Trypanosoma cruzi-induced activation of functionally distinct αβ and γδ CD4- CD8- T cells in individuals with polar forms of Chagas' disease

Abstract

CD4(-) CD8(-) (double-negative [DN]) T cells have recently been shown to display important immunological functions in human diseases. They express γδ or αβ T-cell receptors that recognize lipid/glycolipid antigens presented via the nonclassical major histocompatibility complex molecules of the CD1 family. We recently demonstrated that while αβ DN T cells serve primarily to express inflammatory cytokines, γδ DN T cells express mainly interleukin-10 (IL-10) in patients with cutaneous leishmaniasis. We also demonstrated a correlation between DN T cells and the expression of gamma interferon in the acute phase of Trypanosoma cruzi experimental infection. In this work, we sought to investigate whether αβ or γδ DN T cells display distinct immunoregulatory potentials in patients with polar forms of human Chagas' disease. Our data showed that in vitro infection with T. cruzi leads to expansion of DN T cells in patients with the indeterminate and severe cardiac clinical forms of the disease. However, while αβ DN T cells primarily produce inflammatory cytokines in both forms of the disease, γδ DN T cells display a marked, significant increase in antigen-specific IL-10 expression in indeterminate patients relative to cardiac patients. Finally, higher frequencies of the IL-10-producing γδ DN T cells were correlated with improved clinical measures of cardiac function in the patients, suggesting a protective role for these cells in Chagas' disease. Taken together, these data show distinct functional characteristics for αβ and γδ DN T cells associated with distinct morbidity rates and clinical forms in human Chagas' disease.

FIG. 1.

T. cruzi activation of peripheral blood cells induces expansion of αβ and γδ double-negative (CD4⁻ CD8⁻) T cells. Whole blood cells from noninfected controls (N), indeterminate chagasic patients (I), and dilated cardiac chagasic patients (DC) were incubated overnight as described in Materials and Methods with either medium alone (MED) or with live T. cruzi parasites (TRP) and then analyzed for the frequency of αβ and γδ CD4⁻ CD8⁻T cells using flow cytometry. Panel A shows the average frequencies for each group ± standard deviations. The numbers of individuals in each group were as follows: N, seven; I, seven; and DC, five. Statistical significance is indicated in each graph, with differences between groups indicated by common numbers. Comparisons between groups were performed using a Tukey-Kramer comparison of all pairs, and comparisons within groups (MED versus TRP) were performed using a paired t test, as described in Materials and Methods. All patients were meticulously classified based on clinical criteria as described in Materials and Methods. Panel B shows representative dot plots from an indeterminate patient and gating for analysis of αβ and γδ CD4⁻ CD8⁻T cells. Both anti-CD4 and anti-CD8 antibodies were conjugated with CyChrome, allowing identification of the DN T cells using specific antibodies against both αβ and γδ T-cell receptors conjugated with FITC. The gates used for determining the percentage of DN T cells and further analysis of cytokine expression in the DN T-cell populations are shown.

FIG. 2.

T. cruzi activation of peripheral blood cells induces specific inflammatory cytokine production by αβ and γδ DN (CD4⁻ CD8⁻) T cells from both indeterminate and dilated cardiac chagasic patients. Whole blood cells from noninfected controls (N), indeterminate chagasic patients (I), and dilated cardiac chagasic patients (DC) were incubated overnight with either medium alone (MED) or with live T. cruzi parasites (TRP) and then analyzed for the frequency of αβ or γδ DN T cells producing specific cytokines using flow cytometry, as described in Materials and Methods. The data represent the average for each group ± standard deviations. The numbers of individuals in each group were as follows: N, seven; I, seven; and DC, five. The top panel shows the average percentage of IFN-γ-producing cells within αβ or γδ DN T cells from individual cultures without (MED) or with (TRP) stimulus. The middle panel shows the same for TNF-α-producing cells within αβ or γδ DN T cells, and the bottom panel shows the values for IL-17-producing cells within αβ or γδ DN T cells. Statistical significance is indicated in each graph, with differences between groups indicated by common numbers. Comparisons between groups were performed using a Tukey-Kramer comparison of all pairs, and comparisons within groups (MED versus TRP) were performed using a paired t test, as described in Materials and Methods. All patients were meticulously classified based on clinical criteria, as described in Materials and Methods.

FIG. 3.

γδ DN (CD4⁻ CD8⁻) T cells from indeterminate chagasic patients display a biased down-modulatory profile following stimulation with T. cruzi. Whole blood cells from noninfected controls (N), indeterminate chagasic patients (I), and dilated cardiac chagasic patients (DC) were incubated overnight with either medium alone (MED) or with live T. cruzi (TRP) and then analyzed using flow cytometry for the frequency of αβ or γδ DN T cells producing IL-10, as described in Materials and Methods. The data represent the average for each group ± standard deviations. The numbers of individuals in each group were as follows: N, seven; I, seven; and DC, five. Panel A shows the average percentage of IL-10-producing cells within the αβ or γδ DN T-cell population from individual cultures without (MED) or with (TRP) stimulus for each group. Statistical significance is indicated in each graph, with differences between groups indicated by common numbers. Comparisons between groups were performed using Tukey-Kramer comparison of all pairs, and comparisons within groups (MED versus TRP) were performed using a paired t test, as described in Materials and Methods. Panel B shows representative dot plots and gating for analysis of αβ and γδ CD4⁻ CD8⁻ T cells producing IL-10. Both anti-CD4 and anti-CD8 antibodies were conjugated with CyChrome, allowing identification of the DN T cells using specific antibodies against both αβ and γδ T-cell receptors conjugated with FITC. The gates used for determining the percentage of DN T cells producing IL-10 were then determined in a histogram using anti-IL-10 conjugated with PE. The percentages of cells producing IL-10 from cultures either with medium alone or with T. cruzi stimulation were determined, as described in Materials and Methods. All patients were meticulously classified based on clinical criteria, as described in Materials and Methods.

FIG. 4.

Higher frequencies of IL-10-producing γδ DN (CD4⁻ CD8⁻) T cells are correlated with better heart function in chagasic patients. The frequency of γδ DN T cells producing IL-10 following stimulation with T. cruzi was calculated from a group of chagasic patients who had associated detailed clinical data measuring ventricular function. These measurements were the left ventricular ejection fraction (LVEF) and left ventricular diastolic diameter (LVDD). The higher the LVEF, the better the ventricular function, and the lower the LVDD, the better the ventricular function. Panel A shows Pearson's correlation plots between the frequency of IL-10-producing γδ DN T cells and LVEF or LVDD. Both plots demonstrate a highly significant correlation between higher frequencies of IL-10-producing γδ DN T cells and better ventricular function. In contrast, in panel B, no correlation is seen between IL-10-producing αβ DN T cells and measurements of ventricular function. Clinical data for a total of nine chagasic patients were used in this analysis. Statistical significance (P value) is indicated in each graph together with the r² value.