses were marginal and rarely reached significant levels ( fig . 2c ) . interestingly , gad35za secreted ifn- , il-5 , and il-13 upon antigenic challenge ( table 1 ) , but failed to produce il-4 or il-10 ( table 1 ) . the expression of il-4 , il-10 , and tgf- was also undetectable by rt - pcr in antigen - challenged gad35za t cells ( data not shown ) . therefore , although susceptibility to autoimmune disease in nod mice has often been linked to a poor th2 response , as judged by modest il-4 responses , here we found nod mice to be quite capable of generating both il-5 and il-13 specific responses to p524 . the t - cell clone gad35za produces il-13 , il-5 , and ifn- * i - a  restricted t clones gad35za and ova-1 were plated ( 5  10/well ) with irradiated syngeneic spleen cells ( 5  10/well ) in the presence or absence of cognate antigen , 10 g / ml of p524 - 538 or 50 g / ml of ovalbumin , respectively . forty - eight hours later , the supernatants were collected and cytokine levels determined by elisa , as described in research design and methods , with background levels subtracted ( t cells + antigen - presenting cells ) . to demonstrate that p524-reactive t cells could indeed alter the spontaneous p530-specific response via release of il-5 or il-13 we performed experiments in transwell plates where the upper and lower chambers are separated by a semipermeable membrane . nave nod spleen cells were added to the lower chamber , and gad35za t cells and irradiated syngeneic spleen cells were added to the upper chamber . p524 - 543 was added to the upper chamber along with monoclonal antibody to il-5 , il-13 , or ifn- , or an igg control monoclonal antibody ( fig . the p524 - 543specific ifn- response among the spleen cells recovered from the lower chamber was reduced by 80% when cultured in the presence of gad35za t cells ( fig . 3 , control vs. pbs ) , consequently confirming that the inhibitory effect induced by the p524-reactive cells was mediated by a soluble factor . importantly , this inhibition was completely neutralized by antibodies to il-13 but not by anti il-13 produced by p524 - 538reactive t cells can antagonize the endogenous th1 response to p530 - 543 . gad35za clonotypic t cells were plated ( 5  10 ml ) with irradiated syngeneic spleen cells in the upper chamber of a semipermeable transwell , whereas spleen cells from nave 6- to 8-week - old nod mice were plated ( 1  10/ml ) in the lower chamber . pbs , rat igg ( 10 g / ml ) , monoclonal antibody ( 10 g / ml ) to il-5 , il-13 , or ifn- was added to the upper wells . gad65 peptide 524 - 543 ( diagonal striped bar ) or concanavalin a ( cona ) ( black ) was then added to all upper chambers at a final concentration of 10 g / ml or 2.5 g / ml , respectively . the control well contained the nod spleen cells plus peptide antigen in the lower chamber , but no gad35za cells in the upper chamber . three days later , spleen cells in the lower chamber were recovered and analyzed for ifn- spot - forming cells ( as in fig . the p524 - 543specific results represent the mean and sd of triplicate wells and are representative of three experiments . to determine whether il-13 alone could mimic the suppressive effect of gad35za t cells , mouse ril-13 , rifn- , ril-5 , or ril-4 was added to nave nod spleen cells during in vitro challenge with the peptide p524 - 543 . 4a ) , whereas neither ril-4 , ril-5 , nor rifn- had a similar inhibitory effect ( fig . the ril-13mediated inhibition was not the result of cellular toxicity , since there was no reduction in the number of spleen cells recovered from wells cultured in the presence of this cytokine ( data not shown ) . a : pooled spleen cells ( 8  10/ml ) from three 6-week - old female nod mice were stimulated in vitro with 10 g / ml of p524 - 543 in the presence or absence of recombinant cytokine ( 10 g / ml ) ( ifn- , il-4 , il-5 , or il-13 ) . three days later , the cells were resuspended and challenged with p530 - 543pulsed irradiated spleen cells . forty - eight hours later , the supernatants were tested by elisa for the presence of ifn-. the results are representative of five experiments . b and c : clonotypic spleen t cells from nod.bdc2.5.tcr.tg mice ( b ) or cd4 t cells purified from nod mice ( c ) were stimulated in triplicate on anti - cd3coated plates ( 1.0 g / ml , black bar ) in the presence or absence of murine ril-13 ( 0.2 or 1.0 ng / ml ) . two days later , the wells were pulsed with h - thymidine , then harvested and counted after an additional 18 h of culture . the percentages in the chart indicate the reduction in proliferation ( counts per minute [ cpm ] ) compared with cells stimulated in the absence of ril-13 . the p values were calculated using student t test ( * p = 0.05 ; * * p < 0.005 ; * * * p < 0.0005 ) . to establish whether il-13 could directly suppress the activities of islet - reactive t cells , nod.bdc2.5 tcr clonotypic spleen cells were cultured in wells coated with anti - cd3 in the presence or absence of ril-13 . even in the presence of robust antigen receptor engagement , bdc2.5 , clonotypic proliferation was inhibited by up to 87% when stimulated in the presence of ril-13 ( fig . although this approach should eliminate influences by antigen - presenting cells , to further confirm that ril-13 was having a direct impact on t cells and that the inhibitory response was not peculiar to the bdc2.5 clonotypic cells , cd4 t cells were purified from the spleens of nave nod mice and stimulated with anti - cd3 in the presence of ril-13 . similar results were observed when the experiment was performed with purified nod.bdc2.5.tcr.tg cd4 t cells ( data not shown ) . because il-13 seemed to directly regulate the t cells , it was necessary to demonstrate that nod and bdc2.5 clonotypic t cells expressed il-13r1 and il-4r , which heterodimerize to form the functional complex for il-13mediated signaling ( 17,18 ) . cdna produced from purified nod or nod.bdc2.5.tcr.tg cd4 splenocytes ( nave or activated ) was analyzed by quantitative real - time pcr using primers specific for mouse il-13r1 , il-4r , or il-13r2 . il-13r1 and il-13r1 mrna expression was reduced following tcr engagement in both sets of t cells ( fig . 5a ) , but il-13r1 and il-4r were not detectable in the el-4 thymoma control cell line ( fig . next , we were able to validate the expression of il-13r1 chain on nod t cells , as approximately 5% of the tcrcd4 cells in the spleens of untreated mice were positive for the cytokine receptor ( fig . these findings support the contention that il-13 can deliver influential signals directly to effector t cells . il-13r2 , a decoy receptor that negatively regulates il-13 signaling ( 19 ) , was not detected in any of the samples above ( data not shown ) . a : cdna produced from purified nod or bdc2.5.tcr.tg cd4 cells , before or after culture on anti - cd3coated plates ( 72 h ) was evaluated by real - time pcr to detect expression of il-4r mrna ( upper panel ) or il-13r1 mrna ( lower panel ) , relative to gapdh . transcripts for gapdh were detected , whereas mrna for il-13r1 or il-4r was not detected ( n.d . ) . b : nod splenocytes were stained with antibodies to tcr , cd4 , and il-13r1 ( upper plot ) or isotype control ( lower plot ) . the numbers in the upper right quadrant indicate percentages of tcr cells that are also positive for the designated parameters . the plots shown are a representative experiments performed six times with similar results . in human cells , ligation of il-13 to the high affinity il-13r1/il-4r heterodimer activates janus kinase 2 tyrosine kinase and subsequently the phosphorylation of stat6 ( 20 ) . here , we show that tcr t cells recovered from the plns of young nod mice respond to il-13 by increasing the phosphorylation of stat6 ( figs . 6a and b ) , demonstrating that the il-13r1 expressed on the t cells was functional and capable of transmitting intracellular signals following ligand binding . a : serum - starved cells collected from the plns of three 4-week - old nod mice were cultured in the absence ( middle plot ) or presence of ril-13 ( 1 ng / ml , left and right plots ) for 30 min , and then stained with antibodies to tcr and the phosphorylated form of stat6 ( pstat6 ) ( right plot ) or isotype control ( left plot ) . b : the mean number of tcr / pstat6 cells was calculated from three independent experiments . the early stage of insulitis , at 45 weeks of age , is characterized by peri - insulitis with very limited -cell destruction . this benign period is followed by stages of progressively enhanced islet destruction and infiltration at 912 weeks of age , and sets the stage for the extensive -cell destruction that gives rise to hyperglycemia . to explore the possibility of il-13mediated t - cell regulation in the context of natural type 1 diabetes , we compared patterns of il-13r1 expression on t cells in 4-week - old mice and 9- to 11-week - old mice . we found that the frequency of il-13r1 t cells was enhanced in the plns of the young mice , compared with their ilns or spleens ( fig . strikingly , there were significantly more il-13r1expressing t cells in the plns of young mice compared with the older animals ( fig . , there was no significant difference between 4- and 9-week - old mice in the frequency of il-13r1 t cells in the ilns or splenic populations ( fig . 7b ) . although the actual percentages of il-13r1 t cells varied , in individual experiments ( containing equal numbers of young and older mice ) the percentage of receptor positive cells was always significantly higher in the younger mice ( fig . il-13r1 expression on t cells in the plns is enhanced in young prediabetic nod mice . ilns , plns , and spleens from 4- or 9-week - old nod mice were analyzed by fluorescence - activated cell sorter for tcr and il-13r1 expression . a : representative density plots from 4-week - old ( top plots ) and 9-week - old ( bottom plots ) female nod tissues . the plots shown are ungated populations , with the numbers indicating the percentages of cells in each quadrant . b : quantitative analysis of il-13r1 t cells in individual 4-week - old or 911-week - old nod mice , female ( f ) and male ( m ) , or female c57bl/6 controls  each marker indicates an individual mouse . the means were calculated from seven experiments performed under identical conditions ; statistical analysis was performed using student t test ( * * p < 0.005 ) . c : the pancreas was collected from three nod mice , individuals in ( b ) , fixed , processed , and stained as described in the research design and methods . at least 50 islets were evaluated for the severity of leukocyte infiltration : normal ( filled bar ) , peri - insulitis ( horizontal stripes ) , and invasive ( diagonal stripes ) . d : plns from female nod mice immunized at 3 weeks of age with p524 - 538/ifa or hel 1125/ifa , were collected at 9 weeks of age and analyzed by flow cytometry . statistical analysis was performed using student t test ( * p < 0.05 , * * p < 0.005 ) . n.s . the findings above offer the possibility that the loss of il-13ra1 t cells in the plns of nod mice could correlate with the increased severity of insulitis . female nod mice bred and housed in our facility have a diabetes incidence rate of greater than 80% , whereas less than 20% of the male counterparts progress to hyperglycemia . when we evaluated 4- and 9-week old male nod mice ( littermates of the females ) we found that both young and adult males displayed a pattern similar to that of 4-week - old females  significantly more il-13r1 t cells in the plns than in the corresponding inguinal or splenic lymphoid compartments ( fig . conversely , the frequency of il-13r1 t cells in ilns and spleens from the older male mice was not different from that of older female nod mice or c57bl/6 control mice ( fig . an analysis of the pancreatic tissue harvested from the same groups of animals revealed that the level of invasive insulitis was highest among the older female mice , whereas tissues from the older male mice contained more normal islets and far fewer islets with the invasive form of inflammation ( fig . 7c ) . because treatment with p524 - 538 ameliorates insulitis and type 1 diabetes in young nod mice , we wanted to discover whether immunization with p524 - 538 could also alter the expression of il-13r1 on t cells in the plns of postinsulitis female nod mice . we found that number of il-13r1 t cells in the plns of 9-week - old female mice treated with control peptide was significantly lower than that expressed on similar cells in young mice ( fig . 7d ) . on the other hand , at 9 weeks of age mice treated with the gad65 peptide retained a frequency of il-13r1 t cells in plns similar to that of 4-week - old mice ( fig . p524 - 543 and p530-specific ifn-producing cells were prevalent in the spleens of nave prediabetic nod mice ( fig . 1 ) , but were undetectable in healthy balb / c or c57bl/6 mice ( data not shown ) . however , treatment of young 2-week - old nod mice with p524 - 538/ifa led to a significant reduction in the endogenous p530-specific ifn- response , regardless of whether p524 - 543 or p530 was used in the challenge ( fig . in addition to blocking hyperglycemia ( 10 ) , treatment with p524 enhanced or primed an il-5 response to p524 - 543 , which was restricted to the p524 epitope and could not be recalled by the p530 peptide ( fig . these observations indicate that p524/ifa primed a p524-specific th2 response while concomitantly antagonizing p530-specific th1 cells ; this correlated well with prior observations of lost proliferative responses to p530 in p524-treated mice ( 9 ) . furthermore , these findings support the hypothesis that the active induction of p524-reactive t cells directly regulates the activities of endogenously primed gad65-specific t cells . spleen cells pooled from three 6-week - old female nod mice were stimulated in vitro with peptide p524 - 543 ( 10 g / ml ) in 24-well plates . five days later , the cells were resuspended in ifn- elispot plates ( 1  10 cells / well ) with the indicated concentration of peptide gad65 530 - 543 ( p530 ) . the number of spot - forming cells was determined using a stereomicroscope , and the results are representative of four experiments . a and b : six 2-week - old nod mice were immunized once ( 1 ) or twice ( 2 ) with 100 g of p524538 or pbs emulsified in ifa . four weeks later , the spleen cells were collected and analyzed ( as in fig . 1 ) to enumerate ( a ) ifn- spot - forming cells or ( b ) il-5 spot - forming cells , in response to challenge with p524 - 543 ( black bar ) , p530 - 543 ( checkered ) , p524 - 538 ( vertical stripes ) , or concanavalin a ( cona ) ( diagonal stripes ) . background responses to control peptide hel 1125 have been subtracted . the results are representative of two experiments that yielded similar findings . in all experiments , the mean response in peptide - treated mice was compared with control mice ( ifa alone ) using student t test ( * * p < 0.005 ; * * * p < 0.0005 ) . the percentages noted in the chart indicate the reduction in spot - forming cells compared with ifa - immunized control animals . c : three 4-week - old nod mice were immunized once with 100 g of p524 - 538/ifa . the individual spleens ( p524 ) were collected 12 days later and challenged in vitro with p524 - 538 ( 10 g / ml , black bar ) . forty - eight hours later , il-4 , il-5 , il-13 , il-10 , and ifn- levels in the supernatants were measured by elisa . a th0 cell line stimulated with anti - cd3 ( 1.0 g / ml , diagonal stripes ) served as a positive control . the bars represent the mean concentration detected in cultures from the three mice ( minus medium controls ) . the data are representative of four experiments . because it was possible that the observed suppression of the p530 th1 responses was mediated through a regulatory cytokine , we wanted to determine whether p524 preferentially induced a cytokine(s ) known to regulate diabetes in nod mice . although il-13 and il-5 were consistently detected in recall responses of 4-week - old nod mice immunized with p524/ifa , il-4 , il-10 , and ifn- , specific responses were marginal and rarely reached significant levels ( fig . 2c ) . interestingly , gad35za secreted ifn- , il-5 , and il-13 upon antigenic challenge ( table 1 ) , but failed to produce il-4 or il-10 ( table 1 ) . the expression of il-4 , il-10 , and tgf- was also undetectable by rt - pcr in antigen - challenged gad35za t cells ( data not shown ) . therefore , although susceptibility to autoimmune disease in nod mice has often been linked to a poor th2 response , as judged by modest il-4 responses , here we found nod mice to be quite capable of generating both il-5 and il-13 specific responses to p524 . the t - cell clone gad35za produces il-13 , il-5 , and ifn- * i - a  restricted t clones gad35za and ova-1 were plated ( 5  10/well ) with irradiated syngeneic spleen cells ( 5  10/well ) in the presence or absence of cognate antigen , 10 g / ml of p524 - 538 or 50 g / ml of ovalbumin , respectively . forty - eight hours later , the supernatants were collected and cytokine levels determined by elisa , as described in research design and methods , with background levels subtracted ( t cells + antigen - presenting cells ) . to demonstrate that p524-reactive t cells could indeed alter the spontaneous p530-specific response via release of il-5 or il-13 we performed experiments in transwell plates where the upper and lower chambers are separated by a semipermeable membrane . nave nod spleen cells were added to the lower chamber , and gad35za t cells and irradiated syngeneic spleen cells were added to the upper chamber . p524 - 543 was added to the upper chamber along with monoclonal antibody to il-5 , il-13 , or ifn- , or an igg control monoclonal antibody ( fig . the p524 - 543specific ifn- response among the spleen cells recovered from the lower chamber was reduced by 80% when cultured in the presence of gad35za t cells ( fig . 3 , control vs. pbs ) , consequently confirming that the inhibitory effect induced by the p524-reactive cells was mediated by a soluble factor . importantly , this inhibition was completely neutralized by antibodies to il-13 but not by anti il-13 produced by p524 - 538reactive t cells can antagonize the endogenous th1 response to p530 - 543 . gad35za clonotypic t cells were plated ( 5  10 ml ) with irradiated syngeneic spleen cells in the upper chamber of a semipermeable transwell , whereas spleen cells from nave 6- to 8-week - old nod mice were plated ( 1  10/ml ) in the lower chamber . pbs , rat igg ( 10 g / ml ) , monoclonal antibody ( 10 g / ml ) to il-5 , il-13 , or ifn- was added to the upper wells . gad65 peptide 524 - 543 ( diagonal striped bar ) or concanavalin a ( cona ) ( black ) was then added to all upper chambers at a final concentration of 10 g / ml or 2.5 g / ml , respectively . the control well contained the nod spleen cells plus peptide antigen in the lower chamber , but no gad35za cells in the upper chamber . three days later , spleen cells in the lower chamber were recovered and analyzed for ifn- spot - forming cells ( as in fig . 1 ) . the p524 - 543specific results represent the mean and sd of triplicate wells and are representative of three experiments . to determine whether il-13 alone could mimic the suppressive effect of gad35za t cells , mouse ril-13 , rifn- , ril-5 , or ril-4 was added to nave nod spleen cells during in vitro challenge with the peptide p524 - 543 . 4a ) , whereas neither ril-4 , ril-5 , nor rifn- had a similar inhibitory effect ( fig . the ril-13mediated inhibition was not the result of cellular toxicity , since there was no reduction in the number of spleen cells recovered from wells cultured in the presence of this cytokine ( data not shown ) . a : pooled spleen cells ( 8  10/ml ) from three 6-week - old female nod mice were stimulated in vitro with 10 g / ml of p524 - 543 in the presence or absence of recombinant cytokine ( 10 g / ml ) ( ifn- , il-4 , il-5 , or il-13 ) . three days later , the cells were resuspended and challenged with p530 - 543pulsed irradiated spleen cells . forty - eight hours later , the supernatants were tested by elisa for the presence of ifn-. the results are representative of five experiments . b and c : clonotypic spleen t cells from nod.bdc2.5.tcr.tg mice ( b ) or cd4 t cells purified from nod mice ( c ) were stimulated in triplicate on anti - cd3coated plates ( 1.0 g / ml , black bar ) in the presence or absence of murine ril-13 ( 0.2 or 1.0 ng / ml ) . two days later , the wells were pulsed with h - thymidine , then harvested and counted after an additional 18 h of culture . the percentages in the chart indicate the reduction in proliferation ( counts per minute [ cpm ] ) compared with cells stimulated in the absence of ril-13 . the p values were calculated using student t test ( * p = 0.05 ; * * p < 0.005 ; * * * p < 0.0005 ) . to establish whether il-13 could directly suppress the activities of islet - reactive t cells , nod.bdc2.5 tcr clonotypic spleen cells were cultured in wells coated with anti - cd3 in the presence or absence of ril-13 . even in the presence of robust antigen receptor engagement , bdc2.5 , clonotypic proliferation was inhibited by up to 87% when stimulated in the presence of ril-13 ( fig . although this approach should eliminate influences by antigen - presenting cells , to further confirm that ril-13 was having a direct impact on t cells and that the inhibitory response was not peculiar to the bdc2.5 clonotypic cells , cd4 t cells were purified from the spleens of nave nod mice and stimulated with anti - cd3 in the presence of ril-13 . similar results were observed when the experiment was performed with purified nod.bdc2.5.tcr.tg cd4 t cells ( data not shown ) . because il-13 seemed to directly regulate the t cells , it was necessary to demonstrate that nod and bdc2.5 clonotypic t cells expressed il-13r1 and il-4r , which heterodimerize to form the functional complex for il-13mediated signaling ( 17,18 ) . cdna produced from purified nod or nod.bdc2.5.tcr.tg cd4 splenocytes ( nave or activated ) was analyzed by quantitative real - time pcr using primers specific for mouse il-13r1 , il-4r , or il-13r2 . il-13r1 and il-13r1 mrna expression was reduced following tcr engagement in both sets of t cells ( fig . 5a ) , but il-13r1 and il-4r were not detectable in the el-4 thymoma control cell line ( fig . next , we were able to validate the expression of il-13r1 chain on nod t cells , as approximately 5% of the tcrcd4 cells in the spleens of untreated mice were positive for the cytokine receptor ( fig . 5b ) . these findings support the contention that il-13 can deliver influential signals directly to effector t cells . il-13r2 , a decoy receptor that negatively regulates il-13 signaling ( 19 ) , was not detected in any of the samples above ( data not shown ) . a : cdna produced from purified nod or bdc2.5.tcr.tg cd4 cells , before or after culture on anti - cd3coated plates ( 72 h ) was evaluated by real - time pcr to detect expression of il-4r mrna ( upper panel ) or il-13r1 mrna ( lower panel ) , relative to gapdh . transcripts for gapdh were detected , whereas mrna for il-13r1 or il-4r was not detected ( n.d . ) . b : nod splenocytes were stained with antibodies to tcr , cd4 , and il-13r1 ( upper plot ) or isotype control ( lower plot ) . the numbers in the upper right quadrant indicate percentages of tcr cells that are also positive for the designated parameters . the plots shown are a representative experiments performed six times with similar results . in human cells , ligation of il-13 to the high affinity il-13r1/il-4r heterodimer activates janus kinase 2 tyrosine kinase and subsequently the phosphorylation of stat6 ( 20 ) . here , we show that tcr t cells recovered from the plns of young nod mice respond to il-13 by increasing the phosphorylation of stat6 ( figs . 6a and b ) , demonstrating that the il-13r1 expressed on the t cells was functional and capable of transmitting intracellular signals following ligand binding . a : serum - starved cells collected from the plns of three 4-week - old nod mice were cultured in the absence ( middle plot ) or presence of ril-13 ( 1 ng / ml , left and right plots ) for 30 min , and then stained with antibodies to tcr and the phosphorylated form of stat6 ( pstat6 ) ( right plot ) or isotype control ( left plot ) . b : the mean number of tcr / pstat6 cells was calculated from three independent experiments . the early stage of insulitis , at 45 weeks of age , is characterized by peri - insulitis with very limited -cell destruction . this benign period is followed by stages of progressively enhanced islet destruction and infiltration at 912 weeks of age , and sets the stage for the extensive -cell destruction that gives rise to hyperglycemia . to explore the possibility of il-13mediated t - cell regulation in the context of natural type 1 diabetes , we compared patterns of il-13r1 expression on t cells in 4-week - old mice and 9- to 11-week - old mice . we found that the frequency of il-13r1 t cells was enhanced in the plns of the young mice , compared with their ilns or spleens ( fig . strikingly , there were significantly more il-13r1expressing t cells in the plns of young mice compared with the older animals ( fig . , there was no significant difference between 4- and 9-week - old mice in the frequency of il-13r1 t cells in the ilns or splenic populations ( fig . although the actual percentages of il-13r1 t cells varied , in individual experiments ( containing equal numbers of young and older mice ) the percentage of receptor positive cells was always significantly higher in the younger mice ( fig . il-13r1 expression on t cells in the plns is enhanced in young prediabetic nod mice . ilns , plns , and spleens from 4- or 9-week - old nod mice were analyzed by fluorescence - activated cell sorter for tcr and il-13r1 expression . a : representative density plots from 4-week - old ( top plots ) and 9-week - old ( bottom plots ) female nod tissues . the plots shown are ungated populations , with the numbers indicating the percentages of cells in each quadrant . b : quantitative analysis of il-13r1 t cells in individual 4-week - old or 911-week - old nod mice , female ( f ) and male ( m ) , or female c57bl/6 controls  each marker indicates an individual mouse . the means were calculated from seven experiments performed under identical conditions ; statistical analysis was performed using student t test ( * * p < 0.005 ) . c : the pancreas was collected from three nod mice , individuals in ( b ) , fixed , processed , and stained as described in the research design and methods . at least 50 islets were evaluated for the severity of leukocyte infiltration : normal ( filled bar ) , peri - insulitis ( horizontal stripes ) , and invasive ( diagonal stripes ) . d : plns from female nod mice immunized at 3 weeks of age with p524 - 538/ifa or hel 1125/ifa , were collected at 9 weeks of age and analyzed by flow cytometry . statistical analysis was performed using student t test ( * p < 0.05 , * * p < 0.005 ) . the findings above offer the possibility that the loss of il-13ra1 t cells in the plns of nod mice could correlate with the increased severity of insulitis . female nod mice bred and housed in our facility have a diabetes incidence rate of greater than 80% , whereas less than 20% of the male counterparts progress to hyperglycemia . when we evaluated 4- and 9-week old male nod mice ( littermates of the females ) we found that both young and adult males displayed a pattern similar to that of 4-week - old females  significantly more il-13r1 t cells in the plns than in the corresponding inguinal or splenic lymphoid compartments ( fig . conversely , the frequency of il-13r1 t cells in ilns and spleens from the older male mice was not different from that of older female nod mice or c57bl/6 control mice ( fig . an analysis of the pancreatic tissue harvested from the same groups of animals revealed that the level of invasive insulitis was highest among the older female mice , whereas tissues from the older male mice contained more normal islets and far fewer islets with the invasive form of inflammation ( fig . 7c ) . because treatment with p524 - 538 ameliorates insulitis and type 1 diabetes in young nod mice , we wanted to discover whether immunization with p524 - 538 could also alter the expression of il-13r1 on t cells in the plns of postinsulitis female nod mice . we found that number of il-13r1 t cells in the plns of 9-week - old female mice treated with control peptide was significantly lower than that expressed on similar cells in young mice ( fig . 7d ) . on the other hand , at 9 weeks of age mice treated with the gad65 peptide retained a frequency of il-13r1 t cells in plns similar to that of 4-week - old mice ( fig . ongoing clinical trials have provided encouraging data suggesting that treatment with the gad65 protein will be beneficial in prolonging -cell function in recent - onset human diabetes ( 21,22 ) . nevertheless , the molecular mechanisms that govern gad65-induced regulation of insulitis and -cell destruction remain unclear . initially , immune deviation via the enhancement of il-4driven th2 response seemed a likely explanation for the improved outcome observed in neonatal nod mice successfully treated with gad65 protein or peptides ( 10,23,24 ) . this contention was strengthened by data indicating that nod mice had deficient il-4 responses , possibly as the result of reduced numbers of il-4producing natural killer t cells ( 2527 ) and/or inadequate tcr signaling cascades ( 28 ) . however , the significance of il-4 in modulating islet autoimmunity has been challenged in subsequent studies using il-4deficient nod mice ( 29 ) or examining the role of natural killer t cells in autoimmunity ( 30 ) . although neutralizing antibodies provide strong evidence for il-10 as an influential cytokine , in many cases the correlation between reduced islet pathology in nod mice and the appearance of regulatory cytokines such as il-4 and il-10 is indirect and does not address the mechanism(s ) by which the target cells are engaged . here , we show that an alternate th2-like cytokine , il-13 , is able to directly regulate gad65-specific th1 cell  mediated autoimmunity . importantly , il-13r1 expression on t cells may have an impact on the effector functions of -cell  specific t cells and islet injury during the early stage of type 1 diabetes , a period characterized by benign insulitis ( 31 ) . it is of particular interest that a distinct epitope on gad65 is able to incite il-13producing cells , either as p524 - 538 or as a determinant processed from p524 - 543 . prolonged administration of ril-13 prevents or delays the onset of insulitis and diabetes in young nod mice , and reduces the incidence in those with preexisting insulitis ( 32 ) . this th2 cytokine has also been shown to have protective effects in other models of inflammatory autoimmune disease ( 33,34 ) . however , until recently ( 3537 ) , t cells were thought to be devoid of il-13r1 expression and therefore could not be directly regulated by il-13 ; the influence of the cytokine on inflammatory autoimmune disease has largely been attributed to its effect on il-13r1 monocytes and their potential to prevent the expansion of autoreactive t cells ( 34 ) . our data , and that of others ( 36,37 ) , support a role for il-13induced signal transduction in th cells that produces altered gene expression and effector functions . il-13 can specifically induce phosphorylation of the transcription factor stat6 in t cells , as well as increase the expression of gata-3 ( 37 ) . our inhibition experiments with the p524-specific t - cell clone gad35za clearly showed that il-13 suppressed native th1 responses that we observed in nod mice . the regional enhancement of il-13r1 expression on t cells in the draining nodes of the pancreas was specific to 4-week - old female nod mice and our 9-week - old male nod that show resistance to invasive insulitis and diabetes . this age - related loss of il-13r1 receptor expression may contribute to the collapse of islet - specific tolerance , which has been reported to instigate and perpetuate diabetes in the nod mice ( 5 ) . although our report focuses on cd4 t cells , we also detected il-13r1 expression on cd4-tcr cells . it remains to be determined whether treatment with p524 or ril-13 is able to modulate the activities of islet - specific cd8 t cells . ( 35 ) show that il-4 signaling through the il-4r/il-13r1 heterodimer induces apoptosis in neonatal il-13r1expressing th1 cells upon antigenic challenge . we found that exposure to il-13 does not directly reduce the number of autoreactive t cells in vitro , and systemic delivery of the cytokine over several weeks was well tolerated in nod mice ( 32 ) , collectively suggesting the induction of anergy or a functional shift in the inflammatory milieu , rather than cell death , as the basis for our findings . th1-associated ifn- and th17-associated il-17 can contribute to inflammation , and the lack of either cytokine has been shown to reduce or ameliorate inflammatory autoimmune responses in several murine models of disease ( 3840 ) . upon exposure to either p524 - 538specific t cells or ril-13 , we detect a reduction in ifn- production by autoreactive p530 - 543specific th1 cells . we found that the t - cell clone gad35za was able to inhibit the spontaneous th1 responses to a dominant gad65 epitope that has been associated with progression of autoimmune diabetes in wild - type nod mice ( 10,41,42 ) . interestingly , il-13deficient mice exhibit increased proliferation of antigen specific cd4 and cd8 effector t cells ( 34 ) , and prophylactic treatment with il-13 reduces the ability of splenic cells to produce ifn- in response to concanavalin a or anti - cd3 . because ifn- is reported to antagonize the generation of il-17producing th17 cells ( 43 ) , it was possible that suppression of ifn- could promote a shift toward a th17-dominated response . however , we did not find that the il-13mediated reduction of ifn- resulted in production of il-17 ( data not shown ) . furthermore , our previous data show that p524 - 538 immunization blocks the progression to diabetes in nod mice , which is not supportive of the conclusion that the treatment skews the milieu toward a th17 phenotype . collectively , our data confirm the reports that t cells are able to express il-13r1 ( 37 ) , but suggest that expression is not restricted to th17-polarized cd4 t cells in nod mice . although clone gad35za ameliorated diabetes when transferred to young nod mice ( 9 ) , disease protection was not elicited upon clone transfer to older nod with pronounced insulitis , or when cotransferred with diabetogenic splenocytes ( data not shown ) , suggesting that p524 - 538specific regulation of autoreactive t cells is most effective in the early stages of pancreatic autoimmunity . this loss of regulatory activity could be related to the temporal loss of il-13r1 expression we observed in the plns . although the antigenic specificity of the il-13r1 t cells is unknown , our data show that the il-13producing t cells ( p524 - 538 ) and il-13responding t cells ( -cell specific ) are distinct . therefore , since -cell  specific t cells are present in the peripheral blood of patients with type 1 diabetes ( 44 ) and of at - risk individuals long before the onset of hyperglycemia ( 45 ) , as well as being detected in the periphery of diabetes - free first - degree relatives ( 46 ) , it would be of interest to discover whether differential expression of the il-13r1 on such t cells could serve as a biomarker ( 47 ) for progressive -cell destruction and signal the onset of hyperglycemia . this could be accomplished most efficiently if staining for the receptor is combined with the soluble major histocompatibility complex : peptide technology that helped foster the identification , enumeration , and isolation of -cell  specific t cells in humans ( 45,48 ) . a biomarker for progressive immune dysregulation could provide the underpinnings for earlier introduction of gad65 immunotherapy and the obvious implications for improved benefits .
objectiveimmunotherapy using peptides from the -cell antigen gad65 can preserve glucose homeostasis in diabetes - prone nod mice ; however , the precise mechanisms that arrest islet - reactive t cells remain unresolved . 
 our previous work revealed that a dominant gad65 epitope contained two overlapping i - ag7restricted determinants , 524 - 538 and 530 - 543 , with the former associated with amelioration of hyperglycemia . here 
 , we sought to discover whether p524 - 538specific t cells could directly regulate islet - reactive t cells.research design and methodsprediabetic nod mice were used to determine the relationship between peptide p524 - 538induced interleukin ( il)-13 and regulation of islet autoimmunity . 
 pancreatic lymph node ( pln ) cells from mice at distinct stages of islet inflammation , peri - insulitis versus invasive insulitis , were harvested to establish the expression pattern of il-13 receptor 1 ( il-13r1 ) on islet - associated t cells.resultspeptide p524 - 538 preferentially induced il-13producing t cells that antagonized the release of -interferon by spontaneously arising gad65 autoimmunity , and recombinant human il-13 inhibited proliferation of islet - reactive clonotypic t cells . 
 a subset of cd4 + t cells in nod and nod.bdc2.5 t cell receptor transgenic mice expressed functional il-13r1 , which induced phosphorylation of signal transducer and activator of transcription 6 in the presence of cognate cytokine . 
 notably , the number of il-13r1 + t cells was heightened in the pln of young nod mice when compared with older female counterparts with advanced insulitis . 
 immunization with p524 - 538 preserved il-13r1 expression on pln t cells.conclusionsil-13 may be important for regulating autoimmunity in the early stages of insulitis , and the loss of il-13r1 on islet - reactive t cells may be a biomarker for fading regional immune regulation and progression to overt diabetes .