T cells responding to Trypanosoma cruzi detected by membrane TNF-α and CD154 in chagasic patients

Abstract

Introduction: Chagas disease is a parasitic infection whose pathogenesis is related to parasite persistence and a dysfunctional cellular immune response. Variability in cytokine secretion among chronic Trypanosoma cruzi-infected patients might preclude the identification of the pool of antigen specific T cells. The goal of this study was to determine the fraction of T cells responding to T. cruzi antigen measured by the expression of membrane TNF-α and CD154.

Methods: A total of 21 chagasic patients, 11 healthy and 5 non-chagasic cardiomyopathy controls were analyzed. PBMCs were short-term cultured in the presence of anti-CD28, anti-CD49d, anti-TNF-α, and TACE (TNF-α converting enzyme) inhibitor either under T. cruzi-lysate or polyclonal stimuli. Cells were stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD154, and analyzed with flow cytometry.

Results: CD4+ and CD8+ T cells in chagasic patients displayed higher percentages of membrane-bound TNF-α+ and CD154+ compared with controls after T. cruzi-antigen stimulation. Both markers displayed a positive correlation in the T cell subpopulations analyzed. Symptomatic chagasic patients were differentiated from asymptomatic patients based on the expression of CD154 and membrane TNF-α in TCD4+ and TCD8+ compartments, respectively.

Conclusions: These results show that both markers could be useful for assessing the pool of antigen-specific T cells in chronic chagasic patients.

Keywords: CD4+ T lymphocytes; CD8+ T lymphocytes; T lymphocyte; chagas disease; flow cytometry.

Figure 1

CD154 and membrane TNF‐α expression among T cell subsets. PBMC from chronic chagasic patients were cultured for 4.5 h and stimulated under the following conditions: polyclonal stimuli or medium alone. Relative size or FSC is displayed in x‐axis. Examples of dots plot for membrane TNF‐α (A) and CD154 expression (B) in CD4+ (upper panel) and CD8+ (lower panel) T cells under medium alone and SEB as indicate in y‐axis. Scatter plot showing correlation of membrane TNF‐α and CD154 on CD4+ T cells (C) and CD8+ T cells (D) in cells from chagasic patients.

Figure 2

Membrane TNF‐α expression and CD154 on CD4+ T cells after Trypanosoma cruzi antigen‐derived stimulus. Percentage of membrane TNF‐α expression for CD4+ T cells in the analyzed groups (A). Examples of density/scatter plots for membrane TNF‐α (x‐axis) and CD4+ T cells (B). Percentage of CD154 expression for CD4+ T cells on the analyzed groups (C). Examples of density/scatter for CD154 (x‐axis) and CD4+ T cells (D). Box and whiskers indicate the median frequency and range of CD154 and membrane TNF‐α expression among CD4+ T cells (25th–75th percentile). CP, Chagasic patient; CP Asympt, Asymptomatic Chagasic Patients; CP Sympt, Symptomatic Chagasic Patients; HC, Healthy Controls; and NCC, Non‐Chagasic Cardiomyopathy.

Figure 3

Membrane TNF‐α and CD154 expression on CD8+ T cells after T. cruzi antigen‐derived stimulus. Percentage of membrane TNF‐α+ expression for CD8+ lymphocytes in the analyzed groups (A). Examples of density/scatter plots for membrane TNF‐α (x‐axis) and CD8+ T cells (B). Percentage of CD154 expression for CD8+ T cells in the analyzed groups (C). Examples of density/scatter for CD154 (x‐axis) and CD8+ T cells (D). Box and whiskers indicate the median frequency and range of CD154 and membrane TNF‐α expression among CD8+ T cells (25th–75th percentile). CP, Chagasic patient; CP Asymptomatic, Asymptomatic Chagasic Patients; CP Symptomatic, Symptomatic Chagasic Patients; HC, Healthy Controls; and NCC, Non‐Chagasic Cardiomyopathy.

Figure 4

Expression and co‐expression of CD107a/b and membrane TNF‐α in CD8+ T lymphocytes. Percentage of membrane TNF‐α (A), CD107a/b (B) and both surface markers (C) in CD8+ T cells from chagasic patients under the following conditions: medium, T. cruzi lysate and SEB stimuli. Examples of cytometry plots of membrane TNF‐α and CD107a/b co‐expression under medium alone (D), SEB stimuli (E) and T. cruzi lysate exposure (F).