Figure 4.
In contrast to phagocytosis, engulfment of living T cells is independent of Annexin V–PS interaction. (A) PS exposure on Th17-skewed cells (day 5 of culture) was determined via Annexin V staining. Mean (± SEM) Annexin V binding was compared between CD25-expressing living CD4+ cells and non-CD25-expressing living CD4+ cells using flow cytometry analysis (n = 3 from two different experiments). (B–G) 24-h co-cultures of organotypic hippocampal slices from CX3CR1GFP pups with pathogenic B6.2D2.CFP.Th17 cells were stained with Image-iT LIVE Red Caspase-3 and Caspase-7 Detection Kit to identify apoptotic cells and imaged over a time period of 20 min with two-photon microscopy. (B) Representative example of the Image-iT LIVE Red Caspase-3 and -7 (red) staining in 24-h co-cultures of organotypic hippocampal slices with B6.2D2.CFP.Th17 cells (blue). Scale bars = 40 µm. Representative of n = 5 co-cultures from four different experiments. (C) Pie chart comparing the distribution (%) of interaction modes between myeloid cells and viable 2D2.CFP.Th17 cells (n = 27 organotypic slices from 17 different experiments) and contingency bars reflecting the respective distribution of engulfment, escape, and engulfment attempt. (D) Representative two-photon images of the 24-h co-cultures of the organotypic hippocampal slices with B6.2D2.CFP.Th17 cells stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit, with or without 10 µg/ml Annexin V treatment (24 h). Relevant areas are magnified (right panel); asterisks mark apoptotic cells (red). Scale bars = 40 µm. Representative of n = 7 co-cultures from three different experiments. (E) Representative two-photon images of the 24-h co-cultures of the organotypic hippocampal slices with B6.2D2.CFP.Th17 cells stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit, with or without 10 µg/ml Annexin V treatment (24 h). Relevant areas are magnified (right panel); number signs mark nonapoptotic T cells (blue). Scale bars = 40 µm, representative of n = 8 co-cultures from five different experiments. (F) Mean (± SEM) percentages of engulfment-related process (Eps; left panel) and full engulfments (right panel) among the detectable cell count (viable T cells and apoptotic cells), without (n = 7 organotypic slices for viable cells from five different experiments; n = 8 organotypic slices for apoptotic cells from three different experiments) and with Annexin V treatment (n = 11 organotypic slices for viable cells from five different experiments, n = 8 organotypic slices for apoptotic cells from three different experiments). (G) Mean (± SEM) T cell motility parameters compared between Caspase-3/7+ and viable B6.2D2.CFP.Th17 cells without (n = 10 organotypic slices, 276 T cells for viable cells from five different experiments; n = 8 organotypic slices, 185 T cells for apoptotic cells from three different experiments) and with (n = 12 organotypic slices, 205 T cells for viable cells from five different experiments; n = 8 organotypic slices, 171 T cells for apoptotic cells from three different experiments) Annexin V treatment. Statistical analysis for A was performed using two-sided Student’s t tests. Statistical analyses for F and G were performed using one-way ANOVA with Tukey’s post hoc test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

In contrast to phagocytosis, engulfment of living T cells is independent of Annexin V–PS interaction. (A) PS exposure on Th17-skewed cells (day 5 of culture) was determined via Annexin V staining. Mean (± SEM) Annexin V binding was compared between CD25-expressing living CD4+ cells and non-CD25-expressing living CD4+ cells using flow cytometry analysis (n = 3 from two different experiments). (B–G) 24-h co-cultures of organotypic hippocampal slices from CX3CR1GFP pups with pathogenic B6.2D2.CFP.Th17 cells were stained with Image-iT LIVE Red Caspase-3 and Caspase-7 Detection Kit to identify apoptotic cells and imaged over a time period of 20 min with two-photon microscopy. (B) Representative example of the Image-iT LIVE Red Caspase-3 and -7 (red) staining in 24-h co-cultures of organotypic hippocampal slices with B6.2D2.CFP.Th17 cells (blue). Scale bars = 40 µm. Representative of n = 5 co-cultures from four different experiments. (C) Pie chart comparing the distribution (%) of interaction modes between myeloid cells and viable 2D2.CFP.Th17 cells (n = 27 organotypic slices from 17 different experiments) and contingency bars reflecting the respective distribution of engulfment, escape, and engulfment attempt. (D) Representative two-photon images of the 24-h co-cultures of the organotypic hippocampal slices with B6.2D2.CFP.Th17 cells stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit, with or without 10 µg/ml Annexin V treatment (24 h). Relevant areas are magnified (right panel); asterisks mark apoptotic cells (red). Scale bars = 40 µm. Representative of n = 7 co-cultures from three different experiments. (E) Representative two-photon images of the 24-h co-cultures of the organotypic hippocampal slices with B6.2D2.CFP.Th17 cells stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit, with or without 10 µg/ml Annexin V treatment (24 h). Relevant areas are magnified (right panel); number signs mark nonapoptotic T cells (blue). Scale bars = 40 µm, representative of n = 8 co-cultures from five different experiments. (F) Mean (± SEM) percentages of engulfment-related process (Eps; left panel) and full engulfments (right panel) among the detectable cell count (viable T cells and apoptotic cells), without (n = 7 organotypic slices for viable cells from five different experiments; n = 8 organotypic slices for apoptotic cells from three different experiments) and with Annexin V treatment (n = 11 organotypic slices for viable cells from five different experiments, n = 8 organotypic slices for apoptotic cells from three different experiments). (G) Mean (± SEM) T cell motility parameters compared between Caspase-3/7+ and viable B6.2D2.CFP.Th17 cells without (n = 10 organotypic slices, 276 T cells for viable cells from five different experiments; n = 8 organotypic slices, 185 T cells for apoptotic cells from three different experiments) and with (n = 12 organotypic slices, 205 T cells for viable cells from five different experiments; n = 8 organotypic slices, 171 T cells for apoptotic cells from three different experiments) Annexin V treatment. Statistical analysis for A was performed using two-sided Student’s t tests. Statistical analyses for F and G were performed using one-way ANOVA with Tukey’s post hoc test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

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