
 ethics statement - this study was in accordance with the ethical 
 standards of the national ethical clearance committee of brazil as well as with the 
 ethical committee for human research of the oswaldo cruz foundation ( cep - fiocruz ) and 
 the evandro chagas clinical research institute ( cep - ipec / fiocruz ) and with the helsinki 
 declaration of 1975 , as revised in 1983 .
patients and controls - the study included healthy subjects ( hs ) ( n = 
 18 ) and two cohorts of cl patients : patients with active disease ( pad ) ( n = 14 ) , showing 
 ulcerated skin lesions , and pcc ( n = 11 ) , evaluated eighty day after the beginning of 
 treatment , at which time clinical cure was defined as full epithelialisation of 
 ulcerated lesions , regression of crust , desquamation and infiltration .
all adults 
 included were between 18 - 60 years of age with a mean age of 35.1  13.2 years .
all hs enrolled in this study were from nonendemic areas in rj , showing neither a 
 previous history of leishmaniasis nor any other comorbidity .
2000 ) and were 
 recruited at the leishmaniasis surveillance laboratory , ipec / fiocruz .
the diagnosis of leishmaniasis was based on clinical and epidemiological evidence ; 
 positive montenegro skin test ( mst ) and parasitological exams .
all patients were 
 submitted to meglumine antimoniate treatment independent of their enrolment in our 
 study , according to the guidelines of the brazilian ministry of health ( svs / ms 2010 ) .
we investigated clinical and 
 epidemiologic information , such as age , sex , number and diameter of lesion at active 
 phase and evolution period .
patients who presented with comorbidity , reactivation of cl , 
 reinfection or nonresponse to antimonial therapy were excluded from the study . 

analysis of the v repertoire and cell phenotyping - venous blood was 
 collected from cl patients and hs in heparinised tubes and peripheral blood mononuclear 
 cells ( pbmc ) were obtained by ficoll - hypaque density gradient centrifugation ( sigma 
 aldrich , usa ) .
v repertoire and cell phenotype staining was performed as previously 
 described ( barral - netto et al .
, we 
 performed six - colour cell surface staining using the iotest beta mark tcr - v repertoire 
 kit ( beckman coulter inc , usa ) , which contains eight tubes with three antibodies in each 
 against the following tcr - v chains : v1 , v2 , v3 , v4 , v5.1 , v5.2 , v5.3 , v7.1 , 
 v7.2 , v8 , v9 , v11 , v12 , v13.1 , v13.2 , v13.6 , v14 , v16 , v17 , v18 , v20 , 
 v21.3 , v22 , v23 belonging to 19 of 26 v human families known .
these 24 v chains 
 cover 70% of the t - lymphocyte tcr - v repertoire in a normal individual .
the staining 
 protocol included one aliquot of the v repertoire kit , which combines tcr antibodies 
 labelled with fluorescein isothiocyanate ( fitc ) , phycoerythrin ( pe ) or both pe and fitc , 
 with one aliquot of each monoclonal antibody anti - cd8 allophycocyanine ( apc ) , 
 anti - cd45ra pe - texas red , anti - cd27 pe - cyanine 7 ( beckman coulter ) and anti - cd3 
 apc - cyanine 7 ( bd biosciences , usa ) .
this antibody combination allowed us to 
 simultaneously identify the v repertoire of total cd8 t - lymphocytes and the 
 v repertoire of nave , lde , cm and em subsets .
the staining was performed in 
 phosphate - buffered saline containing 0.1% sodium azide ( sigma aldrich ) and 2% foetal 
 calf serum ( sigma aldrich ) and incubated for 30 min at 4c , protected from light and 
 finally washed for subsequent acquisition in a flow cytometer . 

flow cytometry - fifty - thousand events were acquired in by beckman 
 coulter cyan adp ( beckman coulter ) or bd facsaria ii ( becton & dickinson , usa ) flow 
 cytometers .
all flow cytometry data were analysed using kaluza 1.2 software ( beckman 
 coulter ) where the distribution of the cd8 t - lymphocyte tcr - v repertoire 
 was determined . in this manner
, the total cd8t - lymphocytes was determined 
 in a cd3 vs. cd8 dot - plot created from a region encompassing lymphocyte population in a 
 side vs. forward dot - plot .
to evaluate nave , lde , cm and em cd8 + 
 t - lymphocyte subsets , a cd27 vs. cd45ra dot - plot was created based on 
 cd3cd8region .
simultaneously , tcr - v repertoire 
 distributions were determined by analysing the frequency of 24 v families , three for 
 each staining tube based on fitc vs. pe dot - plot , gated on five different populations : 
 total , nave , lde , cm and em cd8t - cell subsets ( fig .
1 ) . to ensure confidence in cytofluorimetric v analyses 
 from different dates , the limits for the regions created in v dot - plots , quadrant 
 markers and histograms were set , each time , based on nonstaining cells and negative 
 isotypic controls .
additionally , fluorescence compensation adjustments were performed 
 based on single - labelled samples . 

1:representative flow cytometry protocol to determine v repertoire of total 
 cd8 + t - lymphocyte and subpopulations .
peripheral blood mononuclear cells from 
 cutaneous leishmaniasis patients were stained ex vivo with 
 cd3-allophycocyanine - cyanine ( apc ) 7 , cd8-apc , cd45ra - pe - texas red , 
 cd27-pe - cyanine 7 and 24 different anti - v monoclonal antibodies conjugated 
 with fluorescein isothiocyanate ( fitc ) , phycoerythrin ( pe ) and fitc - pe .
the 
 lymphocytes were gated on forward ( fsc ) vs. side ( ssc ) scatter dot - plot ( a ) and 
 cd8 + t - lymphocytes were defined by double positive staining to cd3 and cd8 
 ( cd3+cd8 + ) ( b ) . based on lymphocyte gate
we analysed the expression of three 
 different v families ( in each of the 8 tubes ) ( c ) and cd8 + t lymphocyte 
 subsets were defined by cd45ra and cd27 staining as late - differentiated 
 effector ( lde ) ( cd45ra+cd27- ) , early - differentiated ( nave ) ( cd27+cd45ra+ ) , 
 late - differentiated effector memory ( em ) ( cd45ra - cd27- ) and central memory ( cm ) 
 ( cd45ra - cd27 + ) ( d ) .
we also performed the v analysis of nave ( e ) , lde ( f ) , em 
 ( g ) and cm ( h ) cd8 + t - lymphocytes . 
 
 statistical analysis - for a comparison of the means between groups , 
 the mann - whitney u nonparametric test was applied .
correlation 
 analyses were performed using spearman s correlation coefficient and were reported with 
 its associated r and p - value . all statistical calculations and graphical representations 
 of data
were obtained using the graphpad prism v.5.0 software ( graphpad software inc , 
 usa ) .

 clinical characteristics of cl patients - all patients were from 
 endemic areas in rj and were included in this study after informed consent and donation 
 of peripheral blood .
all cl patients received glucantimetherapy , as 
 suggested by the brazilian ministry of health ( svs / ms 
 2010 ) and , at the end of therapy , all of them showed epithelialised lesions 
 with an absence of erythema and were considered clinically cured .
the duration of lesion ranged from one - six months and the largest 
 measured diameter of the ulcers varied from 15 - 60 mm ( mean 42.18  12.51 mm ) .
basic 
 demographic and clinical information of the studied groups are summarised in table . 

tableclinical information of patients included in this study  hspadpccvolunteers ( n ) sex ( m / f)18 11/714 9/511 8/3age ( years)29  9.735.7  13.441  15.1lesions ( n)na11diameter of lesion [ ( mm )
mean  sd]na42  12.5141.4  13.98mst ( mm ) duration of disease [ ( months ) median 
 ( range)]na na11.71  3.6 2 ( 1 - 6)12.3  3.68 2 ( 1 - 5)hs : healthy subjects ; m / f : male / female ; mst : montenegro skin test ; na : not 
 applicable ; pad : patients with active disease ; pcc : patients clinical cured ; 
 sd : standard deviation . 
 
hs : healthy subjects ; m / f : male / female ; mst : montenegro skin test ; na : not 
 applicable ; pad : patients with active disease ; pcc : patients clinical cured ; 
 sd : standard deviation . 

definition of tcr - v repertoire of total cd8 
  t - lymphocytes - due to the diversity of the cd8 
 t - lymphocyte tcr repertoire , the antigenic specificity of these cells and the role they 
 play in a cl - associated immune response is of fundamental importance to assess the 
 preferential expression of certain clones , associating them with active disease and the 
 healing process . to this end , we determined the frequency of total cd8 t - lymphocytes 
 expressing 24 tcr v chains in cl pad and pcc as well as in hs . in these three groups , 
 we observed a polyclonal distribution of these v families . in a comparative analysis 
 between groups
, we observed a significant difference in frequencies of total 
 cd8 t - lymphocytes expressing v2 , v9 , v13.2 , v18 and v23 chains in 
 pad compared to hs and/or to pcc ( fig .
2 ) . pad 
 showed lower frequencies of cd8t - cells expressing v2 ( mean 2.5%  1.9% ) 
 and v13.2 regions ( mean 1.8%  1.5% ) compared to hs ( v2 : mean 5.6%  3.1% ; v13.2 : 
 mean 3.8%  1.6% ; p < 0.01 and p < 0.05 , 
 respectively ) .
conversely , pad showed higher frequencies of cd8 
 t - lymphocytes expressing v9 ( mean 2.9%  0.6% ) and v18 ( mean 1.8%  0.4% ) when 
 compared to hs ( v9 : mean 0.6%  0.1% ; v18 : 0.3%  0.1% ; p < 0.05 
 and p < 0.01 , respectively ) .
these results suggest that during the 
 active phase of disease there is a down - modulation in the frequencies of cd8 
 t - lymphocytes expressing v2 and v13.2 and an up - modulation of cd8 
 t - lymphocytes expressing v9 and v18 . in pcc ,
the normal distribution of v18 appears 
 to be restored , whereas frequencies of cd8 t - lymphocytes expressing v9 
 remain high .
in contrast , pcc showed higher frequencies of total cd8 
 t - lymphocytes expressing v18 ( mean : 1.3%  0.4% ) and v23 ( mean 4.1%  1.7% ) when 
 compared to hs ( v18 : mean 0.3%  0.1% ; v23 : mean 1.3%  1% ; p < 0.05 and p < 
 0.01 , respectively ) , whereas the frequency of cd8 t - lymphocytes expressing 
 v13.2 was higher ( 3.6%  0.85% ) when compared to pad ( mean 1.8%  1.5% ; p < 0.05 ) 
 ( fig .
2:24 v chain repertoire profile of total cd8 + t - lymphocytes . a : v chains 
 1 - 7.1 ; b : v chains 7.2 - 13.6 ; c v chains 14 - 23 . statistical analyses were 
 performed by mann - whitney u test .
hs : healthy individuals ( white 
 bars ; n = 18 ) ; pad : patients with active disease ( grey bars ; n = 14 ) ; pcc : 
 patients clinically cured ( black bars ; n = 11 ) ; * : p < 
 0.05 ; * * : p < 0.01 . 
 
 evaluation of tcr - v repertoire of memory , lde and nave cd8 
  t - lymphocyte subsets - to evaluate a link between the type of 
 cd8 t subsets and the diversity of the v repertoire of these cells , we 
 also performed a detailed analysis of the distribution and alterations of v repertoire 
 in nave and lde , cm and em cd8 t - lymphocytes in cl patients and hs .
these 
 frequencies were obtained by a gating strategy according to the expression of cd45ra 
 and/or cd27 ( see subjects , materials and methods ) : cd27cd45ra 
 ( nave ) , cd45racd27(lde ) , cd45racd27- 
 ( em ) and cd45racd27 ( cm ) ( fig .
1 ) . the subset analysis showed a polyclonal distribution of tcr v 
 repertoires ; hence , all 24 v chains were expressed in the tcr of these four 
 subpopulations ( data not shown ) . 

frequencies of lde cd8 
 t - lymphocytes expressing v12 and v22 in cl patients - an analysis of 
 the v repertoire of lde cd8 t - lymphocytes revealed that two v families 
 appeared to expand in the lde cd8 t - lymphocyte subset from cl patients .
pad 
 showed higher frequencies of lde cd8 t - lymphocytes expressing v12 ( mean 
 3.8%  1.5% ) , when compared to both hs ( mean 1.1%  0.8% ; p < 0.001 ) and pcc ( mean 
 2.3%  1.1% ; p < 0.05 ) ( fig .
similarly , 
 the frequency of lde cd8 t - lymphocytes expressing v22 was higher in pad 
 ( mean 2.1%  1.5% ) when compared to hs ( mean 0.2%  0.5% ; p < 0.001 ) and to pcc ( mean 
 1.7%  0.9% ; p < 0.01 ) ( fig .
3:percentage of late - differentiated effector ( lde ) cd8 + t - lymphocyte 
 expressing v12 ( a ) and v22 ( b ) .
hs : healthy 
 subjects ( n = 18 ) ; pad : patients with active disease ( n = 14 ) ; pcc : patients 
 clinically cured ( n = 11 ) ; * : p < 0.05 ; * * : p < 
 0.01 ; * * * : p < 0.001 . 
 

cl patients exhibited higher frequencies of em cd8 
 t - cell subsets expressing v22 and lower frequencies of both em and cm 
 cd8 
  t - cell subsets expressing v2 - evaluating em cd8 
 t - lymphocytes expressing v22 , we observed a pronounced clonal expansion in the active 
 cl , as pad showed higher frequencies ( mean 2.6%  0.1% ) when compared to hs ( mean 0.7%  
 0.1% ; p < 0.001 ) .
notably , pcc ( mean 0.9%  0.3% ) showed similar frequencies of this 
 subset in expressing v22 when compared to hs and lower frequencies compared to pad ( p 
 < 0.01 ) ( fig .
em 
 cd8 t - cells expressing v2 have lower frequencies in pad ( mean 1.6%  
 0.4% ; p < 0.01 ) and also in pcc ( mean 0.9%  0.2 ; p < 0.01 ) when compared to hs 
 ( 3.3%  0.3% ) ( fig .
this contraction of the 
 v2 family was also observed in cm cd8 t - lymphocytes from pcc ( mean 2.4%  
 0.4% ) and in pad ( mean 1.4%  0.3% ) , showing lower frequencies of these cells when 
 compared to hs ( mean 4.5%  0.3% ; p < 0.01 and p < 0.001 , respectively ) ( fig .
another contraction could also be observed 
 in cm cd8 t - lymphocyte clones expressing v 13.2 , as pad presented lower 
 frequencies of these cells ( mean 0.9%  0.2% ) when compared to both hs ( mean 2.2%  
 0.3% ; p < 0.01 ) and pcc ( mean 2.6%  0.3% ; p < 0.001 ) ( fig .
4:percentage of late - differentiated effector memory ( em ) cd8 + t - lymphocyte 
 expressing v2 ( a ) and v22 ( b ) and of central memory ( cm ) cd8 + t - lymphocyte 
 expressing v2 ( c ) and v13.2 ( d ) .
hs : healthy 
 subjects ( n = 18 ) ; pad : patients with active disease ( n = 14 ) ; pcc : patients 
 clinically cured ( n = 11 ) ; * * : p < 0.01 ; * * * : p 
 < 0.001 . 
 
 lower frequencies of cd8 
 t - cells expressing v2 correlate with larger lesion size - due to 
 relationship between clinical features and immune response in cl , we verified whether 
 the frequency of cd8 t - cell expressing v2 , v12 , v13.2 and v22 was 
 associated with the size of the cl lesion .
an analysis of the pad data revealed an 
 inverse correlation between frequencies of em and cm cd8t - cells expressing 
 v2 and lesion size ; the greater the lesion size , the lower the frequency of em and cm 
 cd8 t - lymphocytes expressing v2 ( fig . 
 5 ) .
no statistically significant correlation was observed in pcc and in other 
 evaluated v families . 

5:correlation analysis between lesion size and frequency of effector memory 
 ( em ) ( a ) and central memory ( cm ) ( b ) cd8 + t - lymphocyte expressing v2 .
the 
 lesion size is correlated negatively with frequency of em cm cd8 + t 
 -lymphocytes from patients with active disease ( n = 14 ) .
the central line 
 represents medians values and the graphs show the best fitted lines with 95% 
 confidence interval .
the analysis of the t - cell profiles involved in local or systemic immune responses has 
 been shown to be very important in clarifying many immunoclinical phenomena , including 
 autoimmunity , response to viral or bacterial superantigens , alloimmunity and tumour 
 immunity ( gorski et al .
the establishment 
 of an effective immune response against the protozoanleishmania spp is 
 essential to limit or prevent tissue damage and is responsible for cl outcomes .
an 
 important characteristic of cl is that most patients , especially in rj , have a 
 satisfactory response to antimonial therapy ( de 
 oliveira - neto et al .
therefore , the treatment may be associated with 
 the development of an efficient immune response that is able to control the 
 infection .
cl immune response leads to lesion resolution mainly through cd4 t - helper 1 
 lymphocytes with activation of cd8 t - cells and macrophages / dendritic cells 
 via cytokine production ( da - cruz et 
 al .
this 
 response is characterised by an antigenic presentation with the involvement of 
 particular v families to guide to antigen - specific immunological events , including 
 t - cell activation and the triggering of effector and regulatory mechanisms .
some authors 
 suggest that cd8 t - lymphocytes have a central role in the process of healing 
 cl resolution ( bertho et al .
another study suggests that the high 
 frequencies of cd8 t - lymphocytes in the cl lesion milieu support the 
 hypothesis that these cells may be involved in the healing process of lesions ( da - cruz et al .
2005 ) and the ifn- production of 
 cd8 t - lymphocytes correlates with the control of infection ( rostami et al .
in contrast , some reports 
 have suggested that cytotoxicity may be involved in cl pathology ( novais et al .
2015 ) and others have shown that the high expression of ifn- increases 
 cytotoxic activity , which may be responsible for the lack of control of the inflammatory 
 response in mucosal leishmaniasis ( faria et al . 

thus , faced with the paradoxical role of cd8 t - cells linked 
 to immune responses in cl ( novais & scott 
 2015 ) , we may assume that a particular expansion or contraction of specific v 
 in cd8 t - lymphocyte tcr may be associated with disease control or its 
 progression .
the analysis of individual t - cell subsets based on their tcr expression is a powerful 
 tool for studying t - cell responses , allowing for the evaluation of the diversity and 
 distribution of different clones in peripheral blood from cl patients .
our analysis of 
 the total cd8 t - lymphocyte v repertoire revealed a polyclonal distribution 
 that includes the expression of 24 different v chains by all studied individuals . 
 although all v families included in our study
were expressed by cd8 
 t - cells , we observed a heterogeneity in the frequency of expression of different 
 families that comprise the repertoire , as some chains , such as v3 and v8 , had a 
 tendency to be more frequently expressed in the three studied cohorts , whereas others , 
 such as v16 and v18 , were less frequently expressed .
these data corroborate those 
 reported by others , showing that cd4 and cd8t - lymphocytes from 
 healthy individuals present a nonrandom usage profile of v families ( giacoia - gripp et al .
considering that the v repertoire of healthy individuals may serve as a parameter for 
 comparing the v repertoire in cl patients , we may observe a modulation of the 
 repertoire of cd8 t - lymphocytes both in pad and pcc , suggesting an 
 oligoclonal induction of these lymphocytes . according to our analysis of the total 
 cd8t - cell compartment
, we observed a disturbance in the expression of 
 some clones / families of tcr / v repertoire , such as that of the v2 , v9 , v13.2 , v18 
 and v23 families , which showed lower or higher frequencies in cl patients when compared 
 to hs .
this may indicate that these clones are involved in the response against 
 l. braziliensis .
it is important to highlight that few v chains 
 have been associated with immunopathogenesis of cl to date . in the lesions ofl . 

braziliensis - infected patients , the expansion of v6 has already been 
 observed ( uyemura et al . 1993 ) and , in another 
 report , it was suggested that t - lymphocytes expressing v12 are involved in the cl 
 immune response ( clarncio et al .
moreover , exposure to leishmania guyanensispreferentially induces 
 cd8 t - lymphocytes expressing v14 ( kariminia et al .
in contrast , in chagas disease patients , the 
 preferential expression of v3.1 ( menezes et al . 

2004 ) and v5 ( costa et al . 2000 ) may 
 be associated with the pathology of this disease .
knowing that nave and effector lde , cm and em subsets are inserted in the pool of total 
 cd8 t - lymphocytes , some expansions and contractions of v regions in 
 these subsets may be missed in general population analysis . in this context , a 
 particular evaluation of these cd8t - lymphocyte subset frequencies and their 
 profiles is of great interest to investigate the involvement of specific clones of 
 cd8 t - lymphocyte subsets expressing a particular tcr / v region in the 
 immune response context .
therefore , the disturbance of the total cd8 
 t - lymphocyte repertoire was also observed in at least one of the four analysed 
 cd8 t - lymphocyte subsets , except v18 .
hence , we determined that these 
 repertoire perturbations may be subset - specific and should be studied individually in 
 each subpopulation , as this expression is not observed in total cd8 
 t - lymphocyte populations . proposing to more clearly understand the differentiation 
 status of specific subsets of cd8 t - lymphocytes based on v expression , we 
 performed v repertoire analysis of lde , nave , em and cm cd8 t - lymphocytes . 

the distribution of v-clone cd8t - lymphocyte subsets in the cl patients had 
 not yet been described and an evaluation of differences between the frequencies of these 
 subsets represent one possible method for assessing the role of these cell populations 
 in the cl immune response .
t - lymphocytes directly involved in antigen recognition and specific responses may be led 
 to clonal expansion in response to antigenic presentation or to contraction due to 
 chronic re - stimulation and subsequent t - cell death ( menezes et al . 2004 , keesen et al . 

2011 ) . in the present report , we observed high frequencies of lde 
 cd8 t - lymphocytes expressing v12 and v22 in pad , which led us to 
 hypothesise that this cell clone selection may be related to this phase of the disease 
 and to the lde roles of these cells .
2006 ) that showed that 
 the expansion of cd4 and cd8 t - lymphocytes expressing v12 in cl 
 patients was associated with an in vitro proliferation in response to the l. 
 amazonensis antigen . moreover , in vaccinated individuals , the increase in 
 ifn- production by cd4 and cd8t - lymphocytes was associated with 
 higher frequencies of both t - cells expressing v12 . in a more recent study , increased 
 frequencies of cd4 t - lymphocytes expressing v12 after stimulation of pbmc 
 in infected cl patients with solublel .
these findings suggest that v12 could be expanded by different species of 
 leishmania and could be related to the lde status in cl immune 
 responses ( keesen et al .
taking all these 
 data together , we contend that v12 is actually involved in the 
 anti - leishmania t - cell response and that cd8 
 t - lymphocytes expressing v12 have a central role in the 
 leishmania - specific immune response displaying a lde profile . because pcc showed lower frequencies of lde cd8 t - lymphocytes expressing 
 v12 , we hypothesise that these clones are down - modulated after clinical cure . moreover , 
 we were unable to confirm that cd8 t - lymphocytes expressing v12 were 
 associated with protective or deleterious effects , but the lde cd8 t - cells 
 expressing v12 appear to be selected for during active disease , suggesting an 
 involvement of these clones in the cl immune response .
a similar phenomenon was observed in the lde cd8 t - lymphocyte subpopulation 
 expressing v22 in pad , suggesting that these clones , as well as v12
cd8 + 
 t - cells , may be associated with a lde role during the active phase of cl . 

moreover , these v22-expressing clones may be activated and/or differentiated in em 
 cd8 t - lymphocytes , as we observed an expansion of these effector - memory 
 cells expressing v22 during active disease .
thus , notably , these data indicate that 
 cd8t - cells expressing v22 may be a candidate clone involved in the 
 effector response as well as in the protective immune response in cl .
in contrast , we observed a contraction of em cd8 t - lymphocytes expressing 
 v2 in pad .
( 2006 ) , who showed that total cd8t - lymphocytes expressing 
 v2 , when cultured in the presence of antigens ofl .
amazonensis , had a 
 decrease in their frequencies compared to cells cultured without antigen stimulation . 

these authors attributed such decrease to the apoptosis of these clones after their 
 antigenic stimulation .
remarkably , we also observed this decrease in the frequencies of 
 cm cd8 t - lymphocytes expressing v2 from pad and the frequency of these 
 clones appears to be restored in pcc .
conversely , pcc showed a lower frequency of em 
 expressing v2 when compared to pad and hs , suggesting a down - modulation of these clones 
 in different subsets , depending on the stage of disease .
( 2004 ) identified a decrease in the frequency of 
 activated - cd28-cd8 t - lymphocytes expressing v2 in patients 
 with chagas disease .
although leishmania 
 andtrypanosoma are different parasites and drive 
 different pathologies , it is relevant that activated and/or lde 
 cd8t - lymphocyte expressing v2 may be modulated and have a key role in the 
 immune response occurring in these two distinct pathologies .
an analysis of the cm 
 cd8 t - lymphocyte repertoire revealed observed small frequencies v2 
 cd8 t - cells from pad , suggesting a selected down - modulation of these 
 clones in both memory subsets of cd8t - lymphocytes .
additionally , the largest lesions found in cl patients were correlated to the low 
 frequency found in em and in cm cd8 t - lymphocytes expressing v2 . because 
 lesion size characterises the severity of disease and
is considered to be the most 
 significant clinical feature of cl ( oliveira et al . 

2011 ) , the low frequency of these memory subsets expressing v2 may be 
 associated with the development or maintenance of the lesions .
we still observed low 
 frequencies of both memory subsets expressing v2 in pad , suggesting that these memory 
 cd8 t subsets are down - modulated during active disease .
we therefore 
 hypothesise that cd8 t - lymphocytes expressing v2 are an important clone in 
 the protective immune response . in conclusion , we have demonstrated that distinct cd8 t - lymphocyte subsets 
 expressing specific v families are involved in the immune response of cl patients 
 infected with l. braziliensis .
particular v expansions suggest that 
 determined tcr / v clones of cd8 t - lymphocytes are selected during the cl 
 immune response during disease and/or after antimonial therapy , supporting the 
 hypothesis that cd8 t - lymphocytes expressing v12 and v22 are involved in a 
 lde anti - leishmania immune response .
in contrast , the tcr / v2 
 contractions in memory cd8 t subsets appear to be a result of the 
 down - modulation of these clones during active disease , which was associated with tissue 
 damage .
the identification of antigens inleishmania that activate 
 specific v clones is still lacking and we hope that our results contribute to the 
 definition of preferentially used immunodominant antigens .
we therefore argue that 
 expansions or contractions of cd8t - cells expressing v12 , v22 and v2 play 
 an important role in the anti - leishmania immune response .
moreover , the 
 information gleaned by our study clearly points to specific clonally related cd8 + 
 t subpopulations recruited by the immune system , with the aim of 
 killingleishmania parasites , leading to the resolution of human 
 cl .
 in human cutaneous leishmaniasis ( cl ) , the immune response is mainly mediated by 
 t - cells . 
 the role of cd8 + t - lymphocytes , which are related to healing or 
 deleterious functions , in affecting clinical outcome is controversial . 
 the aim of 
 this study was to evaluate t - cell receptor diversity in late - differentiated effector 
 ( lde ) and memory cd8 + t - cell subsets in order to create a profile of 
 specific clones engaged in deleterious or protective cl immune responses . 
 healthy 
 subjects , patients with active disease ( pad ) and clinically cured patients were 
 enrolled in the study . 
 total cd8 + t - lymphocytes showed a disturbance in 
 the expression of the v2 , v9 , v13.2 , v18 and v23 families . 
 the analyses of 
 cd8+t - lymphocyte subsets showed high frequencies of lde 
 cd8+t - lymphocytes expressing v12 and v22 in pad , as well as 
 effector - memory cd8 + t - cells expressing v22 . 
 we also observed low 
 frequencies of effector and central - memory cd8 + t - cells expressing v2 in 
 pad , which correlated with a greater lesion size . 
 particular v expansions point to 
 cd8 + t - cell clones that are selected during cl immune responses , 
 suggesting that cd8 + t - lymphocytes expressing v12 or v22 are involved in 
 a lde response and that v2 contractions in memory cd8+t - cells are 
 associated with larger lesions . 
SUBJECTS, MATERIALS AND METHODS
RESULTS
DISCUSSION