Expression of TCF7L2 in MS lesions and non-demyelinating inflammatory CNS diseases.

<p>In a subset of early MS lesions increased numbers of TCF7L2 positive cells were detected in the periplaque white matter and in remyelinating lesion areas. However, also in other inflammatory neurological diseases (OND) TCF7L2 positive cells were present. Red dots indicate a subset of tissue samples with high numbers of TCF7L2 expressing cells (see Figure 4) (<b>A</b>). There was no correlation between numbers of TCF7L2 and NOGOA positive cells in the different MS lesion areas. NOGOA positive oligodendrocytes and TCF7L2 expressing cells were quantified in periplaque white matter (PPWM), actively demyelinating (AD), demyelinated (DM) and remyelinating (RM) lesion areas; TCF7L2 expressing cells (dots) and NOGOA expressing cells (squares) from the same lesion area are labelled in the same colour (<b>B</b>). Double stainings revealed that a subset of TCF7L2 positive cells were NOGOA positive oligodendrocytes (arrows). TCF7L2 negative oligodendrocytes are indicated by arrow heads (Double immunohistochemistry for NOGOA (red) and TCF7L2 (black) (<b>C</b>). In inflammatory non demyelinating disease TCF7L2 positive oligodendrocytes and astrocytes were detected (<b>D</b> and <b>E</b>). In <b>D</b> a GFAP and TCF7L2 positive astrocyte (arrow) as well as a GFAP negative TCF7L2 positive cell (arrow head) are depicted (double immunohistochemistry for GFAP (green) and TCF7L2 (red). Additional GFAP and TCF7L2 positive cells are shown in <b>E</b> (arrows) (double immunohistochemistry for GFAP (red) and TCF7L2 (black)).</p

Expression of TCF7L2 during remyelination in the CNS of mice.

<p>Mice were fed with 0.25% cuprizone for six weeks and the numbers of oligodendroglial lineage cells were quantified at the indicated time points. Lowest numbers of OLIG2 positive cells were observed 21 days after onset of cuprizone diet; the increased numbers at the end of the cuprizone diet (at 42 days) suggest a recruitment of OPCs during ongoing demyelination (<b>A</b>). NOGOA is expressed by mature oligodendrocytes; lowest numbers of NOGOA positive cells were as well found at day 21 (B). High numbers of TCF7L2 expressing cells were only detected at day 42 (C).</p

Expression of TCF7L2 during myelination in the CNS of mice.

<p>At P10 single myelinated axons are observed in the lateral parts of the corpus callosum (arrow) (<b>A</b>) whereas in adult mice myelination is complete (<b>B</b>). High numbers of TCF7L2 expressing cells were observed in P10 mice (<b>C</b>), but only few TCF7L2-positive cells were found in adult mice (<b>D</b>) as quantified in the diagrams (<b>E</b> and <b>F</b>). The percentage of OLIG2 positive cells expressing TCF7L2 decreased significantly in adult mice (<b>G</b>).</p

Expression of HDAC2 in MS lesions and control tissue samples.

<p>In control tissue samples (<b>A</b>) as well as in MS lesions (<b>B</b>) numerous NOGOA positive oligodendrocytes were seen which express abundantly HDAC2 (arrows) (double immunohistochemistry for NOGOA (red) and HDAC2 (black)).</p

Myelination and expression of TCF7L2 in the human CNS.

<p>The extent of myelination in human frontal lobes was quantified using a semiquantitative score. Between 30 and 40 weeks of gestation no MBP-positive axons were found. Myelination became first obvious between 0 and 6 months after birth (<b>A</b>). First TCF7L2-positive cells were detected at the end of gestation with maximal numbers between 7 and 12 months after birth. Afterwards, the numbers of TCF7L2-positive cells decreased quickly (<b>B</b>). At 6 months after birth numerous myelinating oligodendrocytes were observed (immunohistochemistry for MBP) (<b>C</b>). Many TCF7L2 positive cells also expressed NOGOA (double immunohistochemistry for NogoA (red) and TCF7L2 (black) (<b>D</b>) but not GFAP (double immunohistochemistry for TCF7L2 (black) and GFAP (red)) (<b>E</b>). TCF7L2 was also expressed in human fetal oligodendrocytes in vitro (green O4, red TCF7L2) (<b>F</b>).</p

Additional file 2: of Relationship of acute axonal damage, Wallerian degeneration, and clinical disability in multiple sclerosis

Axons undergoing Wallerian degeneration are at least in part myelinated in EAE lesions. No co-localization of NPY-Y1R immunoreactivity with myelin proteins, i.e., PLP (A-C), MOG (D-F), MAG (G-I), and CNPase (J-L) was observed by fluorescence double IHC in WT EAE mice, which further confirms that the antiserum against NPY-Y1R applied does not detect an antigen situated within the myelin sheath or myelin ovoids. Insets in (A-C) represent NPY-Y1R+ degenerating fiber(s) in largely intact myelinated tracts, as determined by anti-PLP IHC. Scale bars=(A-L) 200 μm; (insets A-C) 10 μm. (JPG 673 kb

Chemotactic activities for CD45RA^hi Treg and CD45RO^hi Treg subsets.

<p>Chemotactic indices (CI) of Treg from a healthy donor towards CSF supernatants of 11 MS patients as determined by transwell migration assays. Original input PBMC and migrated cells were stained for CD4, CD25, FOXP3, CD95, CD45RO, and CD45RA, and CIs were calculated by dividing percentages of Treg subsets in CD4⁺ T-cells of migrated cells by percentages of Treg in CD4⁺ T-cells in original input PBMCs. Symbols indicate CIs of individual patients. Means are indicated, ** indicates p<0.001.</p

Six-color FACS analysis of lymphocytes derived from CSF and peripheral blood.

<p>Analysis of CSF and PBMC from 17 treatment-naïve MS patients. Isolated lymphocytes were stained with monoclonal antibodies against CD4, CD25, CD127, FOXP3, CD45RO and CD95. <b>(A)</b> Lymphocytes were gated for Treg and the percentage of CD95^hiCD45RO^hi was determined. Treg were increased in CSF when compared to peripheral blood, * indicates p<0.05. <b>(B)</b> Expression of CD95 on Treg from CSF and PBMC derived from the same MS patient on the same day, representative figure.</p

Prevalence of CD4⁺ and FOXP3⁺ cells in MS lesions.

<p>Brain biopsies of 16 MS patients were analyzed by immunohistochemistry for the presence of CD4⁺ and FOXP3⁺ cells. Sections of each biopsy were stained with CD4 mAb, FOXP3 mAb or isotype control mAbs. Whole-slide analysis by complete scanning of the section and computer-aided analysis of positive cells was performed. (<b>A</b>) Representative CD4 immunohistochemistry of MS lesion. Positive cells show red cell surface staining. Bar indicates µm scale in two different magnifications. Tissue was hematoxylin counterstained prior to analysis. (<b>B</b>) Representative FOXP3 immunohistochemistry of MS lesion. Positive cells show brown nuclear staining. Bar indicates µm scale in two different magnifications. Tissue was hematoxylin counterstained prior to analysis. (<b>C</b>) Number of patients with CD4⁺ T cells in the section (0% negative, 100% positive) and number of patients with FOXP3⁺ cells in the analyzed section (31% negative with 69% positive for FOXP3). (<b>D</b>) Number of patients grouped by their content of FOXP3⁺ cells and CD4⁺ cells respectively in the analyzed section: “-“ indicates 0 cells/mm², “(+)” 0-3 cells/mm², “+” >4-15 cells/mm², “++” >15 cells/mm². (<b>E</b>) Number of patients with (+) or without detection of FOXP3⁺ cells (-) in active or inactive MS lesions.</p

Representative six-color FACS analysis of lymphocytes derived from CSF and peripheral blood.

<p>Isolated lymphocytes were stained with monoclonal antibodies against CD4, CD25, CD127, FOXP3, CD45RO and CD95. CD4⁺ cells were gated (G1) and analyzed for FOXP3, CD127 and CD25 <b>(A)</b>. CD4⁺ cells are shown in all other dot plots of Fig. 2A, B in blue and represent 48% of peripheral lymphocytes. <b>(B)</b> CD4 positive cells with significant FOXP3 expression and low to high CD25 expression were included in gate (G2). These cells are shown in all other dot plots of Fig. 2A, B in red and represent 7.1% of CD4 positive cells. Multicolor analysis confirms that the majority of FOXP3 positive cells co-segregate with CD127^loCD25^lo-hi expression. Note: CD127 negative cells accumulate in the first few channels by conventional dot plotting. <b>(C)</b> Pairwise analysis from CSF and PBMC of a treatment-naïve MS patient. Isolated lymphocytes were stained with monoclonal antibodies against CD4, CD25, CD127, FOXP3, CD45RO and CD95. Lymphocytes were gated for CD4⁺CD25⁺CD127^loFOXP3⁺ cells and the percentage of CD95^hiCD45RO^hi was determined.</p