Fig 1: Cr2 KO mice have impaired GC and antibody responses. WT or Cr2 KO mouse spleens and sera were harvested from unimmunized mice (day 0) and from mice immunized with 5 x 107 SRBC 3, 6, or 14 days before and sera and sections prepared as described in Materials and Methods. (A) Representative images of spleen WP regions surrounded with MZ regions (MOMA, red), B cell follicles (B220, green), GCs (Ki67, grey) and FDCs (FDC-M1, blue). (B) Example of day 6 WT image, illustrating regions of interest (ROIs) defined as per example image with each WP ROI defined as the area surrounded by MOMA (red), follicle (F) ROI as B220+ (green) and GC ROI as the Ki67+ (grey) areas, with the center indicated by “x”. GC ROIs were further divided into LZ and DZ based on the density of Ki67 within GC ROIs as highlighted by LineScan of line illustrated by light blue arrow. (C) The average number (± SEM) of B cell follicles and GC regions per white pulp were quantified for WT (black) and Cr2 KO (red) mice. (D) The area of follicle, GC, LZ and DZ ROIs (µm2 ± SEM). (E) IgM and IgG anti-SRBC responses in Cr2 KO mice. Sera were analyzed for IgM or IgG anti- SRBC via ELISA (optical density (O.D.) 405 nm ± SEM). Statistical differences between the groups were determined by two-way ANOVA. Statistical differences between WT and KO mice at each time point is shown above the upper curve and statistical differences between naive and immunized respective mice over the 14 day time course are indicated above horizontal lines (WT= black; KO= red). ***p values < 0.001, ** < 0.01, and * < 0.05. The data shown are 6-9 mice per group pooled from three independent experiments.
Fig 2: Cr2 KO mice have fewer FDCs in GCs than WT mice. WT or Cr2 KO mouse spleens were harvested from unimmunized mice (day 0) and from mice immunized with 5 × 107 SRBC 3, 6, or 14 days before and prepared for confocal microscopy. (A) FDCs in the WP ROIs of WT (black) and KO (red) mice were quantified by FDC-M1 mean fluorescence intensity (MFI ± SEM). (B) Area of all FDCs present in each B cell follicle (µm2 ± SEM). (C) Area of FDCs within GC LZ (left) and DZ (right) regions (µm ± SEM). (D) Percentage of total GC+ follicle FDCs in each ROI 3-, 6- and 14-days post immunization. (E) Representative images of day 6 post immunization WT and Cr2 KO spleen WP regions surrounded with MZ regions (MOMA, red), B cell follicles (B220, green), GCs (Ki67, grey) and FDCs (FDC-M1, blue). ROIs of example images are outlined in middle panels to highlight distribution of GC and non-GC FDCs. Statistical differences between the groups in (A–C) were determined by two-way ANOVA. Statistical differences between WT and Cr2 KO mice at each time point is shown above the upper curve and statistical differences between naive and immunized respective mice over the 14 day time course are indicated above horizontal lines (WT= black; KO= red). ***p values < 0.001, ** < 0.01, and * < 0.05. The data shown are 6-9 mice per group pooled from three independent experiments, unless indicated (as in D).
Fig 3: Expression of CR1/2, Fc?RIIB, and FcµR increases in response to SRBC in WT mice. WT or Cr2 KO spleens were harvested from unimmunized mice (day 0) and from mice immunized with 5 × 107 SRBC 1, 3, 6, or 14 days before and prepared for confocal microscopy Mander’s colocalization co-efficient of total whole image FDCs (FDC-M1) with (A) CR1 (8C12), (B) CR1/2 (8D9), (C) Fc?RIIB (clone 93) and (D) FcµR (MM3). CR1 was also used as a comparative FDC marker for (E) Fc?RIIB and (F) FcµR Mander’s colocalization. M1 is the frequency of FDCs where immunoreceptors are expressed. M2 is the frequency of respective immunoreceptor expression on FDCs. (G) CR1 (8C12), CR1/2 (8D9), Fc?RIIB (clone 93) and FcµR (MM3) mean fluorescence intensity (MFI ± SEM) in WP ROIs of WT (black) and KO (red) mice 14 days post SRBC immunization. Statistical differences between the groups were determined by two-way ANOVA. Statistical differences between WT and KO mice at each time point is shown above the upper curve and statistical differences between naive and immunized respective mice over the 14 day time course are indicated above horizontal lines (WT = black; KO = red). *** p values< 0.001, ** < 0.01, and * < 0.05. The data shown are six to nine mice per group pooled from three independent experiments.
Fig 4: Representative images of CR1/2, FcγRIIB, and FcµR expression in WP of WT and Cr2 KO mice. Representative images of spleens from unimmunized mice (d 0) or from mice immunized 1-14 days before. WP regions, surrounded by metallophilic macrophages (MOMA, red) indicating MZ regions, FDCs (FDC-M1, blue), CR1 (8C12, grey) and immunoreceptors, (A) CR1/2 (8D9, green), (B) FcγRIIB (clone 93, green) and (C) FcµR (MM3, green) are shown.
Fig 5: Cr2 KO FDCs show different organization to WT FDCs in follicles, GCs, and LZ. WT or Cr2 KO mouse spleens were harvested from unimmunized mice (day 0) and from mice immunized with 5 x 107 SRBC 3, 6, or 14 days before and sera and sections prepared as described in Materials and Methods. (A) Representative image of WT mice day 6 with follicle (F), GC and LZ ROIs shown. Organization of FDCs (FDC-M1, blue) positive thresholded areas within (B) B cell follicle (B220, green), (C) GC (Ki67, all grey) and (D) LZ (Ki67, less dense grey) ROIs were evaluated by plotting mean percentage cumulative area within respective areas (as illustrated in Supplementary Figure 1 ). FDC+ regions for each image, randomly distributed within each ROI, served as controls (n=8 per sample image, broken line). Statistical differences between the groups were determined by two-way ANOVA. Statistical differences between experimental sample and randomized controls for respective strains at each time point are shown above the upper curve and statistical differences between naive and immunized respective mice over the 14 day time course are indicated above horizontal lines (WT = black; KO = red). ***p values< 0.001, ** < 0.01, and * < 0.05. The data shown are 6-9 mice per group pooled from three independent experiments.
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