Ex vivo characterization of Breg cells in patients with chronic Chagas disease

Figures

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  Figure 1. Frequency and phenotypic distribution of total B cells in peripheral blood from chronic Chagas patients and non-infected donors according to CD24 and CD38 expression. (a,b) Frequency of B cells (CD3−CD19+) and mean level of CD19 expression (as mean fluorescence intensity; MFI) in the CD3−CD19+ population from CCD patients (G0, G1) and non-infected donors (NI). (c) Representative dot plots from CCD patients and one non-infected donor, showing the gating strategy used to identify B cell subsets. According to CD24 and CD38 expression levels, B cells were sub-gated into: CD19+ CD24highCD38high (immature transitional B cells), CD19+CD24intCD38int (mature naïve B cells), CD19+CD24highCD38− (memory B cells), CD19+CD24lowCD38high (plasmablasts). (d) Frequency of different B cell subsets in total B cells from CCD patients (G0, G1) and non-infected donors (NI). Each dot represents data from one subject; inside G1 group, solid circles correspond to K1 patients, hollow triangle to K2 patient and hollow square to K3 patient. Boxes and whiskers show median value and interquartile range. Statistically significant differences among the groups are indicated (*p < 0.05).

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  Figure 2. Frequency of B cell subsets based on CD24 and CD27 or CD27 and CD38 markers expression in peripheral blood from CCD patients and non-infected donors. (a,b) Representative dot plots from a non-infected donor showing the gating strategy used to determine the subsets within total B cells according to the combined expression of CD24 and CD27 or CD27 and CD38. (c,d) Frequency of B cell subsets in CCD patients (G0, G1) and non-infected donors (NI). Each dot represents data from one subject; inside G1 group, solid circles correspond to K1 patients, hollow triangle to K2 patient and hollow square to K3 patient. Boxes and whiskers show median value and interquartile range.

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  Figure 3. Frequency and phenotypic distribution of B10 cells according to CD24 and CD38 expression in peripheral blood from CCD patients and non-infected donors. (a) Representative dot plots showing the gating strategy used to determine the IL-10+ B cells (B10 cells) in PIB stimulated PBMC. Fluorescence minus one controls (FMO) were used to define the negative populations for each sample. (b,c) Frequency of B10 cells in total B cells and mean fluorescence intensity (MFI) for IL-10+ in B10 cells in CCD patients (G0, G1) and non-infected donors (NI). (d) Representative dot plots showing the gating strategy used. IL-10 expressing B-cells (B10 cells) were gated according to subject- and condition-matched FMO control tubes and were further sub-gated using CD24 and CD38 to identify the phenotypical distribution of these populations. (e) Frequency distribution of B10 cells according to CD24 and CD38 expression in CCD patients (G0, G1) and non-infected donors (NI). Each dot represents data from one subject; inside G1 group, solid circles correspond to K1 patients, hollow triangle to K2 patient and hollow square to K3 patient. Boxes and whiskers show median value and interquartile range.…

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  Figure 4. Phenotypic distribution of B10 cells according to CD27, CD27-CD24 and CD27-CD38 expression in CCD patients and non-infected donors. (a) Histograms showing the gating strategy used to determine CD27− (naïve) and CD27+ (memory) populations within B10 cells in PIB stimulated PBMC. One representative non-infected donor is illustrated. (b) Frequency of CD27− and CD27+ B10 cells in CCD patients (G0, G1) and non-infected donors (NI). (c,d) Representative dot plots from a non-infected donor showing gating strategy used to determine the subsets within total B10 cells based on the combine expression of CD24 and CD27 or CD27 and CD38 in PIB stimulated PBMC. (e,f) Frequency of B10 cell subsets within CD24-CD27 and CD27-CD38 subpopulations in CCD patients (G0 G1) and non-infected donors (NI). Each dot represents data from one subject; inside G1 group, solid circles correspond to K1 patients, hollow triangle to K2 patient and hollow square to K3 patient. Boxes and whiskers show median value and interquartile range. Statistically significant differences among groups are indicated (*p < 0.05).

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  Figure 5. IL-10 producing cells within CD24highCD38high transitional and CD24highCD27+ memory B cell populations in CCD patients and non-infected donors. (a,b) Representative dot plots showing the gating strategy used to determine frequency of IL-10+ cells in CD24highCD38high and CD24highCD27+ B cells. (c,d) Frequencies of IL-10+ cells within transitional (CD24highCD38high) and memory (CD24highCD27+) compartments in CCD patients (G0, G1) and non-infected donors (NI). Each dot represents data from one subject; inside G1 group, solid circles correspond to K1 patients, hollow triangle to K2 patient and hollow square to K3 patient. Boxes and whiskers show median value and interquartile range.

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  Figure 6. Automatic gating performed by flowPeaks algorithm of B cell subsets based on CD19, CD24, CD27 and CD38 expression. (a) Violin plots show the distribution of the expression of each marker for each peak. (b) Spider plots display the median expression for each marker in the different peaks. One graph is shown for each group of CCD patients (G0, G1) and non-infected donors (NI). (c) Representative biaxial dot plots showing the distribution of peaks based on CD24-CD38, CD24-CD27 and CD27-CD38 expression. (d) Box plots showing the percentage of B cells within each peak in G0, G1 and NI groups. (e) Box plots indicate the percentage of B10 cells within each peak in the G0, G1 and NI groups.

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  Figure 7. Automatic gating performed by flowPeaks algorithm of B10 cell subsets based on CD19, CD24, CD27 and CD38 expression. (a) Spider plots display the median expression for each marker in the different peaks. (b) Representative biaxial dot plots showing the distribution of peaks within B10 cells based on CD24–CD38, CD24–CD27 and CD27–CD38 expression. (c) Box plots showing the percentage of cells within each peak in B10 cells in G0, G1 and NI groups.