Amplification of myeloid cell activation enhances engulfment of living T cells. (A) Flow cytometric analysis of lectin CD206 in living myeloid cells (propidium iodide [PI]⁻GFP⁺) in organotypic hippocampal slices prepared from CX3CR1^GFP pups and treated for 24 h with LPS (1 µg/ml; classical activation) or a combination of LPS (1 µg/ml), dexamethasone (5 × 10⁻⁷ M), and IL-4 (10 ng/ml; alternative activation) in comparison to untreated organotypic hippocampal slices (n = 7 controls from four different experiments, 7 LPS-treated slices from four different experiments, 3 LPS/dexamethasone/IL-4–treated slices from two different experiments). (B) 24-h co-cultures of organotypic hippocampal slices from CX3CR1^GFP 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. The organotypic slices were stimulated with LPS (1 µg/ml) or a combination of LPS (1 µg/ml), dexamethasone (5 × 10⁻⁷ M), and IL-4 (10 ng/ml) for 24 h in order to activate myeloid cells. Mean (± SEM) percentages of engulfment among the interactions in classically (LPS) or alternatively (LPS/dexamethasone/IL-4) activated organotypic hippocampal slices with B6.2D2.CFP.Th17 cells (n = 13 classically activated organotypic slices from five different experiments and 8 alternatively activated organotypic slices from three different experiments) normalized to the untreated control group (n = 13 organotypic slices from five different experiments). (C–E) Flow cytometry was performed to analyze CD206 expression on CD45^loCD11b⁺Ly6C/G⁻ myeloid cells in the CNS of EAE diseased mice. (C and D) Representative dot plots of CD206 staining (CD206-BV421) and isotype control (IgG2aκ-BV421) of CD45^loCD11b⁺Ly6C/G⁻ myeloid cells and CD206 staining of CD45^hiCD11b⁺ myeloid cells (representative of n = 12 EAE diseased mice from three different experiments). (C) Quantitative analysis of BV421 staining in CD45^loCD11b⁺Ly6C/G⁻ myeloid cells comparing CD206 staining and isotype control (n = 12 EAE diseased mice from three different experiments; D). (E) Quantification (mean ± SEM) of CD206 percentages of CD45^loCD11b⁺Ly6C/G⁻ or CD45^hiCD11b⁺ myeloid cells using CD206-BV421 corrected by subtraction of the signal for the corresponding isotype control (IgG2aκ-BV421; n = 12 EAE diseased mice from three different experiments). (F–H) Immunohistochemistry was performed to analyze CD206 expression and MHC-II expression on T cell–engulfing myeloid cells in the spinal cord of EAE diseased CX3CR1.GFP mice. (F) A representative example of a CD206-expressing engulfing CX3CR1⁺ cell is shown. Scale bars = 30 µm (upper panel) and 15 µm (lower panel); representative of n = 4 reslices from two different experiments. A slice view of the engulfing cell was magnified (lower panel). (G) A representative example of an MHC-II–expressing (upper panel, scale bar = 8 µm) and a non-MHCII–expressing (lower panel, scale bar = 10 µm) engulfing CX3CR1⁺ cell is shown. Engulfing myeloid cells are marked with dashed circles and engulfed T cells with asterisks. Representative of n = 9 engulfing cells from two different experiments. (H) Columns reflect the counts of MHC-II⁺ and MHC-II⁻ cells among engulfing CX3CR1⁺ cells in EAE diseased mice (n = 9 engulfing cells from two different experiments). Statistical analyses for A and B were performed using one-way ANOVA with Tukey’s post hoc test. Statistical analyses for D and E were performed using two-sided Student’s t test. *, P < 0.05, **, P < 0.01.