Differential expression profile of genes involved in the immune response associated to progression of chronic Chagas disease.

BACKGROUND: Patients with chronic Chagas disease present marked clinical and immunological heterogeneity. During the disease, multiple immune mechanisms are activated to fight the parasite. The purpose of this study was to investigate the expression patterns of genes involved in relevant immunological processes throughout the disease in patients with chronic Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: High-throughput RT-qPCR with QuantStudio 12K Flex real-time PCR system was used to evaluate the expression of 106 immune-related genes in PBMC from a cohort of cardiac Chagas disease patients (CCC I), asymptomatic patients (IND) and healthy donors (HD) after being stimulated with T. cruzi soluble antigens. Principal component analysis (PCA), cluster analysis and volcano plots were used to identify differentially expressed genes. In addition, gene set enrichment analysis (GSEA) was employed to identify the enriched immunological pathways in which these genes are involved. PCA revealed the existence of a statistically divergent expression profile of the 36 genes correlated with PC1 between CCC I patients and HD (p < 0.0001). Differential gene expression analysis revealed upregulation of 41 genes (expression fold-change > 1.5) and downregulation of 14 genes (expression fold-change < 0.66) (p = 8.4x10-13 to p = 0.007) in CCC I patients versus HD. Furthermore, significant differences in the expression level of specific genes have been identified between CCC I and IND patients (8 up and 1 downregulated). GSEA showed that several upregulated genes in CCC I patients participate in immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, cytokines-inflammatory response and IL-10 anti-inflammatory signaling. CONCLUSIONS: Cardiac Chagas disease patients show an antigen-specific differential gene expression profile in which several relevant immunological pathways seem to be activated. Assessment of gene expression profiles reveal unique insights into the immune response that occurs along chronic Chagas disease.

Humoral and Cellular Immune Response in Asymptomatic Dogs with Visceral Leishmaniasis: A Review.

Visceral leishmaniasis is one of the deadliest parasitic diseases in the world and affects both humans and dogs. The host immune response to Leishmania infection plays a critical role in the evolution of canine visceral leishmaniasis (CVL) and consequently in the manifestation of clinical signs. The asymptomatic form of the disease is a major concern in the diagnosis of CVL and in the transmission control of Leishmania infection. Asymptomatic dogs are found in large proportions in endemic areas and are an unquantifiable source of infection. The present review analyzes the possible relationship between the activation of the antigen-specific immune response of the host and resistance or susceptibility to CVL. The review focuses on works that address the characterization of the humoral and cellular immune response profile, at both the functional and phenotypic levels, in infected dogs. Most studies relate the absence of clinical symptomatology to an increased proliferative response and a Th1 cytokine profile. Despite the numerous findings pointing to a differential immune response in asymptomatic dogs, the contradictory results reported in this review highlight the importance of establishing a precise clinical classification of the disease, performing more longitudinal studies, and including a higher number of animals in trials.

CD8+ T Cell Response Quality Is Related to Parasite Control in an Animal Model of Single and Mixed Chronic Trypanosoma cruzi Infections.

Chagas disease (ChD) is a chronic infection caused by Trypanosoma cruzi. This highly diverse intracellular parasite is classified into seven genotypes or discrete typing units (DTUs) and they overlap in geographic ranges, vectors, and clinical characteristics. Although studies have suggested that ChD progression is due to a decline in the immune response quality, a direct relationship between T cell responses and disease outcome is still unclear. To investigate the relationship between parasite control and immune T cell responses, we used two distinct infection approaches in an animal model to explore the histological and parasitological outcomes and dissect the T cell responses in T. cruzi-infected mice. First, we performed single infection experiments with DA (TcI) or Y (TcII) T. cruzi strains to compare the infection outcomes and evaluate its relationship with the T cell response. Second, because infections with diverse T. cruzi genotypes can occur in naturally infected individuals, mice were infected with the Y or DA strain and subsequently reinfected with the Y strain. We found different infection outcomes in the two infection approaches used. The single chronic infection showed differences in the inflammatory infiltrate level, while mixed chronic infection by different T. cruzi DTUs showed dissimilarities in the parasite loads. Chronically infected mice with a low inflammatory infiltrate (DA-infected mice) or low parasitemia and parasitism (Y/Y-infected mice) showed increases in early-differentiated CD8+ T cells, a multifunctional T cell response and lower expression of inhibitory receptors on CD8+ T cells. In contrast, infected mice with a high inflammatory infiltrate (Y-infected mice) or high parasitemia and parasitism (DA/Y-infected mice) showed a CD8+ T cell response distinguished by an increase in late-differentiated cells, a monofunctional response, and enhanced expression of inhibitory receptors. Overall, our results demonstrated that the infection outcomes caused by single or mixed T. cruzi infection with different genotypes induce a differential immune CD8+ T cell response quality. These findings suggest that the CD8+ T cell response might dictate differences in the infection outcomes at the chronic T. cruzi stage. This study shows that the T cell response quality is related to parasite control during chronic T. cruzi infection.

Differential Expression of Immune Response Genes in Asymptomatic Chronic Chagas Disease Patients Versus Healthy Subjects.

Infection by the Trypanosoma cruzi parasite causes Chagas disease and triggers multiple immune mechanisms in the host to combat the pathogen. Chagas disease has a variable clinical presentation and progression, producing in the chronic phase a fragile balance between the host immune response and parasite replication that keeps patients in a clinically silent asymptomatic stage for years. Since the parasite is intracellular and replicates within cells, the cell-mediated response of the host adaptive immunity plays a critical role. This function is mainly orchestrated by T lymphocytes, which recognize parasite antigens and promote specific functions to control the infection. However, little is known about the immunological markers associated with this asymptomatic stage of the disease. In this large-scale analysis, the differential expression of 106 immune system-related genes has been analyzed using high-throughput qPCR in T. cruzi antigen-stimulated PBMC from chronic Chagas disease patients with indeterminate form (IND) and healthy donors (HD) from endemic and non-endemic areas of Chagas disease. This analysis revealed that there were no differences in the expression level of most genes under study between healthy donors from endemic and non-endemic areas determined by PCA and differential gene expression analysis. Instead, PCA revealed the existence of different expression profiles between IND patients and HD (p < 0.0001), dependent on the 32 genes included in PC1. Differential gene expression analysis also revealed 23 upregulated genes (expression fold change > 2) and 11 downregulated genes (expression fold change < 0.5) in IND patients versus HD. Enrichment analysis showed that several upregulated genes in IND patients participate in relevant immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, and cytokine-inflammatory response. The antigen-specific differential gene expression profile detected in these patients and the relevant immunological pathways that seem to be activated could represent potential biomarkers of the asymptomatic form of Chagas disease, helpful to diagnosis and infection control.

A proportion of CD4+ T cells from patients with chronic Chagas disease undergo a dysfunctional process, which is partially reversed by benznidazole treatment.

BACKGROUND: Signs of senescence and the late stages of differentiation associated with the more severe forms of Chagas disease have been described in the Trypanosoma cruzi antigen-specific CD4+ T-cell population. However, the mechanisms involved in these functions are not fully known. To date, little is known about the possible impact of benznidazole treatment on the T. cruzi-specific functional response of CD4+ T cells. METHODOLOGY/PRINCIPAL FINDINGS: The functional capacity of CD4+ T cells was analyzed by cytometric assays in chronic Chagas disease patients, with indeterminate form (IND) and cardiac alterations (CCC) (25 and 15, respectively) before and after benznidazole treatment. An increase in the multifunctional capacity (expression of IFN-γ, IL-2, TNF-α, perforin and/or granzyme B) of the antigen-specific CD4+ T cells was observed in indeterminate versus cardiac patients, which was associated with the reduced coexpression of inhibitory receptors (2B4, CD160, CTLA-4, PD-1 and/or TIM-3). The functional profile of these cells shows statistically significant differences between IND and CCC (p<0.001), with a higher proportion of CD4+ T cells coexpressing 2 and 3 molecules in IND (54.4% versus 23.1% and 4.1% versus 2.4%, respectively). A significant decrease in the frequencies of CD4+ T cells that coexpress 2, 3 and 4 inhibitory receptors was observed in IND after 24-48 months of treatment (p<0.05, p<0.01 and p<0.05, respectively), which was associated with an increase in antigen-specific multifunctional activity. The IND group showed, at 9-12 months after treatment, an increase in the CD4+ T cell subset coproducing three molecules, which were mainly granzyme B+, perforin+ and IFN-γ+ (1.4% versus 4.5%). CONCLUSIONS/SIGNIFICANCE: A CD4+ T cell dysfunctional process was detected in chronic Chagas disease patients, being more exacerbated in those patients with cardiac symptoms. After short-term benznidazole treatment (9-12 months), indeterminate patients showed a significant increase in the frequency of multifunctional antigen-specific CD4+ T cells.

Differential phenotypic and functional profile of epitope-specific cytotoxic CD8+ T cells in benznidazole-treated chronic asymptomatic Chagas disease patients.

One of the greatest challenges in Chagas disease research is the search for tools that will enable the assessment of pharmacological treatment efficacy. A recently described set of serological biomarkers composed of four parasite antigens and established criteria of treatment efficacy allowed the evaluation of the impact of benznidazole treatment a short/medium time after the treatment. In addition, cellular immunological parameters have also been described as potential indicators of the treatment response. The cytotoxic CD8+ T cells specific to five epitopes in the PFR2, PFR3, TcCA-2 and KMP11 antigens have been analysed, and these epitopes have been shown to be recognized, processed and presented in the context of a natural T. cruzi infection. In the present manuscript, we characterized these antigen-specific CD8+ T cells in indeterminate chronic Chagas disease patients both before and after (from 11 to 28 months) benznidazole treatment. The results indicate that there is a differential memory CD8+ T cell profile depending on the antigenic epitope and that the benznidazole treatment modulates the memory, differentiation and senescence phenotypes of the epitope-specific CD8+ T cells. Moreover, in these patients, the reactivity of sera against the referred set of biomarkers was evaluated. The data obtained show that the patients who met the established therapeutic efficacy criteria presented a differential phenotypic profile of the antigen-specific CD8+ T cells even prior to treatment compared to the patients who did not meet the therapeutic efficacy criteria, and this behaviour is associated with a better functionality of these CD8+ T cells.

Evaluation of the circadian rhythm of anti-Leishmania IgG2 and IgA antibodies in serum and saliva of dogs with clinical leishmaniosis.

In this study, the circadian rhythm of IgG2 and IgA specific antibodies in serum and saliva samples of 6 dogs experimentally infected with Leishmania infantum was assessed. Sampling was performed at 8.00, 12.00, 16.00, 20.00, and 00.00 h on two consecutive days. Anti-Leishmania antibody levels in serum were expressed without any correction, whereas in saliva were shown in different ways: without any correction, adjusted by protein concentration and corrected by the salivary flow rate. No significant differences in anti-Leishmania IgG2 antibody levels in serum and saliva samples with or without correction were found. Significant differences were found when anti-Leishmania IgA levels were corrected by the salivary flow rate. In addition, a greater intra-individual variation of antibody levels was observed in saliva than in serum. However, this variation did not modify the serological status of the dogs. Therefore, it could be concluded that there is no circadian rhythm in serum and saliva samples and sampling can be performed at any time of the day.

Immunological exhaustion and functional profile of CD8+ T lymphocytes as cellular biomarkers of therapeutic efficacy in chronic Chagas disease patients.

The lack of useful tools for detection the impact of treatment during the follow-up of chronic Chagas disease treated patients difficult the adequate care to the affected population. The objective of this study was to evaluate the functional response of CD8+ T lymphocyte population, critical for the control of Trypanosoma cruzi infection, as a possible cellular biomarker of treated Chagas disease patients. Thus, we analyzed the antigen-specific CD8+ T-cell response before and after benznidazole treatment in asymptomatic (indeterminate) and cardiac chronic Chagas disease patients. A marked dysfunctional process of the CD8+ T cell population was found in patients with an advanced pathology. Thus, the cardiac patients have a higher co-expression of inhibitory receptors and a lower antigen-specific multifunctional capacity compared with that of asymptomatic patients. Remarkably, benznidazole treatment partially reverses this functional exhaustion process of CD8+ T cells in both asymptomatic and cardiac Chagas disease patients. Thus, the co-expression of inhibitory molecules tends to be reduced after benznidazole treatment, mainly in asymptomatic patients, finding a significant drop in the expression of inhibitory receptors such as PD-1 and 2B4. In addition, the multifunctional antigen-specific response of CD8+ T cells is enhanced after treatment in chronic patients. An increase in the subset of cells with cytotoxic capacity and production of the IFN-γ cytokine was also observed in both treated asymptomatic and cardiac chronic Chagas disease patients. The results derived from this study show the improvement of the functional capacity of CD8+ T cells after treatment which could be have a positive effect on parasitic control. In addition, the phenotypic and functional profile of the CD8+ T cells described could serve as a tool for monitoring the impact of benznidazole treatment.

One-year follow-up of anti-Leishmania antibody concentrations in serum and saliva from experimentally infected dogs.

The quantification of anti-Leishmania antibodies in serum and saliva by a time-resolved immunofluorometric assay is useful for the diagnosis and treatment monitoring of dogs with clinical leishmaniasis. We compared the kinetics of anti-Leishmania IgG2 and IgA antibodies in serum and saliva from 11 Beagle dogs experimentally infected with Leishmania infantum. Most dogs showed detectable concentrations of anti-Leishmania IgG2 earlier in serum (between 3 and 4 months p.i.) than in saliva (between 4 and 6 months p.i.). Overall, a high correlation between concentrations of anti-Leishmania IgG2 in serum and saliva (r = 0.853; P < 0.0001) was observed. The quantification of anti-Leishmania IgA showed less diagnostic value than IgG2, since detectable amounts of IgA were not observed in the saliva of four dogs and in the serum of one dog. In addition, a very low correlation between anti-Leishmania IgA in serum and saliva (r = 0.289; P < 0.001) was observed. Our results indicate that the antibodies against L. infantum in saliva appear approximately 1 month later than in serum, and suggest that there is a threshold for the passing of immunoglobulins from serum to saliva in dogs. These facts should be taken into consideration for a proper interpretation of saliva assays for quantification of antibodies.

A Parasite Biomarker Set for Evaluating Benznidazole Treatment Efficacy in Patients with Chronic Asymptomatic Trypanosoma cruzi Infection.

One of the current greatest challenges of Chagas disease is the establishment of biomarkers to assess the efficacy of drugs in a short period of time. In this context, the reactivity of sera from 66 adults with chronic indeterminate Chagas disease (IND) for a set of four Trypanosoma cruzi antigens (KMP11, PFR2, HSP70, and 3973d) was analyzed before and after benznidazole treatment. The results showed that the reactivity against these antigens decreased at 9, 24, and 48 months after treatment. Moreover, the 42.4% and 68.75% of IND patients met the established standard criteria of therapeutic efficacy (STEC) at 24 and 48 months posttreatment, respectively. Meeting the STEC implied that there was a continuous decrease in the reactivity of the patient sera against the four antigens after treatment and that there was a substantial decrease in the reactivity for at least two of the antigens. This important decrease in reactivity may be associated with a drastic reduction in the parasite load, but it is not necessarily associated with a parasitological cure. After treatment, a positive PCR result was only obtained in patients who did not meet the STEC. The percentage of granzyme B+/perforin+ CD8+ T cells was significantly higher in patients who met the STEC than in those who did not meet the STEC (35.2% versus 2.2%; P < 0.05). Furthermore, the patients who met the STEC exhibited an increased quality of the multifunctional response of the antigen-specific CD8+ T cells compared with that in the patients who did not meet the STEC.

Dynamics of T Cells Repertoire During Trypanosoma cruzi Infection and its Post-Treatment Modulation.

Chagas disease courses with different clinical phases and has a variable clinical presentation and progression. The acute infection phase mostly exhibits a non-specific symptomatology. In the absence of treatment, the acute phase is followed by a chronic phase, which is initially asymptomatic. This chronic asymptomatic phase of the disease is characterized by a fragile balance between the host's immune response and the parasite replication. The loss of this balance is crucial for the progression of the sickness. The virulence and tropism of the T. cruzi infecting strain together to the inflammation processes in the cardiac tissue are the main factors for the establishment and severity of the cardiomyopathy. The efficacy of treatment in chronic Chagas disease patients is controversial. However, several studies carried out in chronic patients demonstrated that antiparasitic treatment reduces parasite load in the bloodstream and leads to an improvement in the immune response against the Trypanosoma cruzi parasite. The present review is mainly focused on the cellular patterns associated to the clinical status and the evolution of the disease in chronic patients, as well as the effectiveness of the treatment related to T. cruzi infection control. Therefore, an emphasis is placed on the dynamics of specific-antigens T cell subpopulations, their memory and activation phenotypes, their functionality and their contribution to pathogenesis or disease control, as well as their association with risk of congenital transmission of the parasite.

Phenotypic and Functional Profiles of Antigen-Specific CD4+ and CD8+ T Cells Associated With Infection Control in Patients With Cutaneous Leishmaniasis.

The host immunological response is a key factor determining the pathogenesis of cutaneous leishmaniasis. It is known that a Th1 cellular response is associated with infection control and that antigen-specific memory T cells are necessary for the development of a rapid and strong protective cellular response. The present manuscript reports the analysis of the functional and phenotypic profiles of antigen-specific CD4+ and CD8+ T cells from patients cured of cutaneous leishmaniasis (CL), patients with an active process of cutaneous leishmaniasis, asymptomatic individuals with a positive Montenegro test and healthy donors (HD). Peripheral blood mononuclear cells (PBMCs) from the patients exhibited a lymphoproliferative capacity after stimulation with total soluble protein from either Leishmania panamensis (SLpA) or Leishmania infantum (SLiA) or with a recombinant paraflagellar rod protein-1 (rPFR1). Higher frequencies of antigen-specific TNAIVE cells, mainly following stimulation with rPFR1, were observed in asymptomatic and cured patients than in patients with active cutaneous leishmaniasis, while T cells from patients with active cutaneous leishmaniasis showed a higher percentage of effector memory T cells (TEM for CD4+ T cells and TEMRA for CD8+ T cells). The amount of antigen-specific CD57+/CD8+ TEMRA cells in patients with active cutaneous leishmaniasis was higher than that in cured patients and asymptomatic subjects. Regarding functionality, a more robust multifunctional CD8+ T cell response was detected in cured patients than in those with active cutaneous leishmaniasis. Moreover, cured patients showed a significant increase in the frequency of cells expressing a Th1-type cytotoxic production profile (IFN-γ+/granzyme-B/+perforin+). Patients with an active leishmaniosis process had a significantly higher frequency of CD8+ T cells expressing the inhibitory CD160 and 2B4 receptors than did cured patients. The expression profile observed in cured patients could be indicative of an imbalance toward a CD8+ Th1 response, which could be associated with infection control; consequently, the determination of this profile could be a useful tool for facilitating the clinical follow-up of patients with cutaneous leishmaniasis. The results also suggest a possible exhaustion process of CD8+ T cells associated with the evolution of Leishmania infection.

Impact of benznidazole treatment on the functional response of Trypanosoma cruzi antigen-specific CD4+CD8+ T cells in chronic Chagas disease patients.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi. The persistence of the parasite is associated with the disease chronicity and the impairment of the cellular immune response. It has been reported that the CD4+CD8+ T cell population expands in chronic Chagas disease patients. Few studies have focused on this subset of cells, and very little is known about the impact of antiparasitic treatment on this population. METHODOLOGY: Thirty-eight chronic Chagas disease patients (20 asymptomatic and 18 symptomatic) and twelve healthy controls were enrolled in this study. Peripheral blood mononuclear cells were stimulated with soluble T. cruzi antigens to analyze the production of cytokines and cytotoxic molecules by CD4+CD8+ T cells before and after benznidazole treatment. Additionally, expression and co-expression of five inhibitory receptors in these patients after treatment were studied using a multiparameter flow cytometry technique. PRINCIPAL FINDINGS: The frequency of CD4+CD8+ T cells was higher in chronic Chagas disease patients compared with healthy donors. Furthermore, a higher ratio of CD4+CD8low/CD4+CD8high subpopulations was observed in chronic Chagas disease patients than in healthy donors. Additionally, CD4+CD8+ T cells from these patients expressed and co-expressed higher levels of inhibitory receptors in direct proportion to the severity of the pathology. Benznidazole treatment reduced the frequency of CD4+CD8+ T cells and decreased the ratio of CD4+CD8low/CD4+CD8high subpopulations. The co-expression level of the inhibitory receptor was reduced after treatment simultaneously with the enhancement of the multifunctional capacity of CD4+CD8+ T cells. After treatment, an increase in the frequency of T. cruzi antigen-specific CD4+CD8+ T cells expressing IL-2 and TNF-α was also observed. CONCLUSIONS: CD4+CD8+ T cells could play an important role in the control of T. cruzi infection since they were able to produce effector molecules for parasite control. Benznidazole treatment partially reversed the exhaustion process caused by T. cruzi infection in these cells with an improvement in the functional response of the T. cruzi antigen-specific CD4+CD8+ T cells.

Performance of Leishmania PFR1 recombinant antigen in serological diagnosis of asymptomatic canine leishmaniosis by ELISA.

BACKGROUND: Leishmania infantum is a protozoan parasite transmitted by phlebotomine sand flies that causes life-threatening disease in humans and dogs. The dog is the primary reservoir of the parasite and early diagnosis of canine leishmaniosis is crucial at the clinical and epidemiological level. The currently available serological tests for CanL diagnostic show limitations therefore the aim of the present study was to investigate the diagnostic performance of an indirect antibody ELISA based on the Leishmania infantum recombinant antigen PFR1 in asymptomatically infected dogs. One hundred fifty-six dogs including Leishmania-free experimental Beagles and pet dogs from England, Scotland and Leishmania-endemic Murcia in Spain, were tested with the assay. The later were also tested with two commercial L. infantum crude antigen ELISAs (INgezim and Civtest, respectively) and a real-time kinetoplast PCR test. RESULTS: Anti-PFR1 antibodies were detected in the four groups of dogs, and the mean log-transformed optical density (OD) values were lowest in Beagles and in dogs from England and highest among dogs from Murcia (p < 0.05). Using the highest OD in beagles as the PFR1 ELISA cut-off point, the estimated seroprevalence was 27% (14-40%) in dogs from Murcia, 4% (0-9%) in dogs from Scotland and 3% (0-8%) in dogs from England (p < 0.05). Seroprevalence in dogs from Murcia according to the INgezim and Civtest ELISAs were 24% (12-37%) and 31% (18-45%), respectively, whilst the prevalence of infection based on PCR in these dogs was 73% (60-86). The percentages of PFR1-positive dogs that tested negative on the INgezim and Civtest ELISAs were 30% and 35%, respectively, and all of them tested positive on the PCR test. Relative to the PCR, the specificity, sensitivity and area under the ROC curve of the PFR1 ELISA were 100%, 36% and 0.74 (0.63-0.86), respectively. CONCLUSIONS: The ability shown by the PFR1 ELISA to detect infected dogs that go undetected by the crude antigen ELISAs is clinically and epidemiologically useful and PFR1 could be considered a candidate for a multi-antigen-based immunoassay for early detection of L. infantum infected dogs.

Expression of inhibitory receptors and polyfunctional responses of T cells are linked to the risk of congenital transmission of T. cruzi.

Congenital T. cruzi infections involve multiple factors in which complex interactions between the parasite and the immune system of pregnant women play important roles. In this study, we used an experimental murine model of chronic infection with T. cruzi to evaluate the changes in the expression of inhibitory receptors and the polyfunctionality of T cells during gestation and their association with congenital transmission rate of T. cruzi infection. The results showed that pregnant naïve mice had a higher percentage of CD4+ and CD8+ T cells that expressed inhibitory receptors than cells from non-pregnant naïve mice. However, in mice chronically infected with T. cruzi, gestation induced a significant decrease in the frequency of T cells that expressed or co-expressed inhibitory receptors, as well as an increase in the frequency of polyfunctional CD4+ and CD8+ T cells. This different behavior may be due to the breakdown in the infected mice of the gestation-induced immune homeostasis, probably to control the parasite load. Remarkably, it was observed that the mothers that transmitted the parasite had a higher frequency of T cells that expressed and co-expressed inhibitory receptors as well as a lower frequency of polyfunctional parasite-specific T cells than those that did not transmit it, even though the parasitemia load was similar in both groups. All together these data suggest that the maternal immune profile of the CD4+ and CD8+ T cells could be a determining factor in the congenital transmission of T. cruzi.

Antiparasitic Treatment Induces an Improved CD8+ T Cell Response in Chronic Chagasic Patients.

Chagas disease is a chronic infection caused by Trypanosoma cruzi, an intracellular protozoan parasite. Chronic chagasic patients (CCPs) have dysfunctional CD8+ T cells that are characterized by impaired cytokine production, high coexpression of inhibitory receptors, and advanced cellular differentiation. Most patients diagnosed in the chronic phase of Chagas disease already exhibit heart involvement, and there is no vaccination that protects against the disease. Antiparasitic treatment is controversial as to its indication for this stage of the disease. There is a lack of biological markers to evaluate the effectiveness of antiparasitic treatment, and little is known about the effect of the treatment on CD8+ T cells. Thus, the aim of the current study was to analyze the early effects of antiparasitic treatment on CD8+ T cells from CCPs with asymptomatic clinical forms of disease. To evaluate the CD8+ T cell subsets, expression of inhibitory receptors, and functionality of T cells in CCPs, PBMCs were isolated. The results showed that treatment of CCPs with the asymptomatic form of the disease induces an increase in the frequency of CD8+ central memory T cells and terminal effector T cells, a decrease in the coexpression of inhibitory receptors, an improved Ag-specific CD8+ T cell response exhibited by the individual production of IFN-γ or IL-2, and a multifunctional CD8+ T cell profile of up to four functions (IFN-γ+IL-2+Perforin+Granzyme B+). These findings suggest that, in CCPs, antiparasitic treatment improved the quality of Ag-specific CD8+ T cell responses associated with a decrease in inhibitory receptor coexpression, which could serve as biomarkers for monitoring the effectiveness of antiparasitic treatment.

A 12-mer repetitive antigenic epitope from Trypanosoma cruzi is a potential marker of therapeutic efficacy in chronic Chagas' disease.

OBJECTIVES: The objective was to characterize a Trypanosoma cruzi repetitive amino acid sequence that can be used as a marker of therapeutic drug efficacy in patients with chronic Chagas' disease. METHODS: Reactivities to the 3973 peptide were measured in 85 patients with Chagas' disease (41 in the asymptomatic stage and 44 in the cardiomyopathy stage) before and 9 and 24 months after benznidazole administration. Additionally, the levels of IL-6 and C-reactive protein were measured in serum samples from patients with cardiomyopathy. RESULTS: In 85% of the asymptomatic patients and 73% of the symptomatic chronic patients, modifications of the reactivity to the 3973 peptide were observed at 9 and 24 months post-benznidazole treatment. Significant variations in reactivities to the total antigens of T. cruzi were not observed at these times. Significant decreases in the reactivity to the 3973 peptide were observed after treatment in 20 of 41 (49%) asymptomatic patients and 15 of 44 (34%) cardiac chagasic patients (P < 0.001). In these patients, the decreases in reactivity at 24 months post-treatment were at least 40% lower than those detected before treatment. No correlations were found of the detected modifications in reactivity to the 3973 peptide after treatment with the levels of C-reactive protein or IL-6. CONCLUSIONS: Decreases in reactivity to the 3973 peptide may be relevant in the post-treatment follow-up of chronic chagasic patients.

Differential phenotypic and functional profiles of TcCA-2 -specific cytotoxic CD8+ T cells in the asymptomatic versus cardiac phase in Chagasic patients.

It has been reported that the immune response mediated by T CD8+ lymphocytes plays a critical role in the control of Trypanosoma cruzi infection and that the clinical symptoms of Chagas disease appear to be related to the competence of the CD8+ T immune response against the parasite. Herewith, in silico prediction and binding assays on TAP-deficient T2 cells were used to identify potential HLA-A*02:01 ligands in the T. cruzi TcCA-2 protein. The TcCA-2-specific CD8+ T cells were functionality evaluated by Granzyme B and cytokine production in peripheral blood mononuclear cells (PBMC) from Chagas disease patients stimulated with the identified HLA-A*02:01 peptides. The specific cells were phenotypically characterized by flow cytometry using several surface markers and HLA-A*02:01 APC-labeled dextramer loaded with the peptides. In the T. cruzi TcCA-2 protein four T CD8+ epitopes were identified which are processed and presented during Chagas disease. Interestingly, a differential cellular phenotypic profile could be correlated with the severity of the disease. The TcCA-2-specific T CD8+ cells from patients with cardiac symptoms are mainly effector memory cells (TEM and TEMRA) while, those present in the asymptomatic phase are predominantly naive cells (TNAIVE). Moreover, in patients with cardiac symptoms the percentage of cells with senescence features is significantly higher than in patients at the asymptomatic phase of the disease. We consider that the identification of these new class I-restricted epitopes are helpful for designing biomarkers of sickness pathology as well as the development of immunotherapies against T. cruzi infection.

Trypanosoma cruzi paraflagellar rod proteins 2 and 3 contain immunodominant CD8(+) T-cell epitopes that are recognized by cytotoxic T cells from Chagas disease patients.

The protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. To date, no vaccine is available for protection against T. cruzi infection. The CD8(+) T cells immune response against specific antigens has shown to efficiently control the spread of the parasite in murine experimental infection. However, data concerning CD8(+) response in Chagas patients are still restricted to a few epitopes. We have studied the existence of immunodominant CD8(+) T cell epitopes in the paraflagellar rod proteins 2 and 3 (PFR2 and PFR3) from T. cruzi in a mouse model, and analyzed their recognition by cytotoxic T lymphocytes from Chagas disease patients. Immunization of C57BL/6-A2/K(b) transgenic mice with plasmids coding for the fusion proteins PFR2-HSP70 and PFR3-HSP70 induced a specific CTL response against two PFRs epitopes (PFR2(449-457) and PFR3(481-489)), and showed specific lysis percentages of 24 and 12, respectively. Moreover, the PFR2(19-28), PFR2(156-163), PFR2(449-457), PFR3(428-436), PFR3(475-482) and PFR3(481-489) peptides were observed to have a high binding affinity to the HLA-A*02:01 molecule. Remarkably, these HLA-A*02:01-binding peptides are successfully processed and presented during natural infection by T. cruzi in the context of MHC class I as evidenced by using peptide-pulsed K562-A2 cells as antigen presenting cells. The T cells from Chagas disease chronic patients specific for PFR2/PFR3 selected CD8(+) epitopes showed a pro-inflammatory cytokine secretion profile (IFN-γ, TNF-α and IL-6). A positive Granzime B secretion was observed in three out of 16 patients in response to PFR2(156-163) and PFR2(449-457) peptides, two out of 11 patients in response to PFR2(19-28) peptide and one out of 14 and 11 patients in response to PFR3(428-436) and PFR3(481-489) peptides, respectively. The PFRs-specific cytotoxic activity in purified PBMC was only detected in patients in the indeterminate phase of the disease.

Identification of HLA-A∗02:01-restricted CTL epitopes in Trypanosoma cruzi heat shock protein-70 recognized by Chagas disease patients.

CD8(+) cytotoxic T lymphocyte (CTL) response is critical for controlling the infection of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Since only a few CD8 antigens have been described in Chagas disease patients, the identification of new class I-restricted epitopes is urgently needed for the development of immunotherapies against T. cruzi infection. In this study, bioinformatic methods were used to predict HLA-A∗02:01-binders, and 30 peptides were selected, synthesized and tested for HLA-A∗02:01 binding. Among them, sixteen peptides with medium-to-high affinity were assayed for their recognition by CTL from HSP70-immunized or T. cruzi-infected transgenic B6-A2/K(b) mice. Our results show that four immunodominant epitopes (HSP70(210-8), HSP70(255-63), HSP70(316-24) and HSP70(345-53)) are contained in the T. cruzi HSP70 antigen. Indeed two of them (HSP70(210-8) and HSP70(316-24)) were also recognized by CTL of HLA-A∗02:01(+) Chagas disease patients, indicating that these peptides are processed and displayed as MHC class I epitopes during the natural history of T. cruzi infection. The HLA-A∗02:01 restriction was evidenced using peptide-pulsed K562-A2 cells as antigen-presenting cells. Both cytotoxic and cytokine-secreting activities were detected in response to the former two peptides and, moreover, 10/12 patients (83%) recognized at least one of these two HSP70-derived CD8(+) epitopes.