Distinct CD4−CD8− (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease

Figures

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  Figure 1. Analysis of double-negative (DN) memory subpopulations, naive and effector cells in indeterminate (IND, n = 6) and cardiac (CARD, n = 6) Chagas patients. (A) Representative dot plots illustrating the selection of DN T-cell subpopulations after selection of singlets: central memory (CM), effector memory (EM), naive (N), and effector cells (EF). Representative plots were performed using data from one IND patient, in non-stimulated culture. (B) Frequencies of DN TCR αβ+ or γδ+ CM, EM, N, and EF cells before (MED) and after in vitro stimulation with live parasite (TRP). (C) t-SNE from combined data obtained from all IND and CARD patients using MED or TRP cultures, in TCR αβ+ or TCR γδ+ DN T cells. Colors correspond to phonograph-guided clustering. (D) Ratio of CM/EM DN T-cell memory subpopulations in IND and CARD, before (MED) and after in vitro stimulation with live parasite (TRP). Results in (B, D) are expressed as percentage in box plot, extending from the 25th to 75th percentile, with a horizontal line at the median with whiskers. Paired or unpaired t-tests were used to compare unstimulated and stimulated cultures or cultures between different groups of patients, respectively.…

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  Figure 2. Analysis of cytokine expression in DN memory subpopulations from indeterminate (IND, n = 6) and cardiac (CARD, n = 6) Chagas patients. (A) Representative density color illustrating the distribution of DN T-cell subpopulations and selection of CM DN T cells, followed by analysis of single and double-positive IFN-γ+ and/or IL-10+ cells. Representative density color was performed using data from one IND patient, after in vitro stimulation with live parasite (TRP). (B) Frequency of IFN-γ+ or IL-10+ in central memory (CM) and effector memory (EM) in DN TCR αβ and DN TCR γδ subpopulations. (C) Frequency of cells co-expressing IFN-γ+ and IL-10+ in CM and EM in DN TCR αβ and DN TCR γδ subpopulations. The results are expressed as percentage in box plot, extending from the 25th to 75th percentile, with a horizontal line at the median with whiskers. Paired or unpaired t-tests were used to compare unstimulated and stimulated cultures or cultures between different groups of patients, respectively. Statistical significance is indicated in each graph.

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  Figure 3. Analysis of activation status of DN T-cell central (CM) and effector (EM) memory subpopulations, naive (N) and effector (EF) cells in indeterminate (IND, n = 6) and cardiac (CARD, n = 6) Chagas patients gauged by CD69 expression, before (MED) and after in vitro stimulation with live parasite (TRP). The graphs (A, B, top) represent the frequency of activated DN T-cell subpopulations in the different groups and under distinct conditions. The results are expressed as percentage in box plot, extending from the 25th to 75th percentile, with a horizontal line at the median with whiskers. Paired or unpaired t-tests were used to compare unstimulated and stimulated cultures or cultures between different groups of patients, respectively. Statistical significance is indicated in each graph. (A, B, bottom panels) Representative histograms of the expression of CD69 in the different DN T-cell subpopulations in IND and CARD. Clear curves show CD69 expresion in MED and gray curves show CD69 expression in TRP.

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  Figure 4. Cluster heatmap analysis of activation marker (CD69) and cytokine expression (IFN-γ and IL-10) in DN T-cell subpopulations from indeterminate (IND) and cardiac (CARD) Chagas patients. (A) Top panels contain the cluster heatmap analysis of non-stimulated cultures (MED) and bottom panels contain the cluster heatmap analysis after in vitro stimulation with live parasite (TRP). The left panels show the distribution of TCR αβ DN T cells, and the right panels the TCR γδ DN T cells. (B) Difusion maps showing the trajectories and relationships of the different DN T-cell subpopulations. The top plots contain all patients grouped together and the bottom plots discriminate IND and CARD, showing distinct trajectories progressing toward EM and CM, respectively.

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  Figure 5. Correlation analysis of the frequency of DN central memory cells (CM—A, C) and DN effector memory cells (EM—B, D) from Chagas patients (n = 11) and clinical parameters of disease severity: left ventricular ejection fraction (LVEF) and left ventricular diastolic diameter (LVDD). The left panels contain the correlation analysis in TCR αβ DN T cells, and the right panels contain the correlation analysis in TCR γδ DN T cells. Correlation analyses were performed using Pearson’s coefficient. p-values and r 2 are indicated in each graph.

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  Figure 6. Analysis of IFN-γ and IL-10 expression by effector memory (EM) DN T-cell subpopulations from cardiac Chagas patients (CARD, n = 4) before and after treatment with anti-CD1d monoclonal antibodies, as described in the Material and Methods. (A) Representative FACS plots showing the gating of CD4−CD8− TCR αβ+ or TCR γδ+ EM cells, followed by histograms of IL-10 expression measured in the presence of media alone (M), media plus anti-CD1d monoclonal antibody (M+block), parasite antigen alone (TRP-SA), or presence of TRP-SA plus anti-CD1d monoclonal antibody (TRP-SA+block). Representative plots were performed using data from one CARD patient, in stimulated culture. Histograms were from cultures stimulated and treated with blocking antibody. (B) Percent frequency of TCR αβ+ or TCR γδ+ EM DN T cells expressing IFN-γ and IL-10 as indicated. (C) Frequency of TCR γδ+ EM DN T cells co-expressing IFN-γ and IL-10. The results are expressed as percentage ratio for each culture condition (media+block/media or TRP-SA+block/TRP-SA) in box plots, extending from the 25th to 75th percentile, with a horizontal line at the median with whiskers. Paired t-tests were used to compare unstimulated and…