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

Abstract

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.

Figure 1. IFN-γ and IL-2-secreting cells in response to Trypanosoma cruzi antigens in T. cruzi-infected children.

PBMC from T. cruzi-infected children were seeded at 4×10⁵ cells/well and stimulated with a T. cruzi lysate from the Brazil strain, class-I-restricted HLA-A01, HLA-A02, HLA-A03, HLA-A24, HLA-B07 and HLA-B44 supertype trans-sialidase peptide pools, class-I-restricted Flu-derived peptides or media alone for 16–20 h. (A) T cell responses to a T. cruzi 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 trans-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 Materials and Methods.

Figure 2. IFN-γ-secreting cells in response to Flu- and tetanus toxoid-derived antigens in uninfected children, uninfected adults and adults with chronic T. cruzi-infection.

PBMC from uninfected children (n = 6), uninfected adults (n = 7) and long-term T.cruzi-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.

Figure 3. Magnitude of CD4⁺ T cell responses in T. cruzi-infected children and adults.

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 T. cruzi-infected children (hatched columns) and ten adults with chronic T. cruzi 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 T. cruzi-infected children and (&) P = 0.03 vs. CD4⁺TNF-α⁺ T cells in T. cruzi-infected adults by Kruskal-Wallis test with Dunn correction; (**) P = 0.004 vs. CD4⁺CD154⁺ in T. cruzi-infected adults and (≈) P = 0.006 vs. CD4⁺TNF-α⁺ T cells in T. cruzi-infected adults by Mann-Whitney U test.

Figure 4. Cytokine/costimulation profile of T. cruzi-specific CD4⁺ T cells in short-term and long-term T. cruzi infections.

PBMC were stimulated with a T. cruzi lysate preparation and cytokine/costimulation coexpression profiles with triple (ITD), double (IT, ID, TD) or single (I, T, D) function were determined in nineteen T. cruzi-infected children (A) and ten adults with chronic T. cruzi 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 T. cruzi-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.