Polyfunctional T cell responses in children in early stages of chronic Trypanosoma cruzi infection contrast with monofunctional responses of long-term infected adults

Background: Adults with chronic Trypanosoma cruzi exhibit a poorly functional T cell compartment, characterized by monofunctional (IFN-γ-only secreting) parasite-specific T cells and increased levels of terminally differentiated T cells. It is possible that persistent infection and/or sustained exposure to parasites antigens may lead to a progressive loss of function of the immune T cells. Methodology/Principal Findings: To test this hypothesis, the quality and magnitude of T. cruzi-specific T cell responses were evaluated in T. cruzi-infected children and compared with long-term T. cruzi-infected adults with no evidence of heart failure. The phenotype of CD4+ T cells was also assessed in T. cruzi-infected children and uninfected controls. Simultaneous secretion of IFN-γ and IL-2 measured by ELISPOT assays in response to T. cruzi antigens was prevalent among T. cruzi-infected children. Flow cytometric analysis of co-expression profiles of CD4+ T cells with the ability to produce IFN-γ, TNF-α, or to express the co-stimulatory molecule CD154 in response to T. cruzi showed polyfunctional T cell responses in most T. cruzi-infected children. Monofunctional T cell responses and an absence of CD4+TNF-α+-secreting T cells were observed in T. cruzi-infected adults. A relatively high degree of activation and differentiation of CD4+ T cells was evident in T. cruzi-infected children. Conclusions/Significance: Our observations are compatible with our initial hypothesis that persistent T. cruzi infection promotes eventual exhaustion of immune system, which might contribute to disease progression in long-term infected subjects.Fil: Albareda, María Cecilia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: de Rissio, Ana María. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Tomas, Gonzalo. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Serjan, Alicia. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Alvarez, María Gabriela. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Viotti, Rodolfo Jorge. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Fichera, Laura Edith. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Esteva, Mónica Inés. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Potente, Daniel Fernando. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Armenti, Alejandro. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Tarleton, Rick L.. University of Georgia; Estados UnidosFil: Laucella, Susana Adriana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Impact of Aetiological Treatment on Conventional and Multiplex Serology in Chronic Chagas Disease

The main criterion for treatment effectiveness in Chagas Disease has been the seronegative conversion of previously reactive serology, generally achieved many years post-treatment. The lack of reliable tests to ensure parasite clearance and to examine the effect of treatment is the main difficulty in evaluating treatment for chronic Chagas disease. Decreases of conventional and non-conventional serological titers can be useful tools to monitor the early impact of treatment. We serially measured changes in antibody levels, including seronegative conversion as well as declines in titers in 53 benznidazole-treated and 89 untreated chronically T. cruzi-infected subjects. Seronegative conversion as well as decreases of titers was significantly higher in treated compared with untreated patients. A strong concordance was found between decreases of titers of conventional and non-conventional serologic tests post-treatment, reaffirming the findings. When seronegative conversion plus decreases of titers were considered altogether, the impact of treatment was higher, in a shorter follow-up period than previously considered. New tools for monitoring the effectiveness of treatment of chronic Chagas disease are necessary, and the results showed in this study is a contribution to researchers and physicians who assist patients suffering from this disease

CD8(+) T-Cell Responses to Trypanosoma cruzi Are Highly Focused on Strain-Variant trans-Sialidase Epitopes

CD8(+) T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8(+) T cells in T. cruzi–infected mice and humans. This analysis demonstrates that in both hosts the CD8(+) T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi–infected mice, with more than 30% of the CD8(+) T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level

Inhibitory Receptors Are Expressed by Trypanosoma cruzi-Specific Effector T Cells and in Hearts of Subjects with Chronic Chagas Disease

We had formerly demonstrated that subjects chronically infected with Trypanosoma cruzi show impaired T cell responses closely linked with a process of T cell exhaustion. Recently, the expression of several inhibitory receptors has been associated with T cell dysfunction and exhaustion. In this study, we have examined the expression of the cytotoxic T lymphocyte antigen 4 (CTLA-4) and the leukocyte immunoglobulin like receptor 1 (LIR-1) by peripheral T. cruzi antigen-responsive IFN-gamma (IFN-γ)-producing and total T cells from chronically T. cruzi-infected subjects with different clinical forms of the disease. CTAL-4 expression was also evaluated in heart tissue sections from subjects with severe myocarditis. The majority of IFN-γ-producing CD4+ T cells responsive to a parasite lysate preparation were found to express CTLA-4 but considerably lower frequencies express LIR-1, irrespective of the clinical status of the donor. Conversely, few IFN-γ-producing T cells responsive to tetanus and diphtheria toxoids expressed CTLA-4 and LIR-1. Polyclonal stimulation with anti-CD3 antibodies induced higher frequencies of CD4+CTAL-4+ T cells in patients with severe heart disease than in asymptomatic subjects. Ligation of CTLA-4 and LIR-1 with their agonistic antibodies, in vitro, reduces IFN-γ production. Conversely, CTLA-4 blockade did not improved IFN-γ production in response to T. cruzi antigens. Subjects with chronic T. cruzi infection had increased numbers of CD4+LIR-1+ among total peripheral blood mononuclear cells, relative to uninfected individuals and these numbers decreased after treatment with benznidazole. CTLA-4 was also expressed by CD3+ T lymphocytes infiltrating heart tissues from chronically infected subjects with severe myocarditis. These findings support the conclusion that persistent infection with T. cruzi leads to the upregulation of inhibitory receptors which could alter parasite specific T cell responses in the chronic phase of Chagas disease

High Throughput Selection of Effective Serodiagnostics for Trypanosoma cruzi infection

The diagnosis of Trypanosoma cruzi infection (the cause of human Chagas disease) is difficult because the symptoms of the infection are often absent or non-specific, and because the parasites themselves are usually below the level of detection in the infected subjects. Therefore, diagnosis generally depends on the measurement of T. cruzi–specific antibodies produced in response to the infection. However, current methods to detect anti–T. cruzi antibodies are relatively poor. In this study, we have conducted a broad screen of >400 T. cruzi proteins to identify those proteins which are best able to detect anti–T. cruzi antibodies. Using a set of proteins selected by this screen, we were able to detect 100% of >100 confirmed positive human cases of T. cruzi infection, as well as suspect cases that were negative using existing tests. This protein panel was also able to detect apparent changes in infection status following drug treatment of individuals with chronic T. cruzi infection. The results of this study should allow for significant improvements in the detection of T. cruzi infection and better screening methods to avoid blood transfusion–related transmission of the infection, and offer a crucial tool for determining the success or failure of drug treatment and other intervention strategies to limit the impact of Chagas disease

Short-term follow-up of chagasic patients after benznidazole treatment using multiple serological markers

<p>Abstract</p> <p>Background</p> <p>Conventional serological tests, using total soluble proteins or a cocktail of recombinant proteins from <it>T. cruzi </it>as antigens, are highly sensitive for Chagas disease diagnosis. This type of tests, however, does not seem to be reliable tools for short- and medium-term monitoring of the evolution of patients after antiparasitic treatment. The aim of the present study was to search for immunological markers that could be altered in the sera from Chagas disease patients after benznidazole treatment, and therefore have a potential predictive diagnostic value.</p> <p>Methods</p> <p>We analyzed the reactivity of sera from chagasic patients during different clinical phases of the disease against a series of immunodominant antigens, known as KMP11, PFR2, HSP70 and Tgp63. The reactivity of the sera from 46 adult Chronic Chagas disease patients living in a non-endemic country without vector transmission of <it>T. cruzi </it>(15 patients in the indeterminate stage, 16 in the cardiomiopathy stage and 16 in the digestive stage) and 22 control sera from non-infected subjects was analyzed. We also analyzed the response dynamics of sera from those patients who had been treated with benznidazole.</p> <p>Results</p> <p>Regardless of the stage of the sickness, the sera from chagasic patients reacted against KMP11, HSP70, PFR2 and Tgp63 recombinant proteins with statistical significance relative to the reactivity against the same antigens by the sera from healthy donors, patients with autoimmune diseases or patients suffering from tuberculosis, leprosy or malaria. Shortly after benznidazole treatment, a statistically significant decrease in reactivity against KMP11, HSP70 and PFR2 was observed (six or nine month). It was also observed that, following benznidazole treatment, the differential reactivity against these antigens co-relates with the clinical status of the patients.</p> <p>Conclusions</p> <p>The recombinant antigens KMP11, PFR2, Tgp63 and HSP70 are recognized by Chagas disease patients' sera at any clinical stage of the disease. Shortly after benznidazole treatment, a drop in reactivity against three of these antigens is produced in an antigen-specific manner. Most likely, analysis of the reactivity against these recombinant antigens may be useful for monitoring the effectiveness of benznidazole treatment.</p

Regulatory T Cells Phenotype in Different Clinical Forms of Chagas' Disease

CD25High CD4+ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3+ CD25HighCD4+ cells that was also IL-10+ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3+ CD25High CD4+ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections

Protective Human Leucocyte Antigen Haplotype, HLA-DRB1*01-B*14, against Chronic Chagas Disease in Bolivia

Chronic Chagas disease consists of four different forms categorized on the basis of their clinical manifestations, namely; cardiac, digestive, cardiodigestive and indeterminate. In Latin America, there are 8–10 million seropositive persons who are at risk of, or have already developed serious clinical complications and who have limited access to effective treatment. The cardiac and digestive forms are characterized by tissue damage caused by persistent infection of Trypanosoma cruzi and are thought to be modulated by host immunity. In our large scale screening for chronic Chagas disease in Santa Cruz, Bolivia, hearts and colons of 229 seropositive patients were examined. We found 31.4% of patients had abnormal electrocardiograms (ECGs), 15.7% presented with megacolon, 5.2% had a combination of abnormal ECG and megacolon, and 58.1% were of indeterminate status. Previously, we attempted to ascertain whether parasite genetic polymorphism might account for the differences in clinical manefestations, by analyzing parasite DNA taken from the same study group (with the addition of a further 62 megacolon post-operational patients). We found no relationships between parasite lineages and clinical disease form. The present study reveals that host HLA polymorphisms associate with clinical manifestations of Chagas