nod.iapp mice were bred in our colony by backcrossing c57bl6.iapp mice ( 6 ) onto the nod background ; these mice have been fully backcrossed to the nod mouse ( 10 generations ) .
all experimental procedures were in accordance with the guidelines of the institutional animal care and use committee , national jewish health .
( 7 ) were used as a source of -cell tumors for the preparation of antigen ; the enrichment of membrane proteins from -cells isolated from nod.riptag adenomas has been previously described ( 8) . in brief , single - cell suspensions of -cell adenomas were prepared by pressing whole - tumor tissue through a mesh screen , followed by several centrifugation steps to obtain a cellular membrane pellet ; this pellet ( -mem ) is highly antigenic for the t - cell clones and serves as a positive control antigen in t - cell assays as well as the source of antigen for further purification .
membrane protein preparations were solubilized in detergent - containing buffer ( 20 mmol / l tris , ph 8.0 , and 1% octyl--glucoside ) , followed by centrifugation at 18,400 g ( 10 min at 4c ) to remove insoluble debris .
size - exclusion chromatography ( sec ) was carried out on a superdex 200 16/60 column ( amersham biosciences ) at 21c ( flow rate : 1 ml / min ; fraction size : 1.25 ml ; and injection volume : 2.0 ml ) , eluting with sec buffer ( 20 mmol / l tris , ph 8.0 , and 150 mmol / l nacl ) .
fractions from sec were assayed for antigenic activity with the t - cell clones , and a peak antigenic fraction ( 950 l ) was acidified with 20 l trifluoroacetic acid before adding 30 l acetonitrile .
a total of 800 l of this mixture was then applied to a reverse - phase high - performance liquid chromatography ( rp - hplc ) mrp - c18 high - recovery protein column ( agilent ) .
a buffer gradient was used to elute proteins from the column , and a total of 36 fractions were collected between 0 and 72 min at a flow rate of 0.75 ml / min and a constant column temperature of 80c .
the antigenicity of islet cells , cellular and biochemical fractions , or peptides was assessed through the ifn- responses of t - cell clones . in 96-well microtiter plates ,
assay cultures contained 2  10 responder t cells , 2.5  10 nod peritoneal exudate cells as antigen - presenting cells , and -cell antigen .
ifn- production by the responder t cells was determined by elisa ( bd biosciences ) .
ifn- standards were added to the assay plates at concentrations between 0 and 100 ng / ml , but concentrations of the standard > 50 ng / ml were above the assay detection range .
an aliquot of -mem or whole nod islet cells were used as positive controls ; test wells contained sec or rp - hplc fractions , peptides , or islet cells .
the protein - identification strategy we have previously described ( 5 ) was used for mass spectrometry .
proteins were digested with trypsin , and extracted peptides were resolved by chromatography online on a c18 column and a 1200 series hplc system ( agilent technologies ) .
analysis was carried out with a 6340 lcms ion - trap mass spectrometer in the national jewish health facility .
raw data were extracted and used to search the swiss - prot or national center for biotechnology information databases with the spectrum mill search engine ( rev a.03.03.038 sr1 ; agilent technologies ) .
data were evaluated and protein identifications were considered significant if the following confidence thresholds were met : individual peptide scores of at least 10 and scored percent intensity of at least 70% . a reverse ( random ) database search was simultaneously conducted , and manual inspection of spectra was used to validate the match of the spectrum to the predicted peptide fragmentation pattern .
cell cultures of bdc-2.5 and bdc-5.2.9 were expanded in interleukin-2 , and after harvesting , t cells ( 1  10 ) were injected intraperitoneally into age - matched 6- to 14-day - old nod or nod.iapp recipient mice .
mice were monitored on a daily basis for urine glucose using diastix ( bayer ) .
the presence of urine glucose was followed by blood glucose monitoring using a onetouch ultra glucometer ( lifescan ) .
mice were considered diabetic when blood glucose levels were > 18 mmol / l ( 324 mg / dl ) . for disease - transfer experiments ,
nod , nod.scid , and nod.rip-tag mice were bred and maintained in the biological resource center at national jewish health .
nod.iapp mice were bred in our colony by backcrossing c57bl6.iapp mice ( 6 ) onto the nod background ; these mice have been fully backcrossed to the nod mouse ( 10 generations ) .
all experimental procedures were in accordance with the guidelines of the institutional animal care and use committee , national jewish health .
nod.rip-tag mice ( 7 ) were used as a source of -cell tumors for the preparation of antigen ; the enrichment of membrane proteins from -cells isolated from nod.riptag adenomas has been previously described ( 8) . in brief , single - cell suspensions of -cell adenomas were prepared by pressing whole - tumor tissue through a mesh screen , followed by several centrifugation steps to obtain a cellular membrane pellet ; this pellet ( -mem ) is highly antigenic for the t - cell clones and serves as a positive control antigen in t - cell assays as well as the source of antigen for further purification .
membrane protein preparations were solubilized in detergent - containing buffer ( 20 mmol / l tris , ph 8.0 , and 1% octyl--glucoside ) , followed by centrifugation at 18,400 g ( 10 min at 4c ) to remove insoluble debris .
size - exclusion chromatography ( sec ) was carried out on a superdex 200 16/60 column ( amersham biosciences ) at 21c ( flow rate : 1 ml / min ; fraction size : 1.25 ml ; and injection volume : 2.0 ml ) , eluting with sec buffer ( 20 mmol / l tris , ph 8.0 , and 150 mmol / l nacl )
. fractions from sec were assayed for antigenic activity with the t - cell clones , and a peak antigenic fraction ( 950 l ) was acidified with 20 l trifluoroacetic acid before adding 30 l acetonitrile .
a total of 800 l of this mixture was then applied to a reverse - phase high - performance liquid chromatography ( rp - hplc ) mrp - c18 high - recovery protein column ( agilent ) .
a buffer gradient was used to elute proteins from the column , and a total of 36 fractions were collected between 0 and 72 min at a flow rate of 0.75 ml / min and a constant column temperature of 80c .
the antigenicity of islet cells , cellular and biochemical fractions , or peptides was assessed through the ifn- responses of t - cell clones . in 96-well microtiter plates ,
assay cultures contained 2  10 responder t cells , 2.5  10 nod peritoneal exudate cells as antigen - presenting cells , and -cell antigen .
ifn- production by the responder t cells was determined by elisa ( bd biosciences ) .
ifn- standards were added to the assay plates at concentrations between 0 and 100 ng / ml , but concentrations of the standard > 50 ng / ml were above the assay detection range .
an aliquot of -mem or whole nod islet cells were used as positive controls ; test wells contained sec or rp - hplc fractions , peptides , or islet cells .
the protein - identification strategy we have previously described ( 5 ) was used for mass spectrometry .
proteins were digested with trypsin , and extracted peptides were resolved by chromatography online on a c18 column and a 1200 series hplc system ( agilent technologies ) .
analysis was carried out with a 6340 lcms ion - trap mass spectrometer in the national jewish health facility .
raw data were extracted and used to search the swiss - prot or national center for biotechnology information databases with the spectrum mill search engine ( rev a.03.03.038 sr1 ; agilent technologies ) .
data were evaluated and protein identifications were considered significant if the following confidence thresholds were met : individual peptide scores of at least 10 and scored percent intensity of at least 70% . a reverse ( random ) database search was simultaneously conducted , and manual inspection of spectra was used to validate the match of the spectrum to the predicted peptide fragmentation pattern .
cell cultures of bdc-2.5 and bdc-5.2.9 were expanded in interleukin-2 , and after harvesting , t cells ( 1  10 ) were injected intraperitoneally into age - matched 6- to 14-day - old nod or nod.iapp recipient mice .
mice were monitored on a daily basis for urine glucose using diastix ( bayer ) .
the presence of urine glucose was followed by blood glucose monitoring using a onetouch ultra glucometer ( lifescan ) .
mice were considered diabetic when blood glucose levels were > 18 mmol / l ( 324 mg / dl ) . for disease - transfer experiments ,
as previously described ( 5 ) , to identify candidate antigens for the t - cell clones , we carried out biochemical fractionation of -cell tumors and mass spectrometric analysis of peak antigenic fractions .
similar to the other clones from the panel , bdc-5.2.9 responds in vitro to pancreatic islet cells or cell extracts of -cell adenomas presented in the context of i - a ( 2 ) .
our starting source of antigen was -cell tumor tissue excised from nod.riptag mice ( 7 ) and separated by differential centrifugation to yield a highly antigenic -cell membrane pellet ( -mem ) ; this preparation also served as the positive control in t - cell activity assays .
after lysis , the membrane pellet was separated by sequential chromatography , first through an sec column and then by rp - hplc .
1 , the presence of antigen was tracked through the ifn- response of the t - cell clone bdc-5.2.9 ( top axis , gray lines ) to protein ( bottom axis , black lines ) in individual fractions .
figure 1a is the chromatogram from sec and shows a single antigenic peak that corresponds to a low  molecular weight protein region .
the antigenic fractions from sec were further purified by rp - hplc , yielding another single peak of antigen activity ( fig .
presence of antigen was determined by measuring the ifn- response ( gray lines ) of the t - cell clone bdc-5.2.9 to small aliquots of individual fractions in the presence of peritoneal exudate cells used as antigen - presenting cells .
the highly enriched and antigenic rp - hplc fractions were subjected to in - solution tryptic digest and were then analyzed on an ion - trap mass spectrometer .
resulting spectra were sequenced and matched to proteins by searching the swiss - prot protein database using the search program spectrum mill .
comparison of the antigen distribution profile with individual protein spectral - intensity values , an approximate indicator for relative protein abundances , singled out iapp as the only candidate .
other proteins did not match the antigen distribution profile ; for example , secretogranin 1 was detected only in nonantigenic fractions 11 and 14 .
we identified two iapp peptides , representing 28% amino acid coverage of prepro - iapp and 70% coverage of the 37-residue peptide hormone ( fig .
the peptide lanflvr was matched with high confidence and the peptide ssnnlgpvlpptnvgsnty was matched with medium confidence .
antigenic chromatographic fractions were digested with trypsin , separated by rp - hplc , and analyzed by ion - trap mass spectrometry ; the resulting spectra were used to search a protein sequence database . a : proteins identified ( right column ) in the highly purified antigenic rp - hplc fraction no . 12 and neighboring fractions with lower antigen content ( fractions 11 , 13 , and 14 , as shown in fig .
the summarized spectral intensities ( left ) are indicative of the relative abundance of a specific protein in a fraction ; darker shading indicates higher intensities .
b and c : ion - trap mass spectra matching the iapp peptides lanflvr ( b ) and ssnnlgpvlpptnvgsnty ( c ) ( swissprot accession no .
to investigate the peptide(s ) targeted by bdc-5.2.9 , we analyzed a series of overlapping iapp peptides , spanning the entire propeptide region ( fig .
one peptide , ks20 , elicited a vigorous response from bdc-5.2.9 and could stimulate the clone at concentrations as low as 10 ng / ml ( 4.6 nmol / l ) ( fig .
bdc-5.2.9 did not respond to any other iapp peptide tested or to the insulin peptide b923 .
two neighboring peptides , rt20 and gr20 , share either the c- or n - terminus sequence with ks20 , but neither of these peptides can stimulate bdc-5.2.9 , indicating that the t - cell receptor of this clone recognizes the adjoining sequences . an insulin - reactive clone , pd-12.4.4 ( 9 ) ,
the fact that bdc-5.2.9 is stimulated by very low concentrations of the iapp peptide ks20 suggests that this peptide may contain the natural ligand for bdc-5.2.9 .
the hormone peptide is highlighted ( white lettering on black background ) and the region identified by ion - trap mass spectrometry is underlined .
b : ifn- responses of bdc-5.2.9 and the insulin - reactive clone pd-12.4.4 to various concentrations of the peptide antigens iapp ks20 (  ) , gr20/rt20 (  ) , ins b923 (  ) , or the -cell membrane ( -mem ) positive control (  ) .
although bdc-5.2.9 did not respond at all to the insulin peptide b923 or the iapp - peptides gr20 and rt20 , it responded to iapp ks20 at concentrations as low as 0.01 g / ml . the insulin - reactive t - cell clone pd-124.4 responded to ins b923 and to the -mem control but not to any iapp peptides .
data are representative of at least three separate experiments . to further validate iapp as the antigen for the bdc-5.2.9 t - cell clone
, we analyzed ifn- responses to islet cells from either nod ( iapp ) or nod.iapp mice .
the t - cell clone bdc-2.5 was used as a positive control because it responds to a different -cell antigen , chga ( 5 ) . as illustrated in fig .
4 , both clones responded to antigen present in wild - type islets from nod mice ( iapp ) ( fig .
4a ) , and the chga - reactive clone bdc-2.5 also responded to islets from nod.iapp mice ( fig .
in contrast , there was no stimulation of bdc-5.2.9 by iapp - deficient islets ( fig .
4b ) , indicating that islets from iapp mice lack the antigen for this clone .
ifn- response of bdc-2.5 ( chga reactive ) ( a ) and bdc-5.2.9 ( b ) to various numbers of islet cells obtained from nod.iapp (  ) and nod.iapp (  ) mice .
data are representative of three separate experiments , in which each cell concentration was measured in duplicate
. the highly aggressive nature of bdc-5.2.9 in disease induction can be demonstrated by adoptive transfer into young nod and nod.scid mice . to investigate the in vivo islet reactivity of bdc-5.2.9 in iapp - deficient mice , adoptive transfers with this clone were made into young ( aged < 2 weeks ) iapp recipients .
bdc-2.5 was again used as a positive control and rapidly induced disease in both iapp and iapp recipients ( fig .
when bdc-5.2.9 was used in transfers , disease developed in most of the nod.iapp mice within 3 weeks , but there was no diabetes in iapp - deficient mice for the duration of the experiment , up to 45 days .
we also conducted histological analysis of pancreatic sections from iapp - deficient mice to assess leukocyte infiltration and granulation of islet -cells ( fig .
the spontaneous development of insulitis and hyperglycemia in nod.iapp mice is typical and very similar to that in nod.iapp mice .
transfer experiments were terminated when recipients were ~2 months of age and the pancreatic histology of nod.iapp mice receiving bdc-5.2.9 closely resembled that of aged - matched , uninjected iapp controls , with at least 60% of islets being well granulated and no infiltrate apparent .
in contrast , histology from iapp mice that received the bdc-2.5 t - cell clone showed complete degranulation of -cells .
these results further demonstrate that islets lacking iapp do not contain the antigen for bdc-5.2.9 .
a : bdc-2.5 or bdc-5.2.9 t cells ( 1  10 ) were injected intraperitoneally into 6- to 14-day - old nod or nod.iapp mice , which were monitored for hyperglycemia .
nod or nod.iapp mice that received the bdc-2.5 t - cell clone became diabetic within 3 weeks of transfer .
bdc-5.2.9 transferred disease in nod mice but not in nod.iapp mice ( p = 0.002 ) .
data summarize at least three independent experiments for each clone and indicate that bdc-5.2.9 is diabetogenic upon adoptive transfer in nod mice only when iapp is expressed in the islets .
islets in sections of nod.iapp mice that received bdc-5.2.9 showed no infiltrate and were well granulated ; in contrast , islets from mice that received bdc-2.5 were infiltrated and the islets were degranulated .
histology was based on two mice per group for each of the following groups : iapp mice injected with bdc-5.2.9 ; 3-month - old uninjected iapp mice ; and iapp mice injected with bdc-2.5 .
the bar graph summarizes the islet granulation scores : 3 = well - granulated islet -cells ; 2 = 5075% granulated ; 1 = 2050% granulated ; and 0 = no granulation .
as previously described ( 5 ) , to identify candidate antigens for the t - cell clones , we carried out biochemical fractionation of -cell tumors and mass spectrometric analysis of peak antigenic fractions .
similar to the other clones from the panel , bdc-5.2.9 responds in vitro to pancreatic islet cells or cell extracts of -cell adenomas presented in the context of i - a ( 2 ) .
our starting source of antigen was -cell tumor tissue excised from nod.riptag mice ( 7 ) and separated by differential centrifugation to yield a highly antigenic -cell membrane pellet ( -mem ) ; this preparation also served as the positive control in t - cell activity assays .
after lysis , the membrane pellet was separated by sequential chromatography , first through an sec column and then by rp - hplc .
1 , the presence of antigen was tracked through the ifn- response of the t - cell clone bdc-5.2.9 ( top axis , gray lines ) to protein ( bottom axis , black lines ) in individual fractions .
figure 1a is the chromatogram from sec and shows a single antigenic peak that corresponds to a low  molecular weight protein region .
the antigenic fractions from sec were further purified by rp - hplc , yielding another single peak of antigen activity ( fig .
presence of antigen was determined by measuring the ifn- response ( gray lines ) of the t - cell clone bdc-5.2.9 to small aliquots of individual fractions in the presence of peritoneal exudate cells used as antigen - presenting cells .
the highly enriched and antigenic rp - hplc fractions were subjected to in - solution tryptic digest and were then analyzed on an ion - trap mass spectrometer .
resulting spectra were sequenced and matched to proteins by searching the swiss - prot protein database using the search program spectrum mill .
comparison of the antigen distribution profile with individual protein spectral - intensity values , an approximate indicator for relative protein abundances , singled out iapp as the only candidate .
other proteins did not match the antigen distribution profile ; for example , secretogranin 1 was detected only in nonantigenic fractions 11 and 14 .
we identified two iapp peptides , representing 28% amino acid coverage of prepro - iapp and 70% coverage of the 37-residue peptide hormone ( fig .
the peptide lanflvr was matched with high confidence and the peptide ssnnlgpvlpptnvgsnty was matched with medium confidence .
antigenic chromatographic fractions were digested with trypsin , separated by rp - hplc , and analyzed by ion - trap mass spectrometry ; the resulting spectra were used to search a protein sequence database . a : proteins identified ( right column ) in the highly purified antigenic rp - hplc fraction no . 12 and neighboring fractions with lower antigen content ( fractions 11 , 13 , and 14 , as shown in fig .
the summarized spectral intensities ( left ) are indicative of the relative abundance of a specific protein in a fraction ; darker shading indicates higher intensities .
b and c : ion - trap mass spectra matching the iapp peptides lanflvr ( b ) and ssnnlgpvlpptnvgsnty ( c ) ( swissprot accession no .
to investigate the peptide(s ) targeted by bdc-5.2.9 , we analyzed a series of overlapping iapp peptides , spanning the entire propeptide region ( fig .
one peptide , ks20 , elicited a vigorous response from bdc-5.2.9 and could stimulate the clone at concentrations as low as 10 ng / ml ( 4.6 nmol / l ) ( fig .
3b ) . bdc-5.2.9 did not respond to any other iapp peptide tested or to the insulin peptide b923 .
two neighboring peptides , rt20 and gr20 , share either the c- or n - terminus sequence with ks20 , but neither of these peptides can stimulate bdc-5.2.9 , indicating that the t - cell receptor of this clone recognizes the adjoining sequences .
an insulin - reactive clone , pd-12.4.4 ( 9 ) , was used as a negative control and showed no response to iapp peptides .
the fact that bdc-5.2.9 is stimulated by very low concentrations of the iapp peptide ks20 suggests that this peptide may contain the natural ligand for bdc-5.2.9 .
the hormone peptide is highlighted ( white lettering on black background ) and the region identified by ion - trap mass spectrometry is underlined .
b : ifn- responses of bdc-5.2.9 and the insulin - reactive clone pd-12.4.4 to various concentrations of the peptide antigens iapp ks20 (  ) , gr20/rt20 (  ) , ins b923 (  ) , or the -cell membrane ( -mem ) positive control (  ) .
although bdc-5.2.9 did not respond at all to the insulin peptide b923 or the iapp - peptides gr20 and rt20 , it responded to iapp ks20 at concentrations as low as 0.01 g / ml . the insulin - reactive t - cell clone pd-124.4 responded to ins b923 and to the -mem control but not to any iapp peptides .
to further validate iapp as the antigen for the bdc-5.2.9 t - cell clone , we analyzed ifn- responses to islet cells from either nod ( iapp ) or nod.iapp mice .
the t - cell clone bdc-2.5 was used as a positive control because it responds to a different -cell antigen , chga ( 5 ) . as illustrated in fig .
4 , both clones responded to antigen present in wild - type islets from nod mice ( iapp ) ( fig .
4a ) , and the chga - reactive clone bdc-2.5 also responded to islets from nod.iapp mice ( fig .
4b ) , indicating that islets from iapp mice lack the antigen for this clone .
ifn- response of bdc-2.5 ( chga reactive ) ( a ) and bdc-5.2.9 ( b ) to various numbers of islet cells obtained from nod.iapp (  ) and nod.iapp (  ) mice .
data are representative of three separate experiments , in which each cell concentration was measured in duplicate .
the highly aggressive nature of bdc-5.2.9 in disease induction can be demonstrated by adoptive transfer into young nod and nod.scid mice . to investigate the in vivo islet reactivity of bdc-5.2.9 in iapp - deficient mice , adoptive transfers with this clone were made into young ( aged < 2 weeks ) iapp recipients .
bdc-2.5 was again used as a positive control and rapidly induced disease in both iapp and iapp recipients ( fig .
5a ) . when bdc-5.2.9 was used in transfers , disease developed in most of the nod.iapp mice within 3 weeks , but there was no diabetes in iapp - deficient mice for the duration of the experiment , up to 45 days .
we also conducted histological analysis of pancreatic sections from iapp - deficient mice to assess leukocyte infiltration and granulation of islet -cells ( fig .
the spontaneous development of insulitis and hyperglycemia in nod.iapp mice is typical and very similar to that in nod.iapp mice .
transfer experiments were terminated when recipients were ~2 months of age and the pancreatic histology of nod.iapp mice receiving bdc-5.2.9 closely resembled that of aged - matched , uninjected iapp controls , with at least 60% of islets being well granulated and no infiltrate apparent .
in contrast , histology from iapp mice that received the bdc-2.5 t - cell clone showed complete degranulation of -cells .
these results further demonstrate that islets lacking iapp do not contain the antigen for bdc-5.2.9 .
a : bdc-2.5 or bdc-5.2.9 t cells ( 1  10 ) were injected intraperitoneally into 6- to 14-day - old nod or nod.iapp mice , which were monitored for hyperglycemia .
nod or nod.iapp mice that received the bdc-2.5 t - cell clone became diabetic within 3 weeks of transfer .
bdc-5.2.9 transferred disease in nod mice but not in nod.iapp mice ( p = 0.002 ) .
data summarize at least three independent experiments for each clone and indicate that bdc-5.2.9 is diabetogenic upon adoptive transfer in nod mice only when iapp is expressed in the islets .
islets in sections of nod.iapp mice that received bdc-5.2.9 showed no infiltrate and were well granulated ; in contrast , islets from mice that received bdc-2.5 were infiltrated and the islets were degranulated .
histology was based on two mice per group for each of the following groups : iapp mice injected with bdc-5.2.9 ; 3-month - old uninjected iapp mice ; and iapp mice injected with bdc-2.5 .
the bar graph summarizes the islet granulation scores : 3 = well - granulated islet -cells ; 2 = 5075% granulated ; 1 = 2050% granulated ; and 0 = no granulation .
the activation of autoreactive cd4 t cells through t - cell receptor responses to peptide / major histocompatibility complexes is a major event in autoimmune diseases such as type 1 diabetes .
the identification of -cell antigens , and the peptide epitopes from these antigens , could have important implications for both diagnosis and therapy of type 1 diabetes .
our results reported here demonstrate that iapp is now the third secretory granule protein in islet -cells , after insulin and chga , to be targeted by pathogenic cd4 t cells , and it is possible that other secretory granule proteins ( e.g. , other secretogranins ) also could be antigenic targets in type 1 diabetes .
this peptide hormone is cosecreted with insulin from the -cell granules at a molar ratio of ~1:100 iapp : insulin ( 10 ) .
similar to insulin and chga , iapp is expressed as a larger precursor molecule ( pro - iapp ) , which is then posttranslationally processed within the secretory granules by the enzymes prohormone convertase 2 and carboxypeptidase e ( 11 ) .
iapp is well known as a component of the amyloid plaques found in the pancreatic islets of patients with type 2 diabetes ( 1214 ) . in type 1 diabetes ,
there has been documentation of autoantibodies to iapp in patients , but there was no correlation to disease ( 15 ) . to date , there has been little association of iapp with type 1 diabetes .
( 16 ) in which it was found that a significant proportion of cd8 t cells from hla - a*0201 patients with recent - onset type 1 diabetes respond to a peptide derived from the leader sequence of prepro - iapp .
this group also contributed to a later study in which it was found that a second sequence of prepro - iapp ( ppiapp 917 ) elicited responses from cytotoxic t - lymphocytes of type 1 diabetic patients ( 17 ) .
the other indicator that iapp might have a role in type 1 diabetes , and the first evidence from the nod mouse , was a genetic mapping study in which we reported linkage of the iapp gene with the antigen locus for a cd4 t - cell clone bdc-6.9 ( 18 ) .
although we have not identified an iapp peptide that stimulates this t - cell clone , strong corroborating evidence for iapp as its antigen target was provided by islets from the nod.iapp mouse .
as observed with the bdc-5.2.9 clone , iapp - deficient islets were unable to activate bdc-6.9 ( k.h .
, we have provided definitive evidence that diabetogenic cd4 t cells in the nod mouse react to iapp as an antigen , including the identification of a peptide containing a natural ligand for one of these t cells .
the sequence of secreted iapp is quite similar between mouse and human forms of the molecule , and there is only one amino acid difference between the ks20 mouse and human sequences .
it is not unreasonable , therefore , to assume that human cd4 t cells also may react to iapp peptides presented , for example , by hla - dq8 .
a major function of secretory granule proteins is to participate in the biogenesis of the secretory granules and in the process , these proteins tend to form aggregates ( 19,20 ) .
iapp is of particular interest in this regard because of its propensity to form amyloid deposits in type 2 diabetic patients .
such deposits are not found in type 1 diabetic patients , but it has been demonstrated by others that the initial step in amyloid deposition is the formation of toxic iapp oligomers ( 21 ) .
soluble oligomers of iapp also have been shown to be responsible for activation of the nlrp3 inflammasome that leads to increased interleukin-1 production in type 2 diabetes ( 22 ) . in type 1 diabetes , similar smaller aggregates of iapp
it is quite conceivable that such oligomers would be taken up by antigen - presenting cells and processed in a way that leads to the formation of neoantigenic epitopes .
 objectiveto investigate autoantigens in -cells , we have used a panel of pathogenic t - cell clones that were derived from the nod mouse . 
 our particular focus in this study was on the identification of the target antigen for the highly diabetogenic t - cell clone bdc-5.2.9.research design and methodsto purify -cell antigens , we applied sequential size exclusion chromatography and reverse - phase high - performance liquid chromatography to membrane preparations of -cell tumors . 
 the presence of antigen was monitored by measuring the interferon- production of bdc-5.2.9 in response to chromatographic fractions in the presence of nod antigen - presenting cells . 
 peak antigenic fractions were analyzed by ion - trap mass spectrometry , and candidate proteins were further investigated through peptide analysis and , where possible , testing of islet tissue from gene knockout mice.resultsmass-spectrometric analysis revealed the presence of islet amyloid polypeptide ( iapp ) in antigen - containing fractions . 
 confirmation of iapp as the antigen target was demonstrated by the inability of islets from iapp - deficient mice to stimulate bdc-5.2.9 in vitro and in vivo and by the existence of an iapp - derived peptide that strongly stimulates bcd-5.2.9.conclusionsiapp is the target antigen for the diabetogenic cd4 t - cell clone bdc-5.2.9 . 