Figure 1.
Visualization of a new subset of LN stromal cells.
Confocal images of LN sections from a CD21cre-RFP chimera stained for (A) FDC-M2 (green), CD3 (light blue), and B220 (dark blue) and (B) CD21/35 (green), Collagen IV (white), and B220 (blue). RFP+ cells appear in red. In (B), the star (*) signals the position of the collagen-poor central region of the follicle populated by the CD21+ RFP+ FDC network; arrows indicate the extensions of the conduit network within the follicle and arrowheads point to CD21− RFP+ cells attached to these conduits. The dashed line represents the delineation of the B220 staining. No RFP signal was detected in CD21cre−RFP+ chimeric mice, ruling out a leaky expression of the reporter. Data are representative of three different experiments (two mice per experiment).
Figure 2.
Phenotype of CD21− RFP+ stromal cells.
(A and E) Confocal images of a LN section from a CD21cre-RFP chimera stained for the indicated markers. RFP+ cells appear in red. The dashed line delineates B cell follicle boundary. (B, C, D) CD21− RFP− gp38+ CD31− CD45− FRCs and CD21− RFP+ gp38+ CD31− CD45− cells were sorted by flow cytometry and their transcriptomic profiles were analyzed by microarrays (B and C) or RT-PCR (D). (B) Scatter plot of global comparison of gene expression between CD21− RFP+ cells and FRCs. Each gene in the microarray is represented by a dot with coordinates consisting of average gene expression computed from three independent CD21− RFP+ samples (y-axis) and from the three matched FRC samples (x-axis). Genes with Log2 average expression level at least 1.5-fold higher in CD21− RFP+ cells are shown in red, while genes with Log2 average expression level at least 1.5-fold higher in FRCs are shown in blue. These genes are separated from the other genes by colored lines representing these fold change cutoffs. (C) Heat map for the 50 genes with the most significantly higher expression in CD21− RFP+ cells. A subset of the genes shown in red in panel B was further selected based on a p value<0.05 for differential expression between CD21− RFP+ cells and FRCs, as computed by Limma. Genes (rows) and samples (columns) were clustered by complete linkage hierarchical clustering, using Euclidean distance measure. For each gene, expression levels close to the mean value across all six samples are shown in black, high expression levels in red, and low expression levels in green. (D) Relative expression of Cxcl13, Ccl21, Ccl19, Tgfb1, and Adrb2 mRNA levels quantified by RT-PCR. Data are representative of three different experiments (4–5 mice per sample). (E) Confocal images of a LN B cell follicle and its adjacent T cell area from a CD21cre-RFP chimera stained for CXCL13 (green), CD21/35 (blue), and B220 (white). RFP+ cells appear in red. The dashed line represents the delineation of the B220 staining. Data are representative of two different experiments (two mice per experiment).
Figure 3.
CD21− RFP+ stromal cells are surrounded by inflamed B cell follicles.
CD21cre-RFP chimeras were untreated (A, upper panel) or injected (A, lower panel and B) with an emulsion of CFA/PBS in the ears. (A) Three weeks later, ear draining LNs were sectioned, stained for collagen IV (green), IgD (white), and FDC-M2 expression (blue) and imaged by confocal microscopy. RFP+ cells appear in red. (B) Confocal pictures of an inflamed B cell follicle stained for collagen IV (white), B220 (blue), and CD21/35 (green) expression. Arrows highlight the region of the inflamed B cell follicle enriched in collagen IV. Insert displays high magnification of the boundary between T and B cell areas (dashed line). Data are representative of three different experiments (two mice per experiment).
Figure 4.
Induction of CXCL13 secretion in CD21− RFP+ stromal cells upon B cell follicle enlargement.
CD21cre-RFP chimeras were untreated (A) or injected (B) with an emulsion of CFA/PBS in the ears. Three weeks later, ear draining LNs were sectioned, stained for collagen IV (green), IgD (white), and CXCL13 expression (blue), and imaged by confocal microscopy. RFP+ cells appear in red. Inserts display high magnifications of the boundaries between T and B cell areas (dashed line). In (B), the arrow indicates the area of the enlarged B cell follicle covered by the collagen IV network (conduit system). Single z slices show intracellular staining of CXCL13. (C) CXCL13 mRNA levels in CD21− CD45− RFP+ cells purified from resting and CFA-inflamed LNs relative to FRCs. Data are representative of three different experiments (two mice (A and B) and five mice (C) per experiment).
Figure 5.
Acute B cell deletion abrogates CXCL13 staining in converted CD21− RFP+ stromal cells.
CD21cre-RFP mice were irradiated and reconstituted with bone marrow cells isolated from CD21cre-DTR mice. Reconstituted chimeras were injected with an emulsion of CFA/PBS in the ears. Three weeks later, mice were treated or not with DT for 3 consecutive days. The fourth day, ear draining LNs were harvested and either analyzed by flow cytometry (A) or sectioned and imaged by confocal microscopy (B). (A) Dot plots of ear draining LNs showing B cell deletion upon DT treatment. (B) Confocal images of LN tissue sections stained for B220 (blue) and CXCL13 expression (red). RFP+ cells appear in green. Inserts display high magnifications of the B cell areas. The yellow dashed line delineates the approximate size of the B cell follicle prior DT injection (see Materials and Methods), while the white dashed line delineates the boundary of the residual B cell follicle. Data are representative of three different experiments (two mice per experiment).
Figure 6.
LT-β expression on B cells regulates CXCL13 expression in CD21− RFP+ stromal cells.
CD21cre-RFP μMt−/− mice were irradiated and reconstituted with a mixture of μMt−/− (80%) and Wt (20%) bone marrow cells or a mixture of μMt−/− (80%) and LT-β–deficient bone marrow cells. Reconstituted chimeras were injected with an emulsion of CFA/PBS in the ears. Three weeks later, splenocytes were used to assess the percentage of CD3+ T cells and B220+ B cells in each group of mice by flow cytometry (A). Ear draining LNs were sectioned and stained for CD21/35 (blue), CXCL13 (green), and B220 expression (B). Confocal pictures were taken in B220+ B cell follicles. RFP+ cells appear in red. Arrowheads point to CXCL13+ CD21− RFP+ stromal cells. Data are representative of two different experiments (three mice per experiment).
Figure 7.
CXCR5-deficient B cells fail to access inflamed B cell follicles.
CD21cre-RFP chimeras were untreated (left panel) or injected (right panel) with an emulsion of CFA/PBS in the ears. Three weeks later, recipients were injected with a cohort of CMFDA-labeled Wt-deficient B cells (green) and Celltrace violet–labeled CXCR5-deficient B cells (violet). One day later, ear draining LNs were sectioned, stained for B220 (blue) expression, and imaged by confocal microscopy. RFP+ cells appear in red. Data are representative of two different experiments (three mice per experiment).