t1d is a chronic autoimmune disease where cd4 + and cd8 + t cells recognizing islet autoantigens are likely the mediators of selective destruction of pancreatic islet beta cells .
although direct demonstration of the prominent role of t cells in the disease progression is provided only in animal models , the preclinical period of the disease in humans is marked by the presence of circulating islet - related autoantibodies to beta cell antigens including insulin , glutamic acid decarboxylase ( gad ) , isoforms gad65 and gad67 , the insulinoma - associated antigen ( ia2)/tyrosine phosphatase - like molecule ,   ia-2  or phogrin , and proinsulin .   from the 1990s
onwards several laboratories produced an increasing number of reports regarding the detection of t cells directed against these antigens in the peripheral blood .   
the first attempts employed [ h]thymidine incorporation / proliferation assays setup with pbmc of t1d patients ( at onset or long - standing ) and their  high - risk 
subsequently , elispot assays were implemented for measuring cell - mediated immune ( cmi ) responses in t1d [ 35 ] and immunoblot assays .
costimulatory anti - cd28 antibodies were shown to enhance autoreactive t cell responses to gad65 peptides in t1d patients , while , in a previous set of experiments , the expansion of the gad65 ( whole molecule ) reactive t cells was costimulation dependent in healthy controls , as opposed to t1d patients . nevertheless ,   t cell results were largely inconclusive because autoantigen - specific t cells in an in vitro expansion   could indeed be grown both from patients and controls , as evidenced by 4 international workshops of the  immunology diabetes society  and by   multicenter - blinded control trials , organized under the auspices of the  immune tolerance network 
several explanations were put forward for justifying these difficulties including   their low precursor frequency , the inhability to identify them from the vast excess of t cells , and their low to moderate affinity   to self - antigens .
cytotoxicity assays , set in vitro , offered proofs that t lymphocytes are potentially able to kill target cells also in vivo . to this end
hla - a*0201 restricted cd8 + cytotoxic t lymphocytes , specific for a gad65 decapeptide ( 114123 ) , were first detected in pbmc of recent onset t1d patients and in  high - risk  ( ica+ ) individuals by using the classical [ cr ] release cytotoxicity assay .    in more recent investigations elispot assays revealed ifn- production when pbmc from t1d patients were challenged with proinsulin peptides ( 3039 ; 3442 ; 4150 )   and the amyloid polypeptide precursor protein ( ppiapp513 ) peptides .
the nonapeptides ppiapp917 , igrp152160 , and igrp215223 from the islet - specific glucose-6-phosphatase catalytic subunit related protein   and nonapeptides 172180 and 482490   from the islet autoantigen ia-2   that would bind to and stabilize the hla - a2 molecules were also identified .
mhc tetramer technology was initially introduced to target antigen - specific cd4 + t cells in patients with viral ,   bacterial infections , tumors .   in reference to human autoimmunity class ii tetramers
successfully detected gad65 , proinsulin , ia-2 ,   and preproinsulin   reactive cd4 +   t cells in pbmc of t1d patients ,   low percentages of cd4 + t cells autoreactive to gad65 , the melanocyte differentiation antigen tyrosinase and the testis tumor antigen ny - eso-1 ( epitope 120131 ) in pbmc of healthy individuals , and cd4 + gluten - specific t cells   in pbmc of celiac disease patients .  
the hla class i tetramer technology successfully detected circulating cd8 + t lymphocytes autoreactive to the    melan a autoantigen in patients with vitiligo ,   the pbc - e2 autoantigen in patients with primary biliary cirrhosis ( pbc ) ,   vimentin in patients who were heart transplanted , and   insulin beta chain nonapeptide ( insb10 - 18 ) . in a recent investigation hla - a*0201 gad65 ( 114122 ) pentamers detected an increased percentage of autoreactive t cells in the cd45ro+ subset in t1d patients as compared with controls . in long - standing t1d patients who , after islet transplantation , have a loss of islet allograft and recurrent autoimmunity a high frequency of gad65-specific t cell clones was found within the expanded autoreactive memory t cell compartment .   in the light of all the aforegoing , we attempted in this preliminary study to device a   more sensitive methodology than those currently available for measuring cmi responses and , in particular , gad65 autoreactive t cells in t1d .
preliminary data indicate that it is possible to implement an assay that still will require appropriate standardization before being used in large scale screening programs .   in our protocol , after stimulation of the cells with the gad65 114 - 122 epitope , we successfully detected a percentage of cd8 + gad autoreactive t cells in a sample population including 15 t1d patients ( 9 newly diagnosed and 6 long - standing ) by using hla class i tetramers .
9 hla - a*0201 positive pediatric patients ( 4 males and 5 females , age of onset range 9.2 years to 16.4 years , mean 12.8 years ) were recruited from lazio region at the onset of t1d at the unit of pediatric endocrine autoimmune diseases at the children 's hospital bambino ges , rome ( table 1 ) .
we also included 6 long - term hla - a*0201 positive pediatric t1d patients ( between 8 months to 4 years and 5 months after diagnosis ) .
the control population was recruited at the blood transfusion center of our hospital including 10 hla - a*0201 positive nondiabetic healthy blood donors .  
they had no history of autoimmunity and no   islet - related autoantibodies were detected in their serum . after obtaining informed consent from parents of childrens and normal controls ,
pbmc were separated by ficoll - hypaque   ( histopaque , sigma - aldrich chemical c , st louis , mo , usa ) from 510  ml sodium - heparinized venous blood samples , washed twice in pbs , and   then frozen down in liquid nitrogen .
hla - a2 typing was initially performed by flow cytometry analysis and subsequently confirmed by standard allele - specific pcr . in the initial screening , 1  10 cells were incubated , for 30 minutes in ice , with an anti - hla - a2 mouse mab ( 1 : 10 dilution , one lambda , inc , canoga park , ca , usa ) .
after washing by centrifugation at 1500  rpm for 5 minutes at room temperature ( rt ) in wash buffer [ 0,1% sodium azide , 2% fetal bovine serum ( fbs , hyclone , south logan , ut , usa ) in pbs ] , cells were resuspended in the residual volume ( approximately 50  l )
. 1  l of fluorescent- ( fitc- ) conjugated goat anti - mouse igg ( fc  fragment specific antibody ) ( jackson immunoresearch laboratories inc , west grove , pa , usa ) was added .
cells were then incubated in ice for 30 minutes in the dark , washed in wash buffer , and acquired for the analysis in a becton & dickinson facscalibur flow cytometer equipped with the cell quest software program .
hla - a2 subtyping was carried out using a molecular system ( genovision inc . ,
west chester , pa , usa ) ; high - resolution dna - based hla typing of polymorphic class i loci hla - a , -b , and -c was also carried out according to a reverse line blot system . at the time of tetramer assay , cells were thawed and resuspended , at a density of 1  10/ml , in rpmi-1640 ( gibco / brl , invitrogen , gaithersburg , ca , usa ) , supplemented with 2 
mmol / l l - glutamine , 100  g / ml penicillin / streptomycin , and 10% v / v fbs ; cells were then cultured in the presence of the gad65 peptide aa 114122 ( vmnillqyv ) at a concentration of 30  g / ml , for 4 days in 24-well round - bottomed plates ( falcon , labware bd biosciences , oxnard , ca , usa ) ( 1  10  cells / well ) .  
the gad65 nonapeptide had been selected for its high affinity binding to hla a*0201 in an hla peptide motif search database ( http://www-bimas.cit.nih.gov/molbio/hla_bind ) .  
in parallel experiments control cell cultures were set up by incubating pbmc from the same individual with il-2 ( 25  iu / ml , sigma ) for 4 days , in place of the gad65 peptide , in order to ensure that pbmc would live for the entire culture period prior to the flow cytometry analysis ( vide infra ) . at the end of the 4 days ,
pbmc , either stimulated with the gad65 peptide or incubated with il-2 , were washed in calcium - magnesium free dulbecco 's phosphate - buffered saline 1x ( euroclone , wethersby , west york , uk ) , by centrifugation at 1500  rpm for 5 minutes at rt .  
this washing was introduced to remove the excess of gad65 peptide and il-2 from the culture , so to maximally reduce the risk of nonspecific binding , when pbmc will subsequently be stained with labeled tetramers .
the two sets of pbmc were cultured for additional two days with il-2 ( 25  iu / ml ) in the same medium ( rpmi-1640 , 2  mmol / l l - glutamine , 100  g / ml penicillin / streptomycin , 10% v / v fbs ) .
phycoerythrin ( pe ) labeled tetramers were generated , using either the gad65 peptide ( vmnillqyv ) or the flu peptide ( gilgfvftl ) , known to have a high - affinity binding with hla - a*0201 ( purchased from proimmune limited , oxford , uk ) . on day 6 ,
approximately 5  10 cells , stimulated with the gad65 peptide or cultured with il-2 , were allocated for staining conditions .
cells were washed by centrifugation at 1500  rpm for 5 minutes at rt in wash buffer ( 0.1% sodium azide , 2% fbs in pbs )   and resuspended in the residual volume ( approximately 50  l ) . 1  l of phycoerithrin ( pe ) labeled gad65 peptide ( vmnillqyv ) tetramer was added to each cell preparation and incubated in ice for 30 minutes in the dark , then washed in wash buffer .
1  l of mab anti - human cd8 ( fitc labelled ( becton & dickinson , pharmingen , san diego , ca , usa ) ) and 5  l of mab anti - human cd3 [ allophycocyanin ( apc ) labelled ( becton & dickinson ) ] were added for further staining the different cell preparations . in alternative for cd8 staining ,
1  l of biotinylated mab anti - human cd8 ( 1 : 10 , becton dickinson ) was used followed by streptavidin cychrome ( cys5 ) conjugate ( 1 : 10 , southern biotechnology , birmingham , al , usa ) .  
cells were incubated in ice for at least 30 minutes in the dark , washed twice in wash buffer , and then immediately acquired for the analysis in a beckton & dickinson facscalibur flow cytometer and cellquest software program . in order to verify the sensitivity of the hla - a*0201 tetramer assay , the flu nonapeptide ( gilgfvftl )   was used to stimulate pbmc of one hla - a*0201 normal individual .
this test was performed in order to verify that we could obtain an increased sensitivity of the flu tetramer staining after flu peptide stimulation .
the staining with the flu tetramer was carried out either by direct assay ex vivo   or after stimulation of the same pbmc first with the flu peptide ( 3.5  g / ml ) for 4 days followed , after washing the cells , by an incubation with il-2 for 2 days .
a positive control of stimulation was introduced in all the experiments in order to prove that in vitro stimulation worked in all subjects , including those from whom no gad specific t cells were detected .  
the presence of flu tetramer positive cells was assessed in randomly selected samples by staining after flu peptide stimulation . in order to verify the specificity of the reactivity of the gad65 hla a*0201 tetramer in the detection of gad65 nonapeptide reactive t cells and as control of nonspecific
binding , pbmc of randomly selected t1d patients and controls were cultured separately with the gad65 nonapeptide , the flu nonapeptide , and il-2 .
the flu and gad65 nonapeptides were used at the same concentration ( 1030  g / ml ) . after 4 days , pbmc , cultured in the 3 different conditions , were washed and subsequently incubated with il-2 alone for an additional 2 days .
cells were then recovered , washed , and stained with the gad65 nonapeptide hla a*0201 tetramers and analyzed by flow cytometry .
differences in the percentages of cd3+/cd8+/gad65 reactive t cells between normal controls and t1d patients in each ( il-2 and gad65 peptide )   stimulated pbmc populations   were assessed by mann - whitney - u test .  
variations in the response of pbmc , either treated with il-2 or stimulated with the gad65 peptide within each group ( i.e. , controls versus t1d patients ) , were evaluated by wilcoxon 's test .  
it is well known that hla class i   molecules bind   peptides 810 aminoacids long , but preferentially nonapeptides [ 35 , 36 ] . after database search for nonamers of the gad65 protein sequence ( p48320 , ncbi database ) , we selected peptide gad65 114 - 122 , on the basis of its high score of affinity binding ( 1080.239 ) to hla - a*0201 .
direct staining ex vivo with hla a*0201 flu peptide tetramers of pbmc of a normal hla a*0201 positive individual detected 0.03% of cd3+/cd8+/flu reactive t cells .  
the percentage increased to 0.23% , when pbmc of the same individual were first stimulated for 4 days with the same flu peptide ( data not shown ) .
normal controlsin pbmc of normal individuals no significant variations were found in the percentage of cd3/cd8/gad65 reactive t cells between pbmc cultured with either il-2 or the gad65 peptide ( mann - withney test versus pbmc il-2 treated p = .05 , figure 1 ) . in pbmc of normal individuals
no significant variations were found in the percentage of cd3/cd8/gad65 reactive t cells between pbmc cultured with either il-2 or the gad65 peptide ( mann - withney test versus pbmc il-2 treated p = .05 , figure 1 ) .
td patientsin pbmc of t1d patients the percentage of cd3+/cd8+/gad65 reactive t cells were significantly more pronounced in comparison to those registered with pbmc of controls after stimulation with the same gad65 peptide ( p = .001 t1d versus controls , figures 1 and 2 ) . 

figure 2 shows pbmc of a representative t1d patient treated with il-2 ( panel ( a ) , ( b ) , ( c ) ) and with gad65 peptide ( panel ( d ) , ( e ) , ( f ) ) .   the analysis was conducted only in r1 region ( living cells as evidenced by morphological parameters ) and in the r2 region ( cd3+/cd8 + cells ) .  
the population of cd3+/cd8+/gad65 reactive cells was shown in panel ( f ) ( 4.04% of total cell population).il-2 treatment affected differentially the detection of cd3+/cd8+/gad65 reactive t cells in t1d patients versus control ( p = .07 t1d patients versus controls ) ( figure 1 ) . in pbmc of t1d patients the percentage of cd3+/cd8+/gad65 reactive t cells were significantly more pronounced in comparison to those registered with pbmc of controls after stimulation with the same gad65 peptide ( p = .001 t1d versus controls , figures 1 and 2 ) . 

figure 2 shows pbmc of a representative t1d patient treated with il-2 ( panel ( a ) , ( b ) , ( c ) ) and with gad65 peptide ( panel ( d ) , ( e ) , ( f ) ) .  
the analysis was conducted only in r1 region ( living cells as evidenced by morphological parameters ) and in the r2 region ( cd3+/cd8 + cells ) .  
the population of cd3+/cd8+/gad65 reactive cells was shown in panel ( f ) ( 4.04% of total cell population ) .
il-2 treatment affected differentially the detection of cd3+/cd8+/gad65 reactive t cells in t1d patients versus control ( p = .07 t1d patients versus controls ) ( figure 1 ) .
specificity of the gad65 peptide responsethe percentage of cd3+/cd8+/gad65 reactive t cells estimated in pbmc of single patients after gad65 peptide stimulation was higher than after flu peptide   stimulation or when cultured with il-2 alone ( data not shown ) .  
with pbmc of the normal controls , the percentage of cd3/cd8/gad65 reactive t cells was comparable in the 3 stimulation conditions . in principle , we   assume that   the specific reactivity could be detectable in each single sample only after gad65 peptide stimulation .  
this test is an alternative to control the nonspecific binding of tetramer to t cells with the use of an irrelevant peptide .
the percentage of cd3+/cd8+/gad65 reactive t cells estimated in pbmc of single patients after gad65 peptide stimulation was higher than after flu peptide   stimulation or when cultured with il-2 alone ( data not shown ) .  
with pbmc of the normal controls , the percentage of cd3/cd8/gad65 reactive t cells was comparable in the 3 stimulation conditions . in principle , we   assume that   the specific reactivity could be detectable in each single sample only after gad65 peptide stimulation .  
this test is an alternative to control the nonspecific binding of tetramer to t cells with the use of an irrelevant peptide .
tetramers containing autoantigenic peptides have clinical utility in autoimmunity for diagnostic and , potentially , therapeutic applications [ 1929 ] .  
previous studies [ 19 , 20 ] clearly demonstrated that mhc class ii tetramers can efficiently detect gad65 reactive cd4 + t cells in pbmc of t1d patients .  
gad65-specific hla dr0401-restricted clones could be even derived from a diabetic patient using tetramers as stimulating agent .  
these encouraging   data prompted us to trace gad65 autoreactive t   cells in t1d ; in the present investigation hla - a*0201 tetramers were therefore constructed with a 
natural  peptide ( gad65 ( aa114122 ) , half - time disassociation 1080,239 ) .
this peptide has been used in previous class i tetramer investigations [ 28 , 29 ] and now employed to establish a more sensitive assay .
we could have chosen the gad65 decamer ( aa114123 )   but the peptide ranked low in the affinity binding scale ( half time disassociation , 35.012 ) .  
in addition , the terminal valine in position 122 seems to be relevant for the antigenic presentation of the motif , as the very low - affinity binding of the nonapeptide 115123 to hla a*0201 ( half time disassociation , 0.316 ) demonstrates .
peptides for assembling tetramers   employed in previous investigations in order to increase their affinity binding to the tetramer .
the first is by direct ex vivo staining with labeled peptide / tetramer complexes ;   in the second method pbmc are preincubated and stimulated with the peptide and then stained with the labeled peptide / tetramer or pentamer complexes [ 2529 ] .
we have chosen the second approach , because we believe that preincubation with the peptide might induce a limited specific t cell expansion in vitro and , therefore , may increase the chance to  awake  autoreactive cd8 + t cells .
novel steps in the experimental procedure were designed as follows : ( 1 ) a reduced period of antigen - specific cell expansion ( 4 days rather than 21 days , 28 days , 10 days , time course selected in other hla class i tetramer assays ) ; ( 2 ) an intermediate washing step after cells were stimulated with the gad65 nonapeptide ; ( 3 ) a 2-day incubation with il-2 in the absence of the gad65 nonapeptide ; and ( 4 ) a final washing step , before staining the cells with the labeled tetramers .
in addition , we relied only on the autologous apcs , present in the pool of the pbmc , not prepulsed with the nonapeptide , as it was done in   previous assays , and we set up parallel pbmc cultures either with il-2 alone , an internal cell culture control .  
the short period of peptide stimulation has the advantage that cultures are terminated before clonal expansion and/or  in vitro 
we need also to underline that the   possibility of testing frozen / defrosted pbmc is of paramount importance when planning large retrospective epidemiological investigations .    by applying these novel experimental conditions , the recovery of cd3+/cd8+/gad65 reactive t cells in t1d patients ranged from 0.5% to 15.6% after gad65 peptide stimulation of pbmc . to our knowledge , this is the highest percentage of autoreactive cd8 t cells ever  captured  by hla class i tetramers in pbmc of patients with a human autoimmune disease ( 0,1% to 1% in vitiligo   4,2% in heart transplantation ; up to 0.4% of cd8 + cd45ro+ t cells in newly diagnosed t1d patients ; between 2.6% and 8.3% within the ki-67+cd8 + cell fraction ( 1.65%   + /0.25%   of the total cd8 + lymphocytes ) in islet transplanted t1d patients ) .
this can be achieved by testing pbmc of a large number of t1d patients , normal controls , and especially prediabetic high - risk individuals .
a valid statistical evaluation of results will help to establish an appropriate cutoff value of positivity in the assay .
 type 1 diabetes ( t1d ) is an autoimmune disease , in which pancreatic  cells are destroyed in genetically predisposed individuals . 
 while the direct contribution of autoantibodies to the disease pathogenesis is controversial , it is generally recognised that the mechanism of  cell destruction is mediated by autoreactive t cells that had escaped the thymic selection . 
 we aimed to design a method to detect circulating cd8 + t cells autoreactive against an epitope of the glutamic acid decarboxylase autoantigen , isoform 65 ( gad65 ) ex vivo in t1d patients by using hla class i tetramers . 
 low frequencies of gad65 peptide - specific cd8 + cytotoxic t lymphocytes were detected in peripheral blood lymphocytes ( pbmc ) of normal controls after gad65 peptide - specific stimulation . 
 conversely , their frequencies were significantly higher than in controls in pbmc of t1d patients after gad65 peptide stimulation . these preliminary data are encouraging in order to develop a reliable assay to be employed in large - scale screening studies . 