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.

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 T_NAIVE 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 (T_EM for CD4⁺ T cells and T_EMRA for CD8⁺ T cells). The amount of antigen-specific CD57⁺/CD8⁺ T_EMRA 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.

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. 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. The frequency of CD4⁺CD8⁺ T cells was higher in chronic Chagas disease patients compared with healthy donors. Furthermore, a higher ratio of CD4⁺CD8^low/CD4⁺CD8^high 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⁺CD8^low/CD4⁺CD8^high 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. 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. Chagas disease is a neglected tropical disease caused by the intracellular parasite Trypanosoma cruzi. The persistence of the parasite leads to deterioration of the host immune response, which is known as an exhaustion process. This process affects T cell populations, leading to increased expression of inhibitory receptors that leads to a dysfunctional ability to respond to the parasite. CD4⁺CD8⁺ T cells form a poorly studied population of T cells in the context of Chagas disease. In this study, as in others previously reported, an increase in the frequency of these cells was observed in chronic Chagas disease patients. In addition, CD4⁺CD8⁺ T cells from chronic Chagas disease patients underwent stronger exhaustion processes with more severe disease pathology. A higher level of expression and co-expression of inhibitory receptors was observed in these cells in symptomatic compared with asymptomatic patients. Furthermore, we evaluated whether antiparasitic treatment affected the population of CD4⁺CD8⁺ T cells. Our results showed that after treatment, the functional capacity of these cells against the parasite improved. Concomitantly, a partial reversion of this exhaustion process occurred since the co-expression of inhibitory receptors decreased in CD4⁺CD8⁺ T cells from chronic patients after treatment.

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.