Journal: Scientific Reports

Article Title: Unaltered intravenous prion disease pathogenesis in the temporary absence of marginal zone B cells

doi: 10.1038/s41598-019-55772-w

Figure Lengend Snippet: Displacement of MZ B cells from the splenic MZ by antibody-mediated blockade of the integrins LFA-1 (αLβ2) and α4β1. Mice were treated with anti-αL and anti-α4 mAb (anti-αL + anti-α4), or isotype-matched control-Ig ( n = 4 mice/group), and spleens analysed seven days later. ( A ) Gating strategy used to identify B220 + CD21 hi CD1d hi MZ B cells by flow cytometry. ( B ) The percentage of B cells that were B220 + CD21 hi CD1d hi MZ B cells was significantly reduced in the spleens of anti-αL + anti-α4 treated mice. *** P < 0.001, Student’s t-test; n = 4 mice/group; closed circles, control-Ig; open circles, anti-αL + anti-α4; horizontal bars, median. ( C ) Representative immunostaining of IgM + (violet) and IgD + (green) cells in the spleens of mice from each treatment group. This analysis showed that IgM + MZ B cells were absent from the MZ region of the spleens of anti-αL + anti-α4 treated mice. MZ, marginal zone; F, B-cell follicle; broken line shows the boundary of the MZ; scale bar, 100 µm. ( D ) Representative immunostaining of CD1d + (brown) cells in the spleens of mice from each treatment group. Haematoxylin (blue) was used as a nuclear counterstain. This analysis showed that CD1d + MZ B cells were absent from the MZ region of the spleens of anti-αL + anti-α4 treated mice. WP, white pulp region; broken line shows the boundary of the MZ; scale bar, 500 µm. ( E ) Representative immunostaining shows a similar distribution of MARCO + (left-hand column), SIGNR1 + (middle column) and CD169 + (right-hand column) macrophages in the spleens of mice from each treatment group. Broken line shows the relative positioning of the marginal zinus. Scale bar, 100 µm.

Article Snippet: Sections were then immunostained using the following antibodies: rat anti-mouse CD1d (clone 1B1; Bio-Rad Laboratories); rat anti-mouse CD21/35 (clone 7G6; BD Biosciences); anti-mouse CD45R:B220 (clone RA3-6B2); anti-mouse CD169 (MOMA-1; Bio-Rad Laboratories); Alexa Fluor 488-conjugated anti-mouse IgD (clone 11–26 c.2a; Biolegend); Alexa Fluor 594-conjugated goat anti-mouse IgM (µ chain; ThermoFisher); anti-mouse MARCO (clone ED31; Bio-Rad Laboratories); Armenian hamster anti-mouse SIGNR1 (clone 22D1; eBioscience).

Techniques: Flow Cytometry, Immunostaining

Journal: Scientific Reports

Article Title: Unaltered intravenous prion disease pathogenesis in the temporary absence of marginal zone B cells

doi: 10.1038/s41598-019-55772-w

Figure Lengend Snippet: Temporary displacement of MZ B cells does not affect the early accumulation of PrP Sc upon FDC in the spleen. Mice were treated with anti-αL and anti-α4 mAb (anti-αL + anti-α4), or isotype-matched control-Ig ( n = 4 mice/group), and seven days later injected IV with ME7 scrapie prions. Spleens were analysed at 35, 70 and 105 days after IV prion exposure. ( A ) Immunohistohemical (IHC) analysis shows heavy prion disease-specific PrP d accumulations (upper row) were detected in association with FDC (CD21/CD35 + cells, arrows; middle row) in the B cell follicles (B220 + cells, bottom row) of mice from each treatment group. Haematoxylin (blue) was used as a nuclear counterstain. Scale bar, 100 µm. ( B ) Paraffin-embedded tissue immunoblotting was used to confirmed the presence of prion-specific, relatively PK-resistant, PrP Sc (blue/black, arrows). Scale bar, 100 µm. ( C ) Similar frequencies of PrP Sc + FDC were detected in the spleens of mice from each treatment group. Closed circles, control-Ig; open circles, anti-αL + anti-α4; horizontal bars, median. n = 4 mice/group.

Article Snippet: Sections were then immunostained using the following antibodies: rat anti-mouse CD1d (clone 1B1; Bio-Rad Laboratories); rat anti-mouse CD21/35 (clone 7G6; BD Biosciences); anti-mouse CD45R:B220 (clone RA3-6B2); anti-mouse CD169 (MOMA-1; Bio-Rad Laboratories); Alexa Fluor 488-conjugated anti-mouse IgD (clone 11–26 c.2a; Biolegend); Alexa Fluor 594-conjugated goat anti-mouse IgM (µ chain; ThermoFisher); anti-mouse MARCO (clone ED31; Bio-Rad Laboratories); Armenian hamster anti-mouse SIGNR1 (clone 22D1; eBioscience).

Techniques: Injection, Western Blot