Figure 6.
Modulation of T cell activity alters T cell engulfment. (A–D) Th17-skewed cells (day 5 of culture) were either stimulated for 24 h with anti-CD3 and anti-CD28 or not, with or without Wortmannin. (A) Quantification of the mean fluorescence intensity (MFI; mean ± SEM) of WGA binding on stimulated, living CD4+ T cells with or without Wortmannin (n = 3 from two different experiments). (B) Brefeldin A was added to the T cells 4 h before the analysis in order to inhibit secretion of IL-17. Quantification of IL-17 expression among CD4+ T cells (mean ± SEM; n = 6 from two different experiments). (C) Histogram and quantification (mean ± SEM) of CD44 expression among living CD4+ T cells (n = 6 from two different experiments). (D) Histogram and quantification (mean ± SEM) of CD25 expression among living CD4+ T cells (n = 6 from two different experiments). (E–J) 24-h co-cultures of organotypic hippocampal slices with Th17 cells were treated with Wortmannin. (E) T cell motility parameters of viable B6.2D2.CFP.Th17 cells without (n = 5 organotypic slices, 141 T cells from four different experiments) and with (n = 8 organotypic slices, 228 T cells from four different experiments) Wortmannin treatment (2.5–5 µM; mean ± SEM). (F) Representative images of organotypic hippocampal slices with pathogenic B6.2D2.CFP.Th17 cells with (n = 8 organotypic slices from four different experiments) and without (n = 5 organotypic slices from four different experiments) Wortmannin (2.5–5 µM) treatment. Asterisks mark engulfed T cells. Scale bars = 40 µm. (G) Pie chart showing the distribution (%) of interaction modes between myeloid cells and viable T cells in Wortmannin (2.5–5 µM)–treated co-cultures of organotypic hippocampal slices with B6.2D2.CFP.Th17 cells (n = 8 organotypic slices from four different experiments; upper panel) and contingency bars reflecting the respective distribution of engulfment, escape, and attempt to engulf (lower panel). (H) Mean (± SEM) percentages of engulfment rate among the interactions in Wortmannin (2.5–5 µM)–treated organotypic hippocampal slices (n = 8 organotypic slices from four different experiments) normalized to the untreated control group (n = 5 organotypic slices from four different experiments). (I) 24-h co-cultures of Wortmannin-treated organotypic hippocampal slices from CX3CR1GFP pups with pathogenic B6.2D2.CFP.Th17 cells were stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit to identify apoptotic cells and imaged over a time period of 20 min. Only nonapoptotic T cells were considered for further analyses. Engulfment and escape frequencies were determined among EPs (n = 8 from four different experiments). (J) 24-h co-cultures of Wortmannin-treated or untreated organotypic hippocampal slices from CX3CR1GFP pups with pathogenic B6.2D2.CFP.Th17 cells were stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit to identify apoptotic cells and imaged over a time period of 20 min. Only nonapoptotic T cells were considered for further analyses. Escape frequencies among EPs are shown (n = 5 controls, 8 Wortmannin-treatments from four different experiments). Statistical analyses for A, E, and G–J were performed using two-sided Student’s t tests. Statistical analyses for B–D were performed using one-way ANOVA with Tukey’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001, ns, not significant.

Modulation of T cell activity alters T cell engulfment. (A–D) Th17-skewed cells (day 5 of culture) were either stimulated for 24 h with anti-CD3 and anti-CD28 or not, with or without Wortmannin. (A) Quantification of the mean fluorescence intensity (MFI; mean ± SEM) of WGA binding on stimulated, living CD4+ T cells with or without Wortmannin (n = 3 from two different experiments). (B) Brefeldin A was added to the T cells 4 h before the analysis in order to inhibit secretion of IL-17. Quantification of IL-17 expression among CD4+ T cells (mean ± SEM; n = 6 from two different experiments). (C) Histogram and quantification (mean ± SEM) of CD44 expression among living CD4+ T cells (n = 6 from two different experiments). (D) Histogram and quantification (mean ± SEM) of CD25 expression among living CD4+ T cells (n = 6 from two different experiments). (E–J) 24-h co-cultures of organotypic hippocampal slices with Th17 cells were treated with Wortmannin. (E) T cell motility parameters of viable B6.2D2.CFP.Th17 cells without (n = 5 organotypic slices, 141 T cells from four different experiments) and with (n = 8 organotypic slices, 228 T cells from four different experiments) Wortmannin treatment (2.5–5 µM; mean ± SEM). (F) Representative images of organotypic hippocampal slices with pathogenic B6.2D2.CFP.Th17 cells with (n = 8 organotypic slices from four different experiments) and without (n = 5 organotypic slices from four different experiments) Wortmannin (2.5–5 µM) treatment. Asterisks mark engulfed T cells. Scale bars = 40 µm. (G) Pie chart showing the distribution (%) of interaction modes between myeloid cells and viable T cells in Wortmannin (2.5–5 µM)–treated co-cultures of organotypic hippocampal slices with B6.2D2.CFP.Th17 cells (n = 8 organotypic slices from four different experiments; upper panel) and contingency bars reflecting the respective distribution of engulfment, escape, and attempt to engulf (lower panel). (H) Mean (± SEM) percentages of engulfment rate among the interactions in Wortmannin (2.5–5 µM)–treated organotypic hippocampal slices (n = 8 organotypic slices from four different experiments) normalized to the untreated control group (n = 5 organotypic slices from four different experiments). (I) 24-h co-cultures of Wortmannin-treated organotypic hippocampal slices from CX3CR1GFP pups with pathogenic B6.2D2.CFP.Th17 cells were stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit to identify apoptotic cells and imaged over a time period of 20 min. Only nonapoptotic T cells were considered for further analyses. Engulfment and escape frequencies were determined among EPs (n = 8 from four different experiments). (J) 24-h co-cultures of Wortmannin-treated or untreated organotypic hippocampal slices from CX3CR1GFP pups with pathogenic B6.2D2.CFP.Th17 cells were stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit to identify apoptotic cells and imaged over a time period of 20 min. Only nonapoptotic T cells were considered for further analyses. Escape frequencies among EPs are shown (n = 5 controls, 8 Wortmannin-treatments from four different experiments). Statistical analyses for A, E, and G–J were performed using two-sided Student’s t tests. Statistical analyses for B–D were performed using one-way ANOVA with Tukey’s post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001, ns, not significant.

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