Chagas disease is a chronic infection caused by 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-γIL-2PerforinGranzyme 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.

BackgroundChagas 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.MethodologyThirty-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 findingsThe 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.ConclusionsCD4+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.

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.

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 3973 d ) 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 ϩ /perforin ϩ CD8 ϩ 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 Ͻ 0.05). Furthermore, the patients who met the STEC exhibited an increased quality of the multifunctional response of the antigen-specific CD8 ϩ T cells compared with that in the patients who did not meet the STEC.

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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.

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.

La enfermedad de Chagas y la leishmaniasis, causadas por parásitos protozoarios intracelulares del orden Trypanosomatida, son consideradas dos de las enfermedades tropicales desatendidas más importantes. Estas infecciones conllevan un alto desgaste social, provocando el deterioro de la salud de un gran número de pacientes e incluso su muerte. Los linfocitos T son células fundamentales del sistema adaptativo y son los principales elementos inmunitarios para el control de estas infecciones intracelulares. La presente revisión explora los estudios y resultados obtenidos hasta la fecha del proceso de agotamiento celular durante las infecciones causadas por los parásitos Leishmania spp. y Trypanosoma cruzi. Así, se recoge que la persistente estimulación celular en respuesta a antígenos de estos patógenos conduce a un proceso de pérdida de la capacidad funcional antígeno-específica en las poblaciones de células T CD4+ y CD8+. Numerosos estudios muestran la existencia de una correlación directa entre el nivel de la co-expresión de receptores inhibitorios y la gravedad de estas patologías. Paralelamente, se detecta la pérdida de la capacidad funcional específica de antígeno de estas células T, lo que reduce su potencial linfoproliferativo y su capacidad de producir moléculas protectoras contra estas infecciones. Además, durante el curso de estas infecciones se observa un incremento de la frecuencia de células T de memoria efectora con un grado de diferenciación tardía o terminal. Este balanceo fenotípico, a su vez, afecta a la capacidad funcional de las células T aumentando el número de células con características senescentes y apoptóticas. Así, los estudios realizados hasta la fecha demuestran con certeza la existencia de un proceso de agotamiento que afecta a poblaciones clave para el control parasitario. Sin embargo, actualmente se desconoce con precisión el papel que este proceso de agotamiento juega en el agravamiento de estas patologías. Los medicamentos actuales usados para tratar estas enfermedades protozoarias revierten parcialmente este proceso de agotamiento. Así, tras el tratamiento, numerosos pacientes muestra una reducción en la expresión de receptores inhibitorios y co-expresión de los mismos. También se ha observado una mejoría en la capacidad funcional de las distintas poblaciones de células T, que podría estar relacionada con la reversión del proceso disfuncional. Sin embargo, los estudios realizados hasta la fecha en la evaluación de terapias de bloqueo de los receptores inhibitorios no han conducido a resultados prometedores. Algunos autores proponen evaluar terapias de bloqueo simultáneo de varias vías de señalización, con el fin de ampliar el conocimiento sobre esta herramienta como posible inmunoterapia de control de la infección por los mencionados parásitos. Además, se considera necesario continuar investigando sobre cómo se desencadena exactamente este proceso de agotamiento celular y en qué medida influye en la aparición de la sintomatología de los pacientes y ausencia de control de la infección.