chronic myocardial inflammation can be identified as a specific pathogenic mechanism in a significant proportion of dilated cardiomyopathy ( dcm ) patients , either with viral persistence ( e.g. , coxsackievirus , adenovirus , parvovirus b19/b19v ) or in terms of a post - viral anti - cardiac immunity maintained even after viral elimination .
immunohistologically detected intramyocardial inflammation has been identified as a feature of myocarditis with prognostic and therapeutic relevance [ 35 ] .
the t - cell receptor ( tcr ) is a transmembrane heterodimer that recognizes peptides presented by hla molecules .
adaptive immunity depends on genetic recombination , which produces diverse functional tcr gene rearrangements from a pool of discontinuous segments .
the human tcr- locus consists of 65 v ( variable ) , 2 d ( diversity ) , and 13 j ( joining ) segments . as a result ,
10 different tcr- chain rearrangements are available in the human nave t - cell repertoire .
. chronic activation of the immune system targeting specific antigenic epitopes can also evoke clonal tcr- expansions in the inflamed tissues .
a further mechanism that may contribute to the development of clonal tcr- rearrangements is superantigens ( from bacteria or viruses ) by cross - linking the lateral side of the mhc class ii molecule with the v portion of the tcr , thereby stimulating proliferation of specifically reactive t - cells .
experimental data from both autoimmune   and coxsackievirus myocarditis   have shown restricted tcr- rearrangements involving v - b 8.2 and 10 by cdr3 ( complementary determining region 3 ) spectratyping in rodents .
depletion of the respective clonotypes by antibodies or dna vaccines resulted in significant protection from disease development . in light of these findings from experimental myocarditis models
, we sought to investigate whether clonal tcr- restrictions are detectable also in human dcm .
we furthermore investigated the distribution pattern of clonal tcr- rearrangements by analyzing multiple samples ( 3 each ) obtained from 3 cardiac regions ( 9 samples total ) from explanted dcm hearts ( rv : right ventricle , s : septum , lv : left ventricle ) .
we hypothesized that if clonal tcr- rearrangements target particular , but multiple , epitopes , various clonal tcr- rearrangements with a multifocal pattern might be present in dcm hearts .
normal tonsillar tissues obtained at autopsy were used as polyclonal controls of the trc - b pcr method , whereas the t - cell line molt4 served as control for tcr- clonality . from 17 explanted dcm hearts ( 1 female ; age : 48.913.1 years ; lvef : 19.74.9% ) , each 3 myocardial samples ( ca .
50 mg tissue ) were obtained from random locations of the right ventricle ( rv ; rv 13 ) , the septum ( s ) , and the left ventricle ( lv ) at explantation ( 9 total myocardial samples per case ) .
coronary artery disease was excluded in all these patients by the lack of significant coronary stenosis in coronary angiography , and further possible secondary causes ( e.g. , valvular , hypertensive or systemic diseases ) were excluded .
patients subjected to immunomodulatory treatment such as immunosuppression or antiviral interferon treatment were excluded from this study .
single lv myocardial samples from 6 explanted hearts with non - dcm cardiomyopathy entities ( 1 female ; age : 5310 years ; lvef : 194% ; ischemic cardiomyopathy : n=3 ; hypertrophic non - obstructive cardiomyopathy : n=1 ; transposition of the great vessels and ventricular septum defect : n=2 ) served as controls .
universittsmedizin berlin in the framework of the sonderforschungsbereich tr19 in compliance with the helsinki declaration .
the pcr for the detection of tcr- rearrangements was performed as described by assaf et al . .
in brief , dna was extracted from snap - frozen myocardial tissues using the qiagen dna extraction kit ( qiagen , hilden , germany ) according the manufacturer s instructions .
two hundred ng of genomic dna from each sample was subjected to a semi - nested pcr with 2 separate reactions involving the same v consensus primer ( v pan : 5-ctcgaattct(t / g)t(a / t ) ( c / t)tggta(c / t)c ( g / a)(t / a)ca-3 ; 200 ng ) and 2 j different primer sets ( 200 ng each set ) consisting of 6 ( j1 family ; jfs1a ) and 7 ( j2 family ; jfs2a ) j family - specific primers , respectively , employing high - quality high - performance liquid chromatographic purified oligonucleotides .
thirty cycles were carried out with a primer annealing temperature of 60c ( 40 sec ) for the initial 5 cycles and 57c ( 40 sec ) for the remaining 25 cycles . for re - amplification , an aliquot ( 1% ) of the first 2 reactions was used as a template in 2 additional separate pcrs , comprising 40 cycles each with the same annealing temperature profile as described above . the same v primer ( 200 ng )
was used in combination with 2 nested family - specific j primer mixes ( jfs1 and jfs2 ; 200 ng each set ) .
the conditions for denaturation ( 96c , 15 sec ) and primer extension ( 72c , 40 sec ) remained constant through all cycles of the first and second pcr , whereas the concentration of mgcl2 was 2.5
mmol / l in the first and 1.5 mmol / l in the second amplification .
all reactions were carried out in a final volume of 100 l with 0.8 mmol / l of dntps ( 200 mol / l each ) and 2.5 units taq polymerase ( perkin elmer , weiterstadt , germany ) in a thermal cycler ( tc9600 , perkin elmer , weiterstadt , germany ) .
high - resolution genescan analysis of the pcr products was performed with 5-carboxyflourescein - labeled v primers .
aliquots ( 2 l ) of pcr products were mixed with loading buffer ( 2 l formamide , 0.5 l edta ) , and 0.5 l of the internal size standard ( genescan-500 ) were included for precise determination of the length of the amplificates .
after denaturation for 2 min at 90c , the products were separated on a sequencing gel and analyzed by automatic fluorescence quantification and size determination , using the computer program genescan 672 ( abi 373a , applied biosystems , darmstadt , germany ) . in cases with clonal tcr- rearrangements , the re - amplification was repeated with unlabeled v primers to generate unlabeled pcr products , which , after purification , can be used for direct fluorescence dye terminator cycle sequencing .
the sequencing reactions were analyzed on a 377a dna sequencer ( applied biosystems , darmstadt , germany ) after removal of the unincorporated fluorescence dye .
each sequencing reaction was carried out in both directions using the primers vpan and jfs1 or jfs2 , respectively .
the sequences were identified by comparison to published sequences in the international immunogenetics database ( imgt ; website : http://www.imgt.org/ ) .
statistical analysis was performed using jmp statistical discovery software 4.02 ( sas institute , inc . ,
normal tonsillar tissues obtained at autopsy were used as polyclonal controls of the trc - b pcr method , whereas the t - cell line molt4 served as control for tcr- clonality . from 17 explanted dcm hearts ( 1 female ; age : 48.913.1 years ; lvef : 19.74.9% ) , each 3 myocardial samples ( ca .
50 mg tissue ) were obtained from random locations of the right ventricle ( rv ; rv 13 ) , the septum ( s ) , and the left ventricle ( lv ) at explantation ( 9 total myocardial samples per case ) .
coronary artery disease was excluded in all these patients by the lack of significant coronary stenosis in coronary angiography , and further possible secondary causes ( e.g. , valvular , hypertensive or systemic diseases ) were excluded .
patients subjected to immunomodulatory treatment such as immunosuppression or antiviral interferon treatment were excluded from this study .
single lv myocardial samples from 6 explanted hearts with non - dcm cardiomyopathy entities ( 1 female ; age : 5310 years ; lvef : 194% ; ischemic cardiomyopathy : n=3 ; hypertrophic non - obstructive cardiomyopathy : n=1 ; transposition of the great vessels and ventricular septum defect : n=2 ) served as controls .
universittsmedizin berlin in the framework of the sonderforschungsbereich tr19 in compliance with the helsinki declaration .
the pcr for the detection of tcr- rearrangements was performed as described by assaf et al . .
in brief , dna was extracted from snap - frozen myocardial tissues using the qiagen dna extraction kit ( qiagen , hilden , germany ) according the manufacturer s instructions .
two hundred ng of genomic dna from each sample was subjected to a semi - nested pcr with 2 separate reactions involving the same v consensus primer ( v pan : 5-ctcgaattct(t / g)t(a / t ) ( c / t)tggta(c / t)c ( g / a)(t / a)ca-3 ; 200 ng ) and 2 j different primer sets ( 200 ng each set ) consisting of 6 ( j1 family ; jfs1a ) and 7 ( j2 family ; jfs2a ) j family - specific primers , respectively , employing high - quality high - performance liquid chromatographic purified oligonucleotides .
thirty cycles were carried out with a primer annealing temperature of 60c ( 40 sec ) for the initial 5 cycles and 57c ( 40 sec ) for the remaining 25 cycles .
for re - amplification , an aliquot ( 1% ) of the first 2 reactions was used as a template in 2 additional separate pcrs , comprising 40 cycles each with the same annealing temperature profile as described above . the same v primer ( 200 ng )
was used in combination with 2 nested family - specific j primer mixes ( jfs1 and jfs2 ; 200 ng each set ) .
the conditions for denaturation ( 96c , 15 sec ) and primer extension ( 72c , 40 sec ) remained constant through all cycles of the first and second pcr , whereas the concentration of mgcl2 was 2.5
mmol / l in the first and 1.5 mmol / l in the second amplification .
all reactions were carried out in a final volume of 100 l with 0.8 mmol / l of dntps ( 200 mol / l each ) and 2.5 units taq polymerase ( perkin elmer , weiterstadt , germany ) in a thermal cycler ( tc9600 , perkin elmer , weiterstadt , germany ) .
high - resolution genescan analysis of the pcr products was performed with 5-carboxyflourescein - labeled v primers .
aliquots ( 2 l ) of pcr products were mixed with loading buffer ( 2 l formamide , 0.5 l edta ) , and 0.5 l of the internal size standard ( genescan-500 ) were included for precise determination of the length of the amplificates .
after denaturation for 2 min at 90c , the products were separated on a sequencing gel and analyzed by automatic fluorescence quantification and size determination , using the computer program genescan 672
( abi 373a , applied biosystems , darmstadt , germany ) . in cases with clonal tcr- rearrangements ,
the re - amplification was repeated with unlabeled v primers to generate unlabeled pcr products , which , after purification , can be used for direct fluorescence dye terminator cycle sequencing .
the sequencing reactions were analyzed on a 377a dna sequencer ( applied biosystems , darmstadt , germany ) after removal of the unincorporated fluorescence dye .
each sequencing reaction was carried out in both directions using the primers vpan and jfs1 or jfs2 , respectively .
the sequences were identified by comparison to published sequences in the international immunogenetics database ( imgt ; website : http://www.imgt.org/ ) .
statistical analysis was performed using jmp statistical discovery software 4.02 ( sas institute , inc . , cary , nc , usa ) .
the analysis of dna extracted from tonsils gave rise to polyclonal tcr- rearrangements ( gaussian - like distribution of the pcr amplificates ) , with a size range from 234 to 261 bp as revealed by high - resolution genescan analysis ( figure 1a ) .
in contrast , tcr- pcr employing dna extracted from a t - cell line ( molt-4 ) resulted in a single pcr product , characteristic for the presence of a single tcr- rearrangement ( figure 1b ) . in control
myocardial tissues ( non - dcm hearts ) , the genescan analysis of the tcr- pcr products revealed a gaussian - like size distribution consistent with the presence of polyclonal t - cell populations ( figure 1c ) .
in contrast , clonal tcr- rearrangements were detectable in at least 1 myocardial sample in 9 out of 17 dcm cases ( 3 each from rv , lv , and s ) of the dcm hearts ( figure 1d ) . the demographic data of this subset of dcm patients were not statistically different compared to the remaining dcm patients without detectable clonal tcr- rearrangements ( female : 0 ; age : 51.811.3 years ; lvef : 18.95.2% ) .
a total of 20 of the 81 specimens displayed single dominant tcr- pcr products consistent with the presence of clonal t - cell populations .
there was a non - significant tendency for a higher detection rate of t - cell clonality in the samples from the lv compared with the remaining regions of the dcm hearts ( rv : n=4 , s : n=4 , lv : n=6 ) .
interestingly , the sizes of the dominant pcr products were not identical within the same case or among the respective cases .
the sequence analysis of the clonal tcr- pcr - products ( n=20 ) from the 9 explanted dcm hearts showed an involvement of v19.01 segments in 14 of the dominant amplificates ( 70% ) .
further tcr - v segments involved in clonal tcr- rearrangements of dcm hearts were v6 - 1.01 ( n=1 ) , v6 - 3.01 ( n=2 ) , v6 - 5.01 ( n=1 ) , v10 - 3.02 ( n=1 ) , and v19.03 ( n=1 ) .
clonal tcr- rearrangements were present in only 1 of the 9 samples from the different cardiac regions in 3 dcm hearts ( patient code 05 : v6 - 3.01 , 07 : v10 - 3.02 , and 20 : v6 - 1.01 ) , and the remaining 6/9 dcm cases demonstrated clonally rearranged tcr- genes in at least 2 of the 9 different samples investigated from each individual .
an involvement of the same vb - segment was detected in multiple ( at least 2 ) samples in 5/6 ( 83% ) of the dcm patients with v19.01 tcr - v rearrangement ( patients 02 , 13 , 14 , 16 and 17 ) , and different v segments were found in the remaining identified clones . only 1 out of 6 cases with a clonal v19.01 rearrangement ( code no . 21 )
the results of sequence analysis of clonal tcr- pcr - products identified by genescan analysis in these explanted dcm hearts are summarized in table 1 .
one important finding of our investigation is that the presence of clonal tcr- rearrangements is a disease - associated phenomenon in 9 out of 17 dcm patients , but not in other non - dcm cardiomyopathies .
these first data on the application of this analysis system of clonal tcr- rearrangements are limited by the fact that only single myocardial samples of the control hearts were investigated , but multiple samples ( 3 each ) were analyzed from 3 cardiac regions ( 9 samples total ) from explanted dcm hearts ( rv : right ventricle , s : septum , lv : left ventricle ) .
however , the presence of disease - specific tcr- dominances was also confirmed by different methodological approaches in further investigations
. one major goal of this study was to determine the applicability of the methodology published by assaf et al .
, followed by high - resolution genescan and direct sequencing . secondly , we sought to determine the distribution of detectable clonal tcr- rearrangements in dcm hearts .
analysis of 9 different tissue samples ( 3 each from the rv , s , and lv ) revealed that these clonal t - cell populations were not identically distributed within the respective dcm hearts , albeit with a clear predominance of v 19.01 segments . the clear predominance of the v19.01 segment suggests that a common antigen may evoke and maintain such a selective clonal tcr- expansion in the majority of dcm cases .
clonal tcr- rearrangements with further tcr- segments ( v6 - 1.01 , v6 - 3.01 , v6 - 5.01 , v10 - 3.02 and v19.03 ) were identified less frequently .
however , we were not able to confirm findings by luppi et al on the restricted usage of v 3 , 7 , and 13.1 families
. firstly , this discrepancy may result from different patient cohorts . whereas luppi et al analyzed 3 children with fulminant myocarditis , we analyzed adult dcm patients with a chronic history of symptoms at end - stage heart failure . furthermore , methodological differences are also important to consider .
the cdr3 spectratyping used by luppi et al strongly depends on the pcr results from 24 different tcr- pcrs obtained from healthy controls ( ie , peripheral blood lymphocytes ) , and restricted tcr - v usage is interpreted as the relative increase of distinct pcr products in impaired tissues .
in contrast , a determination of clonal tcr- rearrangements is possible by use of the tcr- pcr system .
this family - specific pcr with multiple primers , however , may be hampered by pseudoclonality issues . nonetheless , this tcr- pcr approach can discriminate between polyclonal and oligoclonal tcr- compositions without the need of interpretations based on a control population .
interestingly , whereas the same vb - segment was involved in multiple ( at least 2 ) samples in 5/6 ( 83% ) of the dcm patients with v19.01 tcr - v rearrangements , different v segments were found with respect to the remaining identified clones ( v6 - 1.01 , v6 - 3.01 , v6 - 5.01 , v10 - 3.02 , v19.03 ) .
these data are consistent with clonal expansion of t - cells at multiple intramyocardial foci , and rather incompatible with a diffuse infiltration of the whole dcm heart with a single t - cell population
. our data may be furthermore compatible with the typically non - homogeneously distributed t - lymphocytic clusters suggestive of focal expansion and myocytolysis in dcm .
different tcr - v skewing in different cardiac regions were also confirmed by cdr3 spectratyping in 2 out of 3 pediatric patients with acute myocarditis .
the clear predominance of the v19.01 segment suggests that a common antigen may evoke and maintain such a selective clonal tcr- expansion in the majority of dcm cases .
clonal tcr- rearrangements with further tcr- segments ( v6 - 1.01 , v6 - 3.01 , v6 - 5.01 , v10 - 3.02 and v19.03 ) were identified less frequently .
the multifocal presence of different t - cell clones might be a decisively important issue for the design of tcr - based immunotherapies in dcm , since such approaches would need to target not just one , but several , different clonal t - cell populations simultaneously . in line with these considerations ,
matsumoto et al reported that only the combined deletion of both tcr - v 8.2 and tcr - v 10 clones proved efficacious in experimental autoimmune myocarditis , while the approach of a single tcr - v deletion did not improve disease severity .
these insights indicate that multiple antigens may be responsible for clonal expansion of reactive t - cells , and warrant further investigations on the respective epitopes .
clonal expansion of t - lymphocytes in dcm may occur in response to specific stimulation by particular epitopes .
potential candidates may be either viral proteins in the framework of antiviral immunity , or myocardial antigens such as in molecular mimicry . given the insights from both viral   and autoimmune myocarditis , both options may also hold true for the appearance of clonal t - cell populations in human dcm .
the focal nature of viral infection and cell - to - cell viral spreading   might be a further factor contributing to the observed multifocal clonal tcr- rearrangements in dcm .
regarding autoimmunity , it is important to consider that deletion of self - reactive t - cells in the thymus is not complete , and a minority of self - reactive t - cells may also surpass peripheral selection mechanisms .
these potentially self - reactive t - cells can be activated when encountering autoantigens , which are usually not accessible to or are ignored by circulating t - cells . with the ensuing tissue destruction in viral myocarditis
, cryptic myocardial antigens can be presented to the immune system , which may evoke local recruitment and clonal expansion of self - reactive t - cells targeting these epitopes .
an association of findings of skewed cdr3 spectratyping with enteroviral protein ( vp-1 ) expression has been reported in myocarditis patients .
these conclusions were drawn by associations to vp-1 ( enteroviral protein-1 ) immunoreactivity , which was reported in all cardiac samples ( 100% ) . however , pcr failed to confirm enteroviral genomes in these tissues .
the presence of enteroviral genomes assessed by pcr , however , is reported in about 1020% of myocarditis and dcm patients .
sensitivity and , especially , specificity of vp-1 immunostaining may be an important issue to solve this discrepancy .
further cardiotropic viruses such as adenovirus and parvovirus b19 ( b19v ) have been identified even more frequently in myocarditis and dcm patients in recent investigations .
these viral genomes were not investigated in the aforementioned study by luppi et al . .
on the other hand , the debate on the pathogenic and prognostic significance of the presence of b19v genomes in myocardial tissues is controversial .
it may be deduced that the antigen - specific t - cells reactive to these proteins might be different from the t - cell clones targeting enteroviral vp-1 , with relevance also for dcm patients with multiple viral infections .
viral infection and anti - cardiac immunity are involved in the pathogenesis and prognosis of dilated cardiomyopathy ( dcm ) .
the disease - associated presence of clonal tcr- rearrangements , as investigated by semi - nested pcr , genescan , and direct sequencing , indicates a pathogenic relevance of this finding in dcm , since it was not detected in non - dcm cardiomyopathies .
the predominance of v 19.01 segments suggests that the immune response in dcm patients targets particular epitopes .
this finding is consistent with immunity targeting particular antigens involved in dcm , which leads to intramyocardial expansion of reactive t - cell clones .
however , the partly heterogenic tcr- populations in various myocardial samples from the respective cases support the notion that t - cell immunity may target multiple epitopes in human dcm .
further analyses are warranted to investigate the prognostic relevance of clonal tcr- rearrangements , and the impact of immunomodulatory treatment strategies in dcm patients .
these insights might also be interesting for the analysis of t - cell - mediated cardiac allograft rejection .
 backgroundviral infection and anti - cardiac immunity are involved in the pathogenesis of dilated cardiomyopathy ( dcm ) . immunity targeting particular antigens 
 may evoke expansion of reactive t - cell clones.material/methodsmyocardial tissues from explanted hearts were investigated for clonal t - cell - receptor- ( tcr- )  rearrangements by an established semi - nested polymerase chain reaction ( pcr ) , followed by high - resolution genescan analysis and direct sequencing . from 17 explanted dcm hearts , 
 3 myocardial samples each were obtained from the right ventricle , the septum , and the left ventricle ( total : 9 myocardial samples per case ) . six explanted hearts with non - dcm cardiomyopathy entities served as controls.resultsgenescan analysis revealed polyclonal tcr- rearrangements in all controls . 
 in contrast , at least 1 myocardial sample in 9 out of 17 dcm hearts ( total : 20 of the 81 dcm specimens ) displayed single dominant tcr- pcr products consistent with the presence of clonal t - cell populations 
 . direct sequencing of the clonal tcr- pcr - products disclosed an involvement of vb 19.01 segments in 14 of the dominant amplificates ( 70% ) . 
 further tcr - v segments involved in clonal tcr- rearrangements of dcm hearts were v 6 - 1.01 ( n=1 ) , v 6 - 3.01 ( n=2 ) , v 6 - 5.01 ( n=1 ) , v 10 - 3.02 ( n=1 ) , and v 19.03 ( n=1).conclusionsthe detectability of clonal tcr- rearrangements indicates a pathogenic relevance of this finding in dcm . 
 the predominance of v 19.01 segments suggests that the immune response in dcm patients targets particular epitopes . 
 however , the partly heterogenic tcr- populations in various myocardial samples from the respective cases support the notion that t - cell immunity may target multiple epitopes in human dcm . 
Background
Material and Methods
Patients
PCR for the detection of clonal TCR- rearrangements
Statistical analysis
Results
Discussion
Conclusions