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Blood, Vol. 111, Issue 8, 4273-7282, April 15, 2008
Itch–/– β and   T cells independently contribute to autoimmunity in Itchy mice
Blood Parravicini et al.
111: 4273
Supplemental materials for: Parravicini et al
Files in this Data Supplement:
- Figure S1. Splenomegaly, lymphadenopathy, and activated CD4+ T cells in Itchy mice (JPG, 115 KB)
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Cells were counted using an automated cell counter and subpopulations analyzed by flow-cytometry. (A) Total cell counts in lymph nodes and spleen. (B) The average ± SD of T cells (TCR- +) in lymph nodes and spleen as a percentage of live lymphocytes is shown (gated using side and forward scatter profiles). (C) The average ± SD of LN CD4+ T cells is expressed as percentage of live lymphocytes and of LN CD4+ T cells subpopulations stained for surface markers CD44 and CD25. (D) The average of total CD8+ T cell counts in lymph nodes, analyzed for the expression of activation marker CD44 is shown. (E) FACS profiles of splenic CD4+ T cell stained with CD44 and CD62L (effector memory CD44hi CD62Llo; central memory CD44hi CD62Lhi; naive CD44lo CD62Lhi). Crosses = WT mice, Open circles = Itch+/− mice, Closed triangles = Itch−/− mice.
- Figure S2. Splenic B cell subpopulations in Itchy Mice show normal relative distribution and no overt signs of activation (JPG, 134 KB)
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Cells were counted using an automated cell counter and subpopulations analyzed by flow-cytometry. (A) Splenic B cells (B220+) are expressed as percentage of live lymphocytes. Average ± SD are shown. Crosses = WT mice, Open circles = Itch+/− mice, Closed triangles = Itch−/− mice. (B) Bars show average ± SD numbers of B cell subsets identified by FACS as previously reported 54, according to the expression of B220, AA4.1 (CD93), IgM, and CD23. (C) Splenic cells from WT (shaded histograms; 2 representative mice) and Itch−/− mice (open histograms; 3 representative mice) were analyzed according forward scatter (FSC) or gated on live cells and stained for CD23, CD21, and IgD. (D) Splenic cells from WT, Itch−/+, and Itch−/− mice gated on lymphocytes using side and forward scatter profiles were stained for mature follicular (IgDhiIgMlo; gate II) and MZ/transitional (IgMhiIgDlo; gate I) B cells. (E) Analysis of splenic B cells gated on IgMhiIgDlo population and stained for CD23 and CD21 to identify MZ B cells. The percentages of B cell subpopulations are indicated next to each gate. Results in representative mice are shown.
- Figure S3 (JPG, 153 KB)
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(A) Auto-reactive IgM in serum of Itch−/− mice. Anti-nuclear Abs (IgM) were detected by ELISA in the sera of WT, Itch−/− mice, or Itch−/− bone-marrow chimeras. (**p<0.01) (B) Increased Ag-specific IgM in Itch−/− mice. Mice were immunized TI-2 antigen TNP-Ficoll and concentration of Ag-specific IgM was measured 4 weeks later by ELISA. Units are µg/ml. Crosses = WT mice, Open circles = Itch+/− mice, Closed triangles = Itch−/− mice. (C) Moderate increase of peritoneal B1 cells in TCR- −/− Itch−/−. Peritoneal cells from TCR-−/− WT, TCR-−/− Itch−/+, and TCR-−/− Itch−/− mice were stained for B2 (IgDhiIgMlo) and B1 (IgMhiIgDlo) cells gated on lymphocytes using side and forward scatter profiles. (D) Presence of IgM deposits in kidneys of adult Itchy mice. Kidney micro-sections (=7µm) of WT (i) and Itch−/− (ii, iii) mice were stained with FITC-conjugated anti-mouse IgM. (E) Presence of IgG deposits in kidney of adult Itchy mice. WT (i) and Itch−/− (ii, iii) kidneys were stained with FITC-conjugated anti-mouse IgG. Alternatively, micro-sections of WT (iv) and Itch−/− (v, vi) were stained with hematoxylin and eosin and observed with a Zeiss Axioplan and Lasersharp 2000 software. Immunofluorescence samples in panel D and E were analyzed by Deltavision Systems (Applied Precision; Olympus IX70 optics and Softworx software). Representative sections in one of five mice showed.
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