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

Distinct Treatment Outcomes of Antiparasitic Therapy in Trypanosoma cruzi-Infected Children Is Associated With Early Changes in Cytokines, Chemokines, and T-Cell Phenotypes

Background: In contrast to adults, Trypanosoma cruzi-infected children have more broadly functional Trypanosoma cruzi-specific T cells, and the total T-cell compartment exhibits fewer signs of immune exhaustion. However, not much is known about the link between immunocompetence and the treatment efficacy for human Chagas disease.Methods: Using cytokine enzyme-linked immunosorbent spot (ELISPOT) polychromatic flow cytometry, cytometric bead assay, multiplex serological assays and quantitative PCR, we evaluated T. cruzi-specific T-cell and antibody immune responses, T-cell phenotypes and parasitemia in children in the early chronic phase of Chagas disease undergoing anti-Trypanosoma cruzi treatment.Results: Treatment with benznidazole or nifurtimox induced a decline in T. cruzi-specific IFN-γ- and IL-2-producing cells and proinflammatory cytokines and chemokines. T-cell responses became detectable after therapy in children bearing T-cell responses under background levels prior to treatment. The total frequencies of effector, activated and antigen-experienced T cells also decreased following anti-T. cruzi therapy, along with an increase in T cells expressing the receptor of the homeostatic cytokine IL-7. Posttreatment changes in several of these markers distinguished children with a declining serologic response suggestive of successful treatment from those with sustained serological responses in a 5-year follow-up study. A multivariate analysis demonstrated that lower frequency of CD4+CD45RA−CCR7−CD62L− T cells prior to drug therapy was an independent indicator of successful treatment.Conclusions: These findings further validate the usefulness of alternative metrics to monitor treatment outcomes. Distinct qualitative and quantitative characteristics of T cells prior to drug therapy may be linked to treatment efficacy

Cytokine/costimulation profile of <i>T. cruzi</i>-specific CD4⁺ T cells in short-term and long-term <i>T. cruzi</i> infections.

<p>PBMC were stimulated with a <i>T. cruzi</i> lysate preparation and cytokine/costimulation coexpression profiles with triple (ITD), double (IT, ID, TD) or single (I, T, D) function were determined in nineteen <i>T. cruzi</i>-infected children (A) and ten adults with chronic <i>T. cruzi</i> infection (B) using the Boolean gating function of FlowJo software. The proportions of CD4⁺ subsets positive for specific cytokines or co-stimulatory molecules were expressed as percentages of total cytokine/costimulatory-positive CD4⁺ T cells. Mean and SD are shown. (C) Summary of the functional composition of the CD4⁺ T cell response. Each slice of the pie represents the fraction of the total response that consists of CD4⁺ T cells positive for the different seven subsets. D) Prevalence of polyfunctional CD4⁺ T cell responses in <i>T. cruzi</i>-infected children (left panel) and adults (right panel). Each slice of the pie represents the percent of subjects with one (1+), two (2+) or three (3+) functions. I, IFN-γ; T, TNF-α; D, CD154. ITD, proportion of CD4⁺ T cells with concomitant expression of IFN-γ, TNF-α and CD154; IT, proportion of CD4⁺ T cells with concomitant expression of IFN-γ and TNF-α; ID, proportion of CD4⁺ T cells with concomitant expression of IFN-γ and CD154; TD, proportion of CD4⁺ T cells with concomitant expression of TNF-α and CD154; I, proportion of CD4⁺ T cells with IFN-γ only production; T, proportion of CD4⁺ T cells with TNF-α only production; D, proportion of CD4⁺ T cells with CD154 only expression.</p

IFN-γ-secreting cells in response to <i>Flu- and tetanus toxoid-derived antigens</i> in <i>uninfected</i> children, uninfected adults and adults with chronic <i>T. cruzi</i>-infection.

<p>PBMC from uninfected children (n = 6), uninfected adults (n = 7) and long-term <i>T.cruzi</i>-infected adults (n = 10) were seeded at 4×10⁵ cells/well and stimulated with class-I-restricted Flu-derived peptides (A), tetanus toxoid (B) or media alone for 16–20 h. Each symbol represents the mean spot number of triplicate wells for each patient with positive ELISPOT responses, as defined in material and methods. Spot counts with media alone were subtracted. Vertical lines depicting median values are shown. No significant differences were found among groups.</p

IFN-γ and IL-2-secreting cells in response to <i>Trypanosoma cruzi</i> antigens in <i>T. cruzi</i>-infected children.

<p>PBMC from <i>T. cruzi</i>-infected children were seeded at 4×10⁵ cells/well and stimulated with a <i>T. cruzi</i> lysate from the Brazil strain, class-I-restricted HLA-A01, HLA-A02, HLA-A03, HLA-A24, HLA-B07 and HLA-B44 supertype <i>trans</i>-sialidase peptide pools, class-I-restricted Flu-derived peptides or media alone for 16–20 h. (A) T cell responses to a <i>T. cruzi</i> lysate preparation. Each circle represents the mean spot number of triplicate wells for each patient with positive ELISPOT responses out of seventeen assessed. Spot counts with media alone were subtracted. Vertical lines depicting median values are shown. IFN-γ-DP, no. of IFN-γ spots in individuals with positive responses for both IFN-γ and IL-2 ELISPOT assays; IFN-γ-SP, no. of IFN-γ spots in individuals with positive ELISPOT responses for IFN-γ alone; IL-2-DP, no. of IL-2 spots in individuals with positive responses for both IFN-γ and IL-2 ELISPOT assays; IL-2-SP, no. of IL-2 spots in individuals with positive ELISPOT responses for IL-2 alone. (*) P<0.01 vs. IL-2-DP; vs. IL-2-SP and vs. IFN-γ-SP by the Kruskal-Wallis test with Dunn correction. (B) Class I-restricted IFN- γ and IL-2 ELISPOT responses to <i>trans</i>-sialidase peptides. Representative positive ELISPOT responses for IFN-γ (grey bars) and IL-2 (white bars) in a single subject are shown. (*) Indicates positive responses, as defined in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002575#s2" target="_blank">Materials and Methods</a>.</p

Characteristics of study population.

<p>Note.</p>A<p>All children were born from <i>T. cruzi</i>-infected mothers.</p>B<p>All children and adult subjects were living in Buenos Aires (non-endemic) by the time of the study.</p>C<p>Number of patients presenting electrocardiographic alterations.</p>D<p>P<0.05, seropositive children born in areas endemic for <i>T. cruzi</i> infection vs. seropositive and seronegative children born in non-endemic areas by Fisher' s exact test.</p>E<p>P<0.001 vs. seropositive and seronegative children, by Kruskal-Wallis test.</p>F<p>P<0.05 vs. seronegative adults, by Kruskal-Wallis test.</p><p>ANE, abnormal findings in electrocardiography not relevant to Chagas disease; RBBB, right bundle branch block; PP, permanent pacemaker; G1, Group 1 of the Kuschnir grading system.</p

Magnitude of CD4⁺ T cell responses in <i>T. cruzi</i>-infected children and adults.

<p>Whole blood was stimulated with an amastigote lysate preparation, and antigen-responsive CD4⁺ T cells were measured using an intracellular staining assay for IFN-γ, TNF-α and CD154. The percentage of total responsive CD4⁺ T cells for each individual function in nineteen <i>T. cruzi</i>-infected children (hatched columns) and ten adults with chronic <i>T. cruzi</i> infection (black columns) is plotted. Boxes and whiskers depicting median and 10th and 90th percentile values are shown. (*) P = 0.03 vs. CD4⁺TNF-α⁺ in <i>T. cruzi</i>-infected children and (&) P = 0.03 vs. CD4⁺TNF-α⁺ T cells in <i>T. cruzi</i>-infected adults by Kruskal-Wallis test with Dunn correction; (**) P = 0.004 vs. CD4⁺CD154⁺ in <i>T. cruzi</i>-infected adults and (≈) P = 0.006 vs. CD4⁺TNF-α⁺ T cells in <i>T. cruzi</i>-infected adults by Mann-Whitney U test.</p

T cell phenotype of total CD4⁺ T cells in children in the early stages of Chagas disease.

<p>Note. (†) Data are expressed as the median (range) percentage of total CD4⁺ T lymphocytes.</p><p>For each phenotype. (*) Mann-Whitney U-test between <i>T. cruzi</i>-infected and uninfected subjects; NS, no significant different; CM, central memory; EM, effector memory; TE, terminally differentiated effector cells.</p

<i>Trypanosoma cruzi</i>-specific T cell responses in children with Chagas disease and uninfected subjects living in non-endemic areas of Argentina, measured by IFN-γ and IL-2 ELISPOT.

A<p>Fisher exact test p = 0.0181 vs. percentage of IFN-γ-only responders in <i>T. cruzi</i>-infected subjects.</p>B<p>Fisher exact test p = 0.0003 vs. percentage of IL-2-only responders in <i>T. cruzi</i>-infected subjects.</p>C<p>Fisher exact test p = 0.0003 vs. percentage of IFN-γ+IL-2 responders in the uninfected group.</p>D<p>Fisher exact test p = 0.23 vs. percentage of responders in the uninfected group.</p