the original goals of the studies from our group were to better understand t - cell 
 responses to t. cruzi in chronically infected subjects and to correlate 
 these responses with cardiac disease severity . the frequency , phenotype and function of 
 t - cells responsive to t. cruzi antigens were measured in adults ( 30 - 70 
 years of age ) and children ( 5 - 16 years of age ) with chronic chagas disease living in 
 areas nonendemic for t. cruzi infection in argentina . the primary 
 findings included an overall low level of detectable t. cruzi - specific 
 t - cells and the predominance of single cytokine [ interferon ( ifn)- only]-producing 
 t - cells in the circulation of adult subjects with long - term t. cruzi 
 infections ( alvarez et al . conversely , children presumed to have shorter - term infections showed parasite - specific 
 t - cell responses that were more robust and more highly functional ( albareda et al . the simultaneous secretion of ifn- 
 and interleukin ( il)-2 was prevalent among t. cruzi - infected children 
 ( albareda et al . 2013 ) , while monofunctional 
 responses were higher in t. cruzi - infected adults compared with 
 infected children ( albareda et al . 2006 , alvarez et al . polyfunctional t - cell responses are important because these cells 
 are optimised for effector function , including higher ifn- secretion on a per - cell 
 basis , more efficient killing by ifn- and tumour necrosis factor ( tnf)- compared with 
 either cytokine alone and il-2-mediated expansion of t - cells in an autocrine or 
 paracrine manner , which could enhance t - cell memory function ( seder et al . 2008 , virgin et al . table i 
 trypanosoma cruzi - specific t - cell responses in children and 
 adults with chagas disease living in nonendemic areas of argentina measured by 
 interferon ( ifn)- and interleukin ( il)-2 elispotpositive elispot responses to t. cruzi 
 lysatepositive responders / total t. 
 cruzi- infected subjects evaluated n / n ( % ) 
 childrenadultsifn- + il-210/17 ( 58.8 ) 
 4/22 ( 18)ifn--only3/17 ( 17.6 ) 
 11/22 ( 50)il-2-only0/17 ( 0)0/22 ( 0 ) 
 a : fisher exact test p = 0.0181 vs. percentage of 
 ifn--only responders in t. cruzi - infected children ; 
 b : p < 0.02 vs. percentage of ifn- + il-2 in adults ; 
 c : p < 0.05 vs. percentage of ifn--only in 
 adults . 
 
 a : fisher exact test p = 0.0181 vs. percentage of 
 ifn--only responders in t. cruzi - infected children ; 
 b : p < 0.02 vs. percentage of ifn- + il-2 in adults ; 
 c : p < 0.05 vs. percentage of ifn--only in 
 adults . other authors have also reported that children in the early phase of chronic t. 
 cruzi infection exhibit ifn- and tnf--secreting cd4 t - cells in 
 response to t. cruzi antigens ( samudio 
 et al . 1998 , sathler - avelar et al . 
 2006 ) ; however , these responses were associated with il-10 production by 
 cd4 t - cells ( sathler - avelar et al . therefore , the maintenance of functional t - cells is proposed to control 
 parasite replication without the induction of tissue damage and these responses appear 
 to be impaired with the length of the infection . il-10 might counteract t - helper ( th)1 
 responses , which , if sustained overtime , might be deleterious for the host ( dutra & gollobb 2008 ) . because phenotypic defects in t - cells might arise along with functional ability loss , 
 the phenotypic profile of t. cruzi - specific t - cells has also been 
 evaluated in chronic chagas disease patients . the pool of parasite - specific 
 ifn--producing cd4 t - cells is primarily composed of cd122 ( the common beta 
 chain of il-2 and il-15 receptors ) low and chemokine receptor 7 low t - cells , while some 
 of these cells express cd127 ( the homeostatic il-7 receptor ) , which is consistent with 
 effector memory t - cells ( tems ) ( figure ) . tems that are characteristic of persistent 
 infections have low proliferative capacity and primarily migrate to peripheral tissues ; 
 however , in contrast to effector t - cells , tems may persist in the host . furthermore , a 
 fraction of t. cruzi - specific t - cells exhibit low expression levels of 
 killer cell lectin - like receptor ( klrg)1 , cd57 and leukocyte immunoglobulin - like 
 receptor ( lir)-1 ( argello et al . 2012 ) , while t - cells expressing the costimulation 
 molecules cd27 and cd28 are enriched ( figure ) ( albareda et al . considering 
 that klrg1 and cd57 expression increases with the rounds of antigen stimulation and that 
 cd27 and cd28 expression is down - regulated ( appay et al . 2008 ) while lir-1 expression is 
 up - regulated during t - cell differentiation ( antrobus et al . 2005 ) , these findings 
 indicate that nave cd4 t - cells may also be recruited for t - cell responses 
 in chronically infected subjects . long - term infection with t. cruzi 
 also results in the up - regulation of cytotoxic t lymphocyte antigen-4 ( figure ) ( argello 
 et al . 2012 ) likely as part of a homeostatic process to control tissue damage , although 
 this up - regulation might damper t - cell responses . the above - described findings are 
 compatible with immune exhaustion , which has described for other chronic parasitic and 
 viral infections ( gigley et al . phenotypic characterization of trypanosma cruzi - specif 
 cd4 + t - cells in chronically infected subjects . peripheral blood mononuclear 
 cells from eight patients were stimulated with 15 ug / ml of t. 
 cruzi lysate from the brazil strain for 16 - 20 h. cd4+interferon 
 ( ifn)-+ t - cells were selected and analysed for the expression of cd127 , 
 chemokine receptor ( ccr)7 , cd122 , killer cell lectin - like receptor ( klrg)1 , 
 cd27 , cd28 , cd57 , immunoglobulin - like receptor ( lir)-1 and cytotoxic t 
 lymphocyte antigen ( ctla)-4 by flow cytometry . means values  standard 
 deviation are shown . notably , dissimilar findings have been reported regarding the association of ifn- and 
 il-10 production in response to t. cruzi antigens with disease severity 
 during the chronic phase of infection in adult subjects ( table ii ) . in contrast to our findings , increased ifn- production after 
 stimulating peripheral blood mononuclear cells ( pbmcs ) with t. cruzi 
 antigens was observed in patients with severe cardiomyopathy compared to seropositive 
 subjects with no signs of cardiac disease ( abel et al . 2012 ) , whereas il-10 production was either detected in 
 asymptomatic patients ( guedes et al . 2014 ) or undetectable ( abel et al . 2001 ) ( table ii ) . similarly , patients with severe cardiomyopathy had higher 
 frequencies of cd3 or cd4 t - cells , including 
 cd4foxp3cd25hi t - cells with the ability to secrete ifn- in 
 response to t. cruzi antigens ( gomes et 
 al . 2012 ) , whereas il-10-secreting 
 cells were found primarily during the asymptomatic phase ( gomes et al . 2003 ) or detected in all clinical groups ( de arajo et 
 al . 2012 ) . in a recent study focused on chagas disease patients with severe 
 cardiomyopathy , increased levels of ifn- and il-10 specific for t. 
 cruzi antigens have been reported ( longhi et 
 al . table iiinterferon ( ifn)- and interleukin ( il)-10 production in response to 
 trypanosoma cruzi antigens in patients with chronic chagas 
 diseasereferencetestsource of t. cruzi antigenincubation timefindingsabel et al . ( 2001)capture elisa with pbmc culture 
 supernatantsrecombinant b13 protein48 hifn- levels in cchd > 
 asymptomatic il-10 undetectable in all clinical groups 
 guedes et al . ( 2012 ) 
 capture elisa with pbmc culture 
 supernatantstrypomastigote lysate ( y 
 strain)48 hifn- levels in cchd > 
 asymptomatic il-10 levels in asymptomatic > cchdgomes et al . ( 2003)facs intracellular cytokineepimastigote / trypomastigote 
 lysate ( y strain)24 h for il-10 6 days for 
 ifn-cd3ifn- 
 in cchd > asymptomatic cd14il-10 in 
 asymptomatic > cchdfonseca et al . ( 2005)elispothla - a02.01-restricted cruzipain/ 
 fl-160 peptides24 hifn- cells in 
 cchdgomes et al . ( 2014)cba with pbmc culture 
 supernatantsepimastigote / trypomastigote 
 lysate ( cl strain)22 hifn- levels in cchd = 
 asymptomatic il-10 levels in asymptomatic > cchdcuellar et al . ( 2009)facs intracellular cytokinerecombinant kmp-11 t. 
 cruzi protein12 hcd4ifn-+ 
 in cchd > asymptomaticde arajo et al . ( 2012)facs intracellular cytokinetrypomastigote lysate ( cl 
 strain)22 htreg ifn- in cchd 
 > asymptomatic treg il-10 in asymptomatic = cchdde melo et al . ( 2012)real time polymerase chain 
 reactionrecombinant cra / fra proteins3 daysifn- expression in asymptomatic 
 = cchd il-10 expression in asymptomatic = cchdalbareda et al . ( 2006)facs intracellular cytokine 
 t. cruzi - infected dendritic cells ( brazil strain)6 hcd8ifn- 
 in asymptomatic > cchddiez et al . ( 2006)elispothla - a02.01-restricted k1 
 peptide24 hifn- lymphocytes in 
 asymptomatic = cchdalvarez et al . ( 2008)elispothla - a02.01-restricted 
 transialidase peptides24 hifn- lymphocytes in 
 asymptomatic > cchdfiuza et al . ( 2009)facs intracellular cytokineepimastigote lysate ( cl 
 strain)22 hcd4cd45roifn- in asymptomatic = cchd 
 cd4cd45roil-10 in asymptomatic = cchd 
 cd8cd45raifn- in asymptomatic = cchdlorena et al . cra / fra proteins24 hcra / fra : 
 cd4ifn- in asymptomatic = cchd 
 cd4il-10 in asymptomatic = cchd cra : 
 cd8ifn- in cchd > asymptomatic 
 cd8il-10 in asymptomatic = cchdlasso et al . ( 2010)facs tetramer / intracellular 
 cytokinehla - a02.01-restricted k1 
 peptide12 hcd8ifn- 
 cells in asymptomatic = cchdlaucella et al . ( 2004)elispotamastigote enriched lysate 
 ( brazil strain)24 hifn- cells in 
 asymptomatic > cchdalbareda et al . ( 2009)facs intracellular cytokineamastigote enriched lysate 
 ( brazil strain)24 hcd4ifn- 
 in asymptomatic > cchdmaran et al . ( 2011)bio - plex with pbmc culture 
 supernatantshla - a02.01-restricted hsp-70 
 peptides30 hifn- levels in asymptomatic = 
 cchdegui et al . ( 2012)bio - plex with pbmc culture 
 supernatantshla - a02.01-restricted pfr2/pfr3 
 peptides30 hifn- levels in asymptomatic > 
 cchdgomes et al . ( 2012)facs intracellular cytokinelive t. cruzi 
 ( cl strain)6 hifn- lymphocytes in 
 asymptomatic > cchd il-10- lymphocytes in asymptomatic 
 > cchdlonghi et al . ( 2014)milli - plex with pbmc culture 
 supernatantsrecombinant p2/cpo proteins1 - 6 daysil-10 levels increased in cchd 
 longhi et al . ( 2014)milli - plex with pbmc culture 
 supernatantsepimastigote lysate ( cl brener , 
 dtu tcvi)1 - 6 daysifn- and il-10 levels increased 
 in cchd 

 a : asymptomatic patients were not assessed ; cba : cytometric 
 bead array ; cchd : chronic chagas heart disease ; cra : cytoplasmatic 
 repetitive antigen ; dtu : discrete type unit ; facs : fluorescence - activated 
 cell sorting ; fra : flagellar repetitive antigen ; hla : human leukocyte 
 antigen ; hsp-70 : heat shock protein-70 ; kmp-11 : kinetoplastid membrane 
 protein-11 ; pbmc : peripheral blood mononuclear cells ; pfr : paraflagellar rod 
 proteins ; treg : regulatory t - cells . a : asymptomatic patients were not assessed ; cba : cytometric 
 bead array ; cchd : chronic chagas heart disease ; cra : cytoplasmatic 
 repetitive antigen ; dtu : discrete type unit ; facs : fluorescence - activated 
 cell sorting ; fra : flagellar repetitive antigen ; hla : human leukocyte 
 antigen ; hsp-70 : heat shock protein-70 ; kmp-11 : kinetoplastid membrane 
 protein-11 ; pbmc : peripheral blood mononuclear cells ; pfr : paraflagellar rod 
 proteins ; treg : regulatory t - cells . in contrast to these findings , comparable frequencies of cd4 t - cells 
 producing ifn- and il-10 were reported in patients with no signs of cardiac disease and 
 those with severe cardiomyopathy ( fiuza et al . cytoplasmatic repetitive antigen ( lafaille 1989 , 
 kriegger 1992 ) induced ifn- , il-4 and tnf- production by cd4 t - cells in 
 patients with or without cardiac disease ( lorena et al . additionally , only subjects in the indeterminate phase of chagas 
 disease presented higher frequencies of cd4il-10 t - cells 
 compared with uninfected controls ; however , these levels did not differ from those found 
 in patients with severe cardiomyopathy . 1992 ) was used as the antigen source , 
 cd4il10 t - cells were detected in both patients in the 
 indeterminate phase of the disease and those presenting cardiomyopathy , while the 
 frequencies of cd4ifn- t - cells remained unaltered compared with 
 uninfected controls ( lorena et al . 2010 ) . in another study , de melo et al . ( 2012 ) reported 
 that most patients in the indeterminate phase of the infection displayed increased ifn- 
 gene expression after stimulation with t. cruzi antigens , while the 
 majority of patients with cardiomyopathy had high levels of il-10 . the use of soluble parasite antigens vs. in vitro infection might also lead to different 
 results . ( 2012 ) recently 
 demonstrated that the short - term in vitro culture of whole blood samples with live 
 t. cruzi parasites led to increased ifn- and 
 il-10 lymphocytes in subjects with no sign of cardiac disease compared 
 with those with severe cardiomyopathy , in contrast to previous reports from the same 
 group showing increased levels of ifn- upon stimulation with an 
 epimastigote / trypomastigote lysate ( gomes et al . little information is available regarding cd8 t - cell responses in subjects 
 with chronic t. 
 cruzi infection primarily because the complexity of the t. 
 cruzi genome has made it difficult to identify individual targets of 
 cd8 t - cell responses in t. cruzi - infected subjects . in 
 an attempt to identify and quantify t - cell responses to defined t. 
 cruzi epitopes , we tested human leukocyte antigen ( hla)-a2.1-restricted 
 cd8 t - cell responses ( hla - a2.1 is one of the most common class i alleles 
 in this population ) to several trans - sialidase - derived epitopes using a combination of 
 hla - a2 tetramers , elispot analysis and intracellular cytokine staining assays ( martin et al . 2008 ) ( table ii ) . the 
 percentage of responders strongly correlated with disease severity , with 60% of 
 individuals in the indeterminate phase , but only 22% of the subjects with cardiomyopathy 
 responding to trans - sialidase peptides ( alvarez et al . t - cell responses were measured using t. cruzi - infected autologous 
 dendritic cells , we observed that subjects with less severe forms of the cardiac disease 
 were more likely to mount t. cruzi - specific ifn- responses than 
 subjects with severe cardiomyopathy ( albareda et al . hla - a201-restricted epitopes derived from 
 kinetoplastid membrane protein ( kmp)-11 ( diez et al . 2011 ) , fl-160 ( fonseca et al . 2005 ) or cruzipain ( fonseca et al . 2005 ) have also been reported as targets of ifn- responses by 
 cd8 t - cells in chronically t. cruzi - infected subjects 
 ( table ii ) . while no differences in the 
 magnitude of t - cell responses specific for kmp-11 and hsp-70-derived peptides according 
 to the clinical stage of the disease were found , ifn- secretion in response to 
 hla - a2.01-restricted paraflagellar rod proteins ( prp)-2 and prp-3-derived peptides was 
 higher in patients in the indeterminate phase of the infection compared with patients 
 with cardiac disease ( egui et al . 2012 ) ( table ii ) . concerning the phenotype of 
 cd8 t - cells specific for t. cruzi antigens , 
 cd8ifn- t - cells predominantly showed an effector memory 
 phenotype were enriched in cd27cd28 cells in response to 
 t. cruzi ( albareda et al . 2006 ) . as a whole , th1 and il-10-producing t - cells can be detected in the different clinical 
 groups of the chronic infection , with a degree of variation according to the 
 t. 
 cruzi antigen source , the patient s location and immunological assays . the quality of t - cell responses might determine the pattern of the cellular response and 
 the severity of the disease in t. cruzi infection . to gain a clearer understanding of the relationship between parasite persistence and 
 t. cruzi - specific t - cell maintenance during chronic chagas disease , 
 we examined the changes in the frequency of t. cruzi - specific t - cells 
 in adult subjects with chronic t. cruzi infection following bz 
 treatment . we showed that the levels of ifn--producing t - cells in response to a 
 t. cruzi lysate preparation significantly decreased over 12 months 
 following bz treatment and that ifn--secreting t - cells fell below the level of 
 detection by 36 months post - treatment ( laucella et al . 2009 ) , along with a significant drop in antibody levels specific for a set of 
 recombinant proteins ( laucella et al . 2009 , viotti et al . 2011 ) as determined by multiplex 
 serological assays ( cooley et al . this latter assay appeared to be more sensitive than conventional 
 serological tests in detecting changes in antibody levels following bz treatment . surprisingly , a proportion of bz - treated , but not untreated , subjects exhibited an 
 initial increase in ifn- responses before the decrease at 12 months post - treatment 
 ( laucella et al . 2009 ) . we hypothesise that 
 this transient rise in ifn--secreting t - cells might be triggered by a release of 
 parasite antigens due to bz - induced parasite death and might enhance the parasiticidal 
 effect of the drug . the most straightforward interpretation of these results is that bz 
 treatment decreases the parasite load , thus diminishing the antigen that is required to 
 maintain the t. 
 cruzi - specific effector t - cells . other studies have measured t - cells responsive to t. cruzi antigens 
 after bz treatment in subjects with chronic t. cruzi infection . of 
 note , these studies focused on the comparison of subjects treated with bz with those 
 untreated or uninfected ; however , these comparisons were not 
 pre - treatment / post - treatment comparisons . a shift towards a type two - modulated profile 
 with a higher level of il-10 production was found in the cd4t - cell 
 compartment , whereas high ifn- expression was observed in cd8 t - cells of 
 t. cruzi - infected children after one year of bz treatment ( sathler - avelar et al . the treatment 
 of chronically t. cruzi - infected adult subjects with no signs of 
 cardiac disease led to increased levels of tnf-cd8 and 
 ifn-cd8 t - cells ( sathler - avelar et al . 2012 ) with unchanged frequencies of 
 cd4il-17 t - cells ( guedes et 
 al . subjects with 
 chronic t. cruzi infection who were treated with bz 14 - 30 years earlier 
 and achieved serological cure showed higher levels of ifn- production specific for 
 t. cruzi compared with patients who had not been cured ( bahia - oliveira et al . 2000 ) . in a mouse model of acute and chronic t. cruzi infections , bz treatment 
 resulted in the emergence of t. cruzi - specific central memory t - cells 
 ( bustamante et al . a reduction in the total numbers of activated and tems after 
 the treatment of infected mice has also been shown ( fernndez et al . 2009 ) . a positive treatment outcome was demonstrated by the 
 observation that treated animals were protected against re - infection ( olivieri et al . altogether , changes in 
 t. cruzi - specific t - cell responses can be potential early indicators 
 of treatment response , even in cases in which cure is not achieved . long - term monitoring 
 of t - cell responses following chemotherapy in relation to the serological evolution by 
 conventional serology , which is currently the only gold standard of treatment efficacy 
 in chagas disease , will allow the usefulness of immunological parameters as surrogate 
 markers of treatment efficacy to be validated . the immunological status of the host might also be a determining factor of treatment 
 success . in this respect , a relationship between treatment success and the immune status 
 pre - therapy has been demonstrated in human immunodeficiency virus positive subjects 
 under highly active antiretroviral therapy ( marchetti et 
 al . that the immune response can be a key factor in the efficacy of treatment against 
 t. cruzi has been documented in experimental acute t. 
 cruzi infections ; however , strong support of this correlation in t. 
 cruzi - infected humans remains lacking . in a murine model of t. cruzi infection , treating ifn- , il-12 , protein , 55-tnf receptor and 
 inducible nitric oxide synthase knockout mice with bz reduced the cure rates to 42% , 35% 
 and 28% , respectively , compared with wild - type animals ( romanha et al . the absence of cd4 t - cells dramatically 
 affected the efficacy of bz treatment , whereas parasitaemia and cure rates were less 
 affected by the absence of cd8 t - cells and b cells ( ferraz et al . the administration of blocking antibodies for 
 il-12 or ifn- concomitant with a suboptimal dose of bz enhanced parasitaemia and 
 accelerated mortality , while treatment efficacy did not change in mice treated with 
 optimal doses of bz ( michailowsky et al . 1998 ) . 
 simultaneous treatment with a suboptimal dose of bz and recombinant il-12 enhanced the 
 efficacy of drug treatment as measured by parasitaemia levels and mouse survival ( michailowsky et al . 1998 ) . likewise , bz treatment during experimental t. cruzi infection in mice 
 enhanced phagocytosis , parasite destruction and cytokine release by macrophages ( murta et al . 1999 ) and led to a partial reversion of 
 spleen and lymph node enlargement observed in t. cruzi - infected 
 untreated mice ( olivieri et al . these 
 findings support the notion that drug treatment responsiveness results from the combined 
 action of the drug and the activation of appropriate immune responses in the host . a throughout knowledge of how drug treatment works might provide guidance regarding how 
 to most effectively treat and monitor therapy outcomes in chronically t. 
 cruzi - infected individuals . providing tools to not only monitor , but also 
 provide early predictions of treatment success or failure will allow the development of 
 new therapeutic options . the notion that improving host immune responses to t. 
 cruzi could enhance treatment efficacy remains unexplored in t. 
 cruzi - infected subjects and deserves future research .
the aim of this review is to describe the contributions of the knowledge of t - cell 
 responses to the understanding of the physiopathology and the responsiveness to 
 etiological treatment during the chronic phase of chagas disease . 
 t - helper ( th)1 and interleukin ( il)-10 trypanosoma 
 cruzi - specific t - cells have been linked to the asymptomatic phase or to 
 severe clinical forms of the disease , respectively or vice versa , 
 depending on the t. cruzi antigen source , the patient s location and 
 the performed immunological assays . 
 parasite - specific t - cell responses are modulated 
 after benznidazole ( bz ) treatment in chronically t. cruzi - infected 
 subjects in association with a significant decrease in t. 
 cruzi - specific antibodies . 
 accumulating evidence has indicated that 
 treatment efficacy during experimental infection with t. cruzi 
 results from the combined action of bz and the activation of appropriate 
 immune responses in the host . however 
 , strong support of this interaction in 
 t. cruzi - infected humans remains lacking . 
 overall , the quality of t - cell 
 responses might be a key factor in not only disease evolution , but also chemotherapy 
 responsiveness . 
 immunological parameters are potential indicators of treatment 
 response regardless of achievement of cure . providing tools to monitor and 
 provide 
 early predictions of treatment success will allow the development of new therapeutic 
 options .