Miriam Postan

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.

Frequency of Interferon-γ-Producing T Cells Specific for Trypanosoma cruzi Inversely Correlates with Disease Severity in Chronic Human Chagas Disease

This study sought to quantify CD8(+) T cell responses to Trypanosoma cruzi and to identify potential links between these responses and the severity of disease in humans. In the majority of patients with Chagas disease, staining with class I major histocompatibility complex tetramers and analysis of interferon (IFN)- gamma ELISPOT responses to a panel of known cytotoxic T lymphocyte target epitopes from T. cruzi failed to identify parasite-specific CD8(+) T cells. However, the frequency of individuals with positive ELISPOT responses was higher in areas of active transmission. Analysis of IFN- gamma ELISPOT responses to a parasite lysate revealed a very high frequency of responders among patients with mild clinical disease and a very low frequency of responders among those with the most severe form of the disease. These data suggest that the frequency of IFN- gamma -producing T cells in patients with chronic Chagas disease is associated with the history of recent exposure and with the clinical status of the patient.

High Throughput Selection of Effective Serodiagnostics for Trypanosoma cruzi infection

Background Diagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies. Methods In this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection. Findings A set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease. Conclusions These results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.

Changes in Trypanosoma cruzi-Specific Immune Responses after Treatment: Surrogate Markers of Treatment Efficacy

BACKGROUND As many as 20 million people are living with Trypanosoma cruzi infection in Latin American, yet few receive any treatment. The major limitation in developing and evaluating potential new drugs for their efficacy is the lack of reliable tests to assess parasite burden and elimination. METHODS Adults volunteers with chronic T. cruzi infection were evaluated clinically and stratified according to the Kuschnir classification. Individuals with group 0 and group 1 clinical status were treated with benznidazole (5 mg/kg per day for 30 days). The changes in T. cruzi-specific T cell and antibody responses, as well as in clinical status, were measured periodically over the 3-5-year follow-up period and were compared with pretreatment conditions and with values in an untreated control group. RESULTS The frequency of peripheral interferon (IFN)-gamma-producing T cells specific for T. cruzi declined as early as 12 months after benznidazole treatment and subsequently became undetectable in a substantial proportion of treated subjects. In addition, decreases in antibody responses to a pool of recombinant T. cruzi proteins also decreased in many of these same subjects. The shift to negative IFN-gamma T cell responses was highly associated with an early increase in IFN-gamma-producing T cells with phenotypic features of effector/effector memory cells in a subset of subjects. Benznidazole treatment also resulted in an increase in naive and early differentiated memory-like CD8(+) T cells in a majority of subjects. CONCLUSIONS Benznidazole treatment during chronic Chagas disease has a substantial impact on parasite-specific immune response that is likely indicative of treatment efficacy and cure.

Chronic Human Infection with Trypanosoma cruzi Drives CD4+ T Cells to Immune Senescence

Previously we found that the frequency of IFN-γ-producing CD8+ T cells specific for Trypanosoma cruzi inversely correlates with disease severity in chronic human Chagas disease along with low levels of IL-2-secreting CD8+ T cells in all clinical stages. This impairment of the parasite-specific T cell responses was associated with phenotypic features of immune senescence of the CD8+ T cell compartment. These data prompted us to address the question of whether the CD4+ T cell compartment also experiences signs of exhaustion. Thus, we performed a functional and phenotypical characterization of T. cruzi-specific and overall CD4+ T cells in chronically infected subjects with different degrees of cardiac dysfunction. The results show an inverse association between disease severity and the frequency of T. cruzi-specific IFN-γ-producing CD4+ T cells. The high expression of CD27 and CD28 with a relative low expression of CD57 found on CD4+IFN-γ+ T cells suggests that the effector T cell pool in chronic T. cruzi infection includes a high proportion of newly recruited T cells, but a low frequency of long-term memory cells. The total CD4+ T cell compartment shows signs of senescence and later stages of differentiation associated with more severe stages of the disease. These findings support the hypothesis that long-term T. cruzi infection in humans might exhaust long-lived memory T cells.

HLA Class I-T cell epitopes from trans-sialidase proteins reveal functionally distinct subsets of CD8+ T cells in chronic Chagas disease.

Background Previously, we identified a set of HLA-A020.1-restricted trans-sialidase peptides as targets of CD8+ T cell responses in HLA-A0201+ individuals chronically infected by T. cruzi. Methods and Findings Herein, we report the identification of peptides encoded by the same trans-sialidase gene family that bind alleles representative of the 6 most common class I HLA-supertypes. Based on a combination of bioinformatic predictions and HLA-supertype considerations, a total of 1001 epitopes predicted to bind to HLA A01, A02, A03, A24, B7 and B44 supertypes was selected. Ninety-six supertype-binder epitopes encoded by multiple trans-sialidase genes were tested for the ability to stimulate a recall CD8+ T cell response in the peripheral blood from subjects with chronic T. cruzi infection regardless the HLA haplotype. An overall hierarchy of antigenicity was apparent, with the A02 supertype peptides being the most frequently recognized in the Chagas disease population followed by the A03 and the A24 supertype epitopes. CD8+ T cell responses to promiscuous epitopes revealed that the CD8+ T cell compartment specific for T. cruzi displays a functional profile with T cells secreting interferon-γ alone as the predominant pattern and very low prevalence of single IL-2-secreting or dual IFN-γ/IL-2 secreting T cells denoting a lack of polyfunctional cytokine responses in chronic T. cruzi infection. Conclusions This study identifies a set of T. cruzi peptides that should prove useful for monitoring immune competence and changes in infection and disease status in individuals with chronic Chagas disease.

Intracellular growth of Trypanosoma cruzi in cardiac myocytes is inhibited by cytokine-induced nitric oxide release

ABSTRACT The effect of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) on Trypanosoma cruzi multiplication and nitric oxide (NO) production in cardiac myocytes was investigated. Cardiac myocyte cultures were obtained from neonatal Wistar rat hearts, infected with T. cruzi, and treated with IL-1β, TNF-α, IFN-γ, or N-monomethyl-l-arginine (l-NAME) for 72 h. Parasite growth was calculated from the number of infected cells in Giemsa-stained smears. Nitric oxide production was determined with the Griess reagent. Inducible nitric oxide synthase (iNOS) expression by cardiac myocytes was detected by Western blot. The results showed that the percentages of cardiac myocytes containing T. cruzi amastigotes in cytokine-treated cultures were significantly lower than in nontreated cultures. The addition of l-NAME reversed the inhibitory effect on parasite growth of IL-1β and TNF-α but not of IFN-γ. Nitrite levels released by T. cruzi-infected and noninfected cardiac myocyte cultures after 72 h of stimulation with IL-1β were significantly higher than those produced upon treatment with TNF-α, IFN-γ, or medium alone, regardless of the infection status. Nitrite levels in TNF-α-stimulated infected cultures were significantly higher than in untreated infected cultures and TNF-α-treated noninfected cultures. l-NAME inhibited IL-1β- but not TNF-α-induced NO production, indicating the presence of iNOS-dependent and iNOS-independent mechanisms for NO formation in this experimental system. iNOS expression was detected in infected and noninfected cardiac myocytes stimulated with IL-1 β and TNF-α but not with IFN-γ. These results suggest an important role for cardiac myocytes and locally secreted cytokines in the control of parasite multiplication in T. cruzi-induced myocarditis.

TGF‐β regulates pathology but not tissue CD8+ T cell dysfunction during experimental Trypanosoma cruzi infection

Infection with the protozoan parasite Trypanosoma cruzi leads to chronic infection, with parasite persistence primarily in muscle tissue. CD8+ T cells isolated from muscle tissue of T. cruzi‐infected mice display decreased production of IFN‐γ in response to T cell receptor engagement. The expression of TGF‐β at the site of CD8+ T cell dysfunction and parasite persistence suggested that this immunoregulatory cytokine might play a role in these processes. Mice expressing a T cell‐specific dominant negative TGF‐β receptor type II (DNRII) were therefore infected with T. cruzi. Infection of DNRII mice resulted in massive CD8+ T cell proliferation, leading to increased numbers but decreased frequencies of antigen‐specific CD8+ T cells in the spleen compared to wild‐type mice. However, TGF‐β unresponsiveness failed to restore effector functions of CD8+ T cells isolated from muscle tissue. Histological examination of skeletal muscle from T. cruzi‐infected DNRII mice revealed an extensive cellular infiltrate, and DNRII mice displayed higher susceptibility to infection. Overall, while TGF‐β does not appear to be responsible for CD8+ T cell unresponsiveness in peripheral tissue in T. cruzi‐infected mice, these data suggest a role for TGF‐β in control of immunopathology in response to T. cruzi infection.

CARDIAC MYOCYTE HYPERTROPHY AND PROLIFERATING CELL NUCLEAR ANTIGEN EXPRESSION IN WISTAR RATS INFECTED WITH TRYPANOSOMA CRUZI

Chagasic cardiomyopathy is a major life-threatening complication of Trypanosoma cruzi infection in human beings. This study focuses on the hypertrophic and hyperplastic mechanisms underlying the structural changes of the heart during experimental infection. Proliferating cell nuclear antigen (PCNA) expression, transversal diameter, nuclear area, and number of nuclei per unit volume were determined in the ventricular myocytes of T. cruzi–infected Wistar rats. PCNA expression was enhanced throughout the inflamed myocardium and in the spared areas of the left ventricular wall and the septum. Myocyte width increased from 26 to 75% at the inflammation-free myocardium (P < 0.0001), whereas it decreased 25% at the inflamed left ventricular wall areas (P < 0.001). Nuclear size increased in the inflammation-free myocardium of the left ventricle and the septum (>10–36%, P < 0.01 and >0.2–32%, P < 0.03, respectively) and decreased at the inflamed areas of the left ventricular wall (10–22%, P < 0.02) with respect to the controls. The number of nuclei per unit volume decreased at the inflamed myocardium regardless of topographical location (36–65%) with respect to the controls (P < 0.0001) and in the inflammation-free myocardium of the right ventricle and the septum (<21–37%, P < 0.002 and <8–39%, P < 0.002, respectively). These results show that the heart responds to T. cruzi infection with DNA repair and cell multiplication in the inflamed sites and with hypertrophy of the unaffected myocardium.

Trypanosoma cruzi: biological characterization of a isolate from an endemic area and its susceptibility to conventional drugs.

We describe some biological and molecular characteristics of a Trypanosoma cruzi isolate derived from a Triatomine captured in Nicaragua. PCR based typification showed that this isolate, named Nicaragua, belonged to the lineage Tc I. Nicaragua infected culture cells were treated with allopurinol, showing different behavior according to the cellular compartment, being cardiomyocyte primary cultures more resistant to this drug. The course of the infection in a mice experimental model and its susceptibility to benznidazole and allopurinol was analyzed. In benznidazole treatment, mice reverted the high lethal effect of parasites during the acute infection, however, a few parasites were detected in the heart of 88% of mice 1 year post-infection. Since T. cruzi is a heterogeneous species population it is important to study and characterize different parasites actually circulating in humans in endemic areas. In this work we show that T. cruzi Nicaragua isolate, is sensitive to early benznidazole treatment.