Figure 1.
The majority of FasL+ splenocytes are B cells.
Splenocytes from naïve mice were stained for surface expression of FasL and the cellular composition of FasL− and FasL+ subsets was examined by flow cytometry. (A) Representative dot plots of splenocytes stained with a T cell marker (CD3) and three independent B cell markers (B220, CD19, and IgM). (B) The frequency (mean ± SEM) of cells staining positive for the markers presented in (A) among FasL+ splenocytes. Data are representative of more than 5 independent experiments with at least three animals per experiment.
Figure 2.
FasL+ B cells are enriched in the CD5+CD1dhigh subset.
(A) Representative histograms showing relative surface expression of markers for B cell subsets in CD19+FasL− and CD19+FasL+ B cells. Bar graphs are the average median fluorescence intensity from replicate animals. Grey histograms represent isotype control antibody staining. (B) FasL− and FasL+ B cells were stained for co-expression of CD5 and CD1d. (C) The frequency of CD5+CD1dhigh cells (mean ± SEM) among FasL− and FasL+ B cells in replicate animals was measured. (D) Reciprocally, the frequency of FasL+ cells among all B cells and CD5+CD1dhigh B cells was measured. Data are representative of four independent experiments using at least 3 animals per experiment. **p<0.01, ***p<0.001, and ****p<0.0001.
Figure 3.
IL-10-producing B cells are not enriched in the FasL+ subset.
FasL− and FasL+ splenic B cells from naïve mice were sorted by FACS and stimulated with PMA, ionomycin, and LPS in the presence of monensin for five hours. (A) Following stimulation, FasL− and FasL+ B cells were fixed and stained for intracellular IL-10. (B) Frequency of IL-10-producing B cells (mean ± SEM) among CD19+FasL− and CD19+FasL+ subsets.
Figure 4.
FasL+ B cells express higher levels of the IL-5 receptor than FasL
− B cells. (A) Histograms comparing surface expression of CD125 in CD19−, CD19+FasL− and CD19+FasL+ cells. (B) Quantification of median fluorescence intensity of CD125 expression in CD19+FasL− and CD19+FasL+ B cells in replicate animals (mean ± SEM). **p<0.01.
Figure 5.
IL-5 and CD40L stimulation expands a population of B cells enriched for FasL expression.
(A) Diagram of B cell co-culture experiments with CD40L-expressing fibroblasts for study of the effects of IL-5 on B cell growth and function. (B) CD19+ B cells were cultured as illustrated in (A) and harvested after five days. Viable B cells recovered from culture were quantified from replicate samples using a hemocytometer and trypan blue exclusion. The dotted line represents the concentration of B cells at the beginning of the experiment. (C) Proliferation of B cells cultured with CD40L-expressing fibroblasts in the presence or absence of IL-5 was assessed by the incorporation of 3H-thymidine. (D) B cells stimulated with CD40L in the presence or absence of IL-5 were stained for FasL, CD5, and CD1d and compared with freshly-isolated CD19+ B cells. (E) Median fluorescence intensity of surface markers stained as in (D) on freshly-isolated CD19+ B cells, B cells stimulated with CD40L, and B cells stimulated with CD40L and IL-5. (F) The frequency of FasL+ B cells among freshly-isolated CD19+ B cells and B cells stimulated with CD40L and IL-5 was measured by flow cytometry as in (D). (G) Cell lysates from equal numbers of freshly-isolated CD19+ B cells and IL-5-stimulated B cells were probed by immunoblot for FasL and β-Actin. Data are representative of more than four independent experiments. ***p<0.001.
Figure 6.
B cells stimulated with IL-5 and CD40L induce antigen-specific apoptosis in CD4+ T cells.
Splenocytes were isolated from naive cII-TCR transgenic mice and stimulated in vitro with Concanavalin A. After three days in culture, CD4+ T cells were negatively-selected by MACS and co-cultured with freshly-isolated CD19+ splenic B cells or stimulated B cells at a 1∶1 ratio. B cells were incubated with anti-FasL blocking antibody or an isotype control antibody for 1 hour and washed with PBS prior to co-culture with CD4+ T cells. In wells with no B cells, anti-FasL antibody or isotype control antibody were directly added to culture wells to assess FasL-mediated “fratricide” between CD4+ T cells. Apoptosis was measured at 18 hours by flow cytometry as the percentage of CD4+cells that were positive for Annexin V staining. (A) Representative plots of gated CD4+ T cells for Annexin V and propidium iodide following co-culture with B cells. (B) Quantification (mean ± SEM) from a single experiment as described in (A). Data are representative of four independent experiments. ***p<0.001 and ****p<0.0001.
Figure 7.
IL-5 enhances IL-10 secretion from CD40L-stimulated B cells.
CD19+ B cells were isolated by MACS from naïve mice and cultured for five days with CD40L-expressing fibroblasts in the presence or absence of IL-5. B cells were then harvested, washed in PBS, and cultured overnight at equivalent cell concentrations (7.5×105 viable cells/mL) in the presence or absence of stimulation with PMA and ionomycin. IL-10 in the culture supernatants was measured by ELISA. (A) IL-10 secretion from B cells after overnight culture with no further stimulation. (B) IL-10 secretion from B cells after overnight culture with PMA and ionomycin. Data are representative of at least three independent experiments (mean ± SEM). ***p<0.001.
Figure 8.
IL-5-mediated induction of killer B cell function, but not FasL expression, is inhibited by IL-4.
CD19+ B cells from naïve mice were cultured for five days with CD40L Fb and IL-4, IL-5, or both cytokines concurrently. (A) The ability of B cells stimulated under these conditions to induce apoptosis in activated CD4+ T cells was then assessed as in Figure 6. Apoptotic cells were identified and enumerated (mean ± SEM) on the basis of positive staining for Annexin V by flow cytometry. (B) B cells were cultured with CD40L-expressing fibroblasts in the presence or absence of IL-4 and IL-5, or in the presence of both cytokines. Cells were harvested after 5 days in culture and cell lysates were probed for FasL and β-Actin proteins as in Figure 5G. ***p<0.001 and ****p<0.0001.