Natural killer (NK) cells with downregulated activating receptors and IL-10 production promote Trypanosoma cruzi T-cell responses in subjects in the Chronic phase of Trypanosoma cruzi infection: An exploratory immunological analysis

Figures

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  Fig 1. Unsupervised high-dimensional analysis of CD56+ NK-cell phenotype in subjects with chronic T. cruzi infection. PBMC were stained with a panel of monoclonal antibodies specific for surface markers and adquired by flow cytometry. Phenograph followed by FlowSOM clustering algorithms was applied on PBMC samples of T. cruzi-infected subjects (G0, n = 11; G1, n = 7; G2-G3, n = 9), and uninfected subjects (n = 15) The percentages of the CD56bright (A) and CD56dim (C) clusters that are higher or lower than 2.5% are shown in white or grey, respectively. Hierarchical clustering heatmaps showing the expression of the indicated proteins in the clusters identified on pre-gated CD56bright (B) or CD56dim (D) NK cells.

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  Fig 2. Unsupervised high-dimensional analysis of CD56+ NK-cell phenotype in T. cruzi infected subjects according to disease severity. Phenograph followed by FlowSOM clustering algorithms was applied on PBMC samples (G0, n = 11; G1, n = 7; G2-G3, n = 9; uninfected controls, UI, n = 15). Percentages of individual CD56bright (A) or CD56dim (B) clusters. Expression of the mean fluorescence intensity (MFI) for individual surface markers (C-D). Data are presented as median with interquartile range (A and B) or box and whiskers with 10-90 percentile (C-D). Differences among groups were evaluated by ANOVA or the Kruskal–Wallis test followed by posttests. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

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  Fig 3. Manual gating strategy for the identification of NK cells with activating and cytotoxic phenotype. PBMC from T. cruzi-infected subjects (G0, n = 16; G1, n = 13; G2-G3, n = 9) and uninfected controls (UI, n = 15) were stained with a panel of monoclonal antibodies specific for NK cell surface markers and adquired by flow cytometry. Manual gating was used to select CD56brightCD16— (A) and CD56dimCD16+ (C) NK cells, which express NKp46/NKG2D but not CD57. (B) Expression of the mean fluorescence intensity (MFI) of NkP46 in A. Data in A and C are presented as median with interquartile range and as box and whiskers in B. Differences among groups were evaluated by ANOVA or the Kruskal–Wallis test followed by posttests. ** P < 0.01, * P < 0.05.

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  Fig 4. NK-cell function in T. cruzi-infected subjects with different degrees of heart disease. PBMC of T. cruzi-infected subjects in the G0 (n = 16), G1 (n = 13), G2/G3 (n = 9) clinical groups, as well as from uninfected subjects (UI, n = 15), were stimulated with PMA/ionomycin for 4 h and analyzed by flow cytometry for intracellular expression of CD107a, granzyme B, IFN-γ, IL-10 and TNF-α. A. Lymphocytes were gated based on forward scatter (FSC) and side scatter (SSC) parameters, and analysed for viable CD14—CD19—CD3—CD56+ cells versus each marker using Zombie Aqua. The coexpression profiles of one to five functions were identified using the Boolean gating function of the FlowJo software. The contribution of each molecule-producing subset to the total NK-cell response was assessed in donors with positive responses in the intracellular staining assay as indicated in the Materials and Methods. Then, the average for each combination out of 31 possible combinations was calculated. (A) The data are summarized by the pie charts, in which each slice of the pie represents the fraction of the total response that consists of CD56+ T-cells positive for one to five functions. The arches indicate…

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  Fig 5. Principal component analysis of NK-cell function in T. cruzi-infected subjects following benznidazole treatment. PBMC were stimulated with PMA/ionomycin for 4 h and analyzed by flow cytometry for intracellular expression of CD107a, granzyme B, IFN-γ, IL-10 and TNF-α. Coexpression profiles with one to five functions using the Boolean gating function of FlowJo software. The contribution of each molecule-producing subset to the total NK-cell response was assessed in donors with positive responses in the intracellular staining assay as indicated in the Materials and Methods. Then, the average for each combination was calculated. The values obtained in unstimulated samples were subtracted. Following log transformation, the principal components were extracted with eigenvalues of 1.0. Factor loadings >0, 4 or <-0.4 were considered as influential mediators (filled symbols). NK cell functional profiles plotted based on the first 2 extracted principal components PC1 and PC2 prior to treatment (circle symbols) or following 48 months post-treatment with benznidazole (triangle symbols). T. cruzi-infected subjects with declining (A, n = 8) and or unchanged (B, n = 5) T. cruzi-specific…

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  Fig 6. No. of interferon IFN-γ-secreting cells in response to T. cruzi lysate before and after depletion of CD56+ NK cells. Whole PBMC, CD56-depleted PBMC or double CD4 and CD8 depleted PBMC using magnetic beads were seeded at a concentration of 4x105 cells/well and stimulated with 10 mg/mL T. cruzi lysate from the Brazil strain for 16–20 hours. Each point represents the mean number of spots in duplicate wells for each patient and experimental condition, connected by a line.* P < 0.05,**P < 0.01, ***, P < 0.001.