Collateral neuronal apoptosis in CNS gray matter during an oligodendrocyte-directed CD8+ T cell attack

Additional Supporting Information may be found in the online version of this article.

Filename	Format	Size	Description
GLIA_20938_sm_SuppFig1.tif		215K	Supporting Information Figure 1. Phenotyping and cytokine (IFNγ) release following in vitro activation of OT-I T cells and MHC I expression in ODC-OVA slices. A, Following MACS-purification of splenocytes from OT-I mice for CD8 the cell suspension contained CD8+ but virtually no CD4+ T cells. B, In vitro stimulated OT-I T cells (dark grey) predominantly showed an CD62Lhigh CD44high CD45RAlow phenotype compared to the CD62Llow CD44low CD45RAhigh phenotype of unstimulated cells (bright grey). C, OT-I T cells displayed an about 35fold increase in IFNγ-secretion into the supernatant after 3 days of stimulation. D, CD8+ OT-I T cells harvested after 6 hours of incubation in WT or ODC-OVA slices did not differ in cell surface expression of the activation marker CD25 or intracellular IFNγ synthesis as revealed by flowcytometry.
GLIA_20938_sm_SuppFig2.tif		1320K	Supporting Information Figure 2. MHC I expression by gray matter ODCs and neurons is augmented by INFγ and tetanus toxin. A, MHC I expression in ODC-OVA slices assessed by RT-PCR after 1, 3, and 6 h incubation with interferon-γ (IFNγ, 100 U/ml) with and without tetrodotoxin (TTX, 1 μM) (left panel) and supernatant from OT-I T cell in vitro stimulation diluted 1:10 or 1:3 with ACSF (right panel) relative to untreated naïve conditions. B, C, MHC I expression (red) can be detected in gray matter areas of ODC-OVA slices on NeuN+ (green) neuronal perikarya (B) and NogoA+ (green) ODCs (C). The nucleus of these cells is stained with DAPI (blue).
GLIA_20938_sm_SuppFig3.tif		1000K	Supporting Information Figure 3. Gray matter astrocytes are not affected during exposure of ODC-OVA brain slices with OT-I T cells. A, Acute living slices from ODC-OVA and WT mice were incubated for 0, 3, 4 and 6 h with activated OT-I T cells. Immunohistochemistry and quantification of activated caspase-3+ astrocytes (GFAP, green) was performed as described in Figure 2. Immediately (A, top panels) and 6 h (A, bottom panels) after incubation with OT-I T cells no caspase-3 activation was detectable in astrocytes of the dentate gyrus of the hippocampus (left panels) and the cortex (right panels) in ODC-OVA slices. Scale bars represent 10 μM. B, Density of activated caspase-3+ astrocytes after 6 h of incubation of ODC-OVA and WT slices with and without OT-I T cells (B, top panels). Time dependence of the density of activated caspase-3+ astrocytes in ODC-OVA slices after incubation with (gray line) and without (black line) OT-I T cells (B, bottom panels). ** indicates statistical significance (p-values < 0.05), ns = not significant (p-values > 0.05).

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