Figure 5.
Engulfment of viable cells is dependent on GlcNAc exposure on activated T cells. (A, C, D, and G–L) Co-cultures of organotypic hippocampal slices from CX3CR1GFP pups with pathogenic CFP-labeled T 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. (A) 24-h co-cultures of organotypic hippocampal slices with Th17 cells were treated with RGDS (1 mM). The pie chart comparing the distribution (%) of interaction modes between myeloid cells and viable B6.2D2.CFP.Th17 cells (n = 5 organotypic slices from four different experiments) and contingency bar reflecting the respective distribution of engulfment, escape, and engulfment attempt are shown. (B) WGA binding to Th17-skewed cells was assessed in the presence or absence of GlcNAc (200 mM) in the staining solution using flow cytometric analysis. Representative histograms (left panel) and mean (± SEM) of the mean fluorescence intensity (MFI, right panel) are shown (n = 5 Th17 cultures from two different experiments). (C) 24-h co-cultures of organotypic hippocampal slices with Th17 cells were treated with GlcNAc (20 mM, competitive GlcNAc). The pie chart shows the distribution (%) of interaction modes between myeloid cells and viable Th17 cells (n = 4 organotypic slices from three different experiments; upper panel), and contingency bar reflects the respective distribution of engulfment and engulfment attempt (lower panel). (D) Mean (± SEM) percentages of engulfment rate among the interactions in GlcNAc (20 mM) treated co-cultures (n = 4 organotypic slices from three different experiments) normalized to the untreated control group (n = 4 organotypic slices from 3 different experiments). (E–H) Naive T cells were skewed into Th17 cells in the presence or absence of GlcNAc (20 mM). At day 3 of culture, cells were further stimulated with antiCD3 and antiCD28 with/without GlcNAc (20 mM). (E) Cells were washed to remove GlcNAc in the supernatant, and WGA binding was assessed (n = 7 Th17 cell cultures from seven different experiments). (F–H) Control Th17-skewed cells and GlcNAc-pretreated (GlcNAc enhanced) Th17-skewed cells were co-cultured with organotypic hippocampal slices. (F) WGA-binding to T cells was assessed after 5h of co-culture (n = 7 Th17 co-cultures from three different experiments). (G) The pie chart shows the distribution (%) of interaction modes between myeloid cells and viable GlcNAc-pretreated Th17 cells (n = 7 organotypic slices from three different experiments; upper panel), and contingency bar reflects the respective distribution of engulfments and engulfment attempts (lower panel). (H) Mean (± SEM) percentages of engulfment rate among the interactions of GlcNAc-pretreated Th17-skewed cells in organotypic hippocampal slices (n = 7 organotypic slices from three different experiments) normalized to the GlcNAc-untreated control group (n = 7 organotypic slices from three different experiments). (I–L) Pie charts (upper panel) show the distribution (%) of interaction modes between myeloid cells and viable B6.2D2.CFP.Th2 cells (n = 3 organotypic slices from three different experiments; I), viable B6.2D2.CFP.T reg cells (n = 4 organotypic slices from four different experiments; J), polyclonal B6.CFP.Th17 cells (Th17 BL/6; n = 6 organotypic slices from 2 different experiments; K), or viable B6.2D2.CFPTh17 cells in co-cultures treated with MOG (100 µg/ml; n = 3 organotypic slices from two different experiments; L), and contingency bars reflect the respective distribution of engulfment, escape, and attempt to engulf (lower panel). (M) Representative visualization of 24-h co-cultures of organotypic hippocampal slices from CX3CR1GFP pups with pathogenic B6.2D2.RFP.Th17.GFP reporter cells, in which IL-17–producing cells are RFP+GFP+ double positive (orange), while non-IL-17–producing cells are RFP+ single positive (red; representative of n = 3 co-cultures from three different experiments; left panel). Scale bar = 30 µm. A myeloid cell (green) engulfing at the same time an IL-17–producing cell (marked with an asterisk) and a non-IL-17–producing cell (marked with a number sign) was magnified (middle panel), and red signal was hidden to visualize the green signal of the IL-17–producing T cell (right panel). Scale bar = 15 µm. Statistical analyses were performed using two-sided Student’s t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Engulfment of viable cells is dependent on GlcNAc exposure on activated T cells. (A, C, D, and G–L) Co-cultures of organotypic hippocampal slices from CX3CR1GFP pups with pathogenic CFP-labeled T 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. (A) 24-h co-cultures of organotypic hippocampal slices with Th17 cells were treated with RGDS (1 mM). The pie chart comparing the distribution (%) of interaction modes between myeloid cells and viable B6.2D2.CFP.Th17 cells (n = 5 organotypic slices from four different experiments) and contingency bar reflecting the respective distribution of engulfment, escape, and engulfment attempt are shown. (B) WGA binding to Th17-skewed cells was assessed in the presence or absence of GlcNAc (200 mM) in the staining solution using flow cytometric analysis. Representative histograms (left panel) and mean (± SEM) of the mean fluorescence intensity (MFI, right panel) are shown (n = 5 Th17 cultures from two different experiments). (C) 24-h co-cultures of organotypic hippocampal slices with Th17 cells were treated with GlcNAc (20 mM, competitive GlcNAc). The pie chart shows the distribution (%) of interaction modes between myeloid cells and viable Th17 cells (n = 4 organotypic slices from three different experiments; upper panel), and contingency bar reflects the respective distribution of engulfment and engulfment attempt (lower panel). (D) Mean (± SEM) percentages of engulfment rate among the interactions in GlcNAc (20 mM) treated co-cultures (n = 4 organotypic slices from three different experiments) normalized to the untreated control group (n = 4 organotypic slices from 3 different experiments). (E–H) Naive T cells were skewed into Th17 cells in the presence or absence of GlcNAc (20 mM). At day 3 of culture, cells were further stimulated with antiCD3 and antiCD28 with/without GlcNAc (20 mM). (E) Cells were washed to remove GlcNAc in the supernatant, and WGA binding was assessed (n = 7 Th17 cell cultures from seven different experiments). (F–H) Control Th17-skewed cells and GlcNAc-pretreated (GlcNAc enhanced) Th17-skewed cells were co-cultured with organotypic hippocampal slices. (F) WGA-binding to T cells was assessed after 5h of co-culture (n = 7 Th17 co-cultures from three different experiments). (G) The pie chart shows the distribution (%) of interaction modes between myeloid cells and viable GlcNAc-pretreated Th17 cells (n = 7 organotypic slices from three different experiments; upper panel), and contingency bar reflects the respective distribution of engulfments and engulfment attempts (lower panel). (H) Mean (± SEM) percentages of engulfment rate among the interactions of GlcNAc-pretreated Th17-skewed cells in organotypic hippocampal slices (n = 7 organotypic slices from three different experiments) normalized to the GlcNAc-untreated control group (n = 7 organotypic slices from three different experiments). (I–L) Pie charts (upper panel) show the distribution (%) of interaction modes between myeloid cells and viable B6.2D2.CFP.Th2 cells (n = 3 organotypic slices from three different experiments; I), viable B6.2D2.CFP.T reg cells (n = 4 organotypic slices from four different experiments; J), polyclonal B6.CFP.Th17 cells (Th17 BL/6; n = 6 organotypic slices from 2 different experiments; K), or viable B6.2D2.CFPTh17 cells in co-cultures treated with MOG (100 µg/ml; n = 3 organotypic slices from two different experiments; L), and contingency bars reflect the respective distribution of engulfment, escape, and attempt to engulf (lower panel). (M) Representative visualization of 24-h co-cultures of organotypic hippocampal slices from CX3CR1GFP pups with pathogenic B6.2D2.RFP.Th17.GFP reporter cells, in which IL-17–producing cells are RFP+GFP+ double positive (orange), while non-IL-17–producing cells are RFP+ single positive (red; representative of n = 3 co-cultures from three different experiments; left panel). Scale bar = 30 µm. A myeloid cell (green) engulfing at the same time an IL-17–producing cell (marked with an asterisk) and a non-IL-17–producing cell (marked with a number sign) was magnified (middle panel), and red signal was hidden to visualize the green signal of the IL-17–producing T cell (right panel). Scale bar = 15 µm. Statistical analyses were performed using two-sided Student’s t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

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