Article Figures & Data
Figures
- FIG. 1.
CT enhances IgA and IgM secretion in ConA-stimulated splenic cells. Splenic cell suspensions were cultured with 2 μg/ml of ConA for 7 days in the presence or absence of 1 μg/ml of CT, LT-IIa, or LT-IIb. Levels of IgA, IgM, and IgG secreted into culture supernatants were examined by ELISA. Results are reported as the arithmetic means ± standard errors of the means of results obtained from three independent experiments. *, **, and ***, significant differences at P values of ≤0.05, ≤0.01, and ≤0.001, respectively, compared to cells stimulated with ConA alone.
- FIG. 2.
CT induces a population of cells expressing a plasma blast phenotype. (A) Splenic cell suspensions depleted of CD138+ cells were cultured with 2 μg/ml of ConA for 4 or 7 days in the presence or absence of 1 μg/ml of CT or LT-IIa. Cells stained for B220 and CD138 were analyzed by FACS for frequencies of viable cells expressing the two cell markers, and the frequencies are given as percentages. (B) CFSE-labeled splenic cell suspensions were cultured with 2 μg/ml of ConA for 2 or 4 days in the presence of 1 μg/ml of CT. Cells were stained with CD138, and the frequencies of dividing CD138+ cells were determined by FACS. (C) Expression of CD19, CD40, and MHC-II on CD138− cells expressing high levels of B220 (gate A) and on B220low, CD138+ cells (gate B) from cultures treated with ConA plus CT for 4 days. (D) Cells from cultures stimulated with ConA plus CT for 7 days were analyzed for intracellular contents of IgA, IgG, and IgM by FACS, and results are given as percentages (means ± standard deviations).
- FIG. 3.
Effect of CT on activation marker expression by isolated B cells. MACS-purified CD19+ cells were cultured for 48 h in the presence or absence of 1 μg/ml of CT. The expression of CD25, CD40, CD54, CD69, CD80, CD86, and MHC-II on these cells was analyzed by FACS. Numbers in each histogram represent the mean fluorescence intensities of the respective cell populations in relative units. Representative data from three independent experiments are shown.
- FIG. 4.
Enhancement of B-cell proliferation by CT. (A) CFSE-labeled splenic cell suspensions were cultured with 2 μg/ml of ConA in the presence or absence of 1 μg/ml of CT. Frequencies of dividing CD19+ cells were determined at day 4 of culture and are given as percentages. (B) CFSE-labeled splenic cell suspensions were depleted of CD4+ cells by MACS and cultured for 4 days with either ConA, ConA plus CT, CT, or culture medium alone. Cells were stained with CD19, and the frequencies of dividing CD19+ cells were analyzed by FACS. Frequencies are given as percentages (means ± standard deviations). Plots were gated on CD19+ cells. (C) Splenic cell suspensions were cultured with 2 μg/ml ConA plus 1 μg/ml CT for 4 days in the presence or absence of 5 mM MDM. Cells stained for B220 and CD138 were analyzed by FACS for frequencies of viable cells expressing the two cell markers.
- FIG. 5.
CT enhances the expression of CD134 and CD154 in ConA-activated CD4+ T cells. Purified preparations of CD4+ T cells were incubated in the presence or absence of 1 μg/ml CT. After washing to remove unbound enterotoxin, cells were stimulated for 6, 24, 48, or 72 h with ConA plus CD11b+ cells and analyzed by FACS for levels of expression of CD134 and CD154. Plots were gated on CD4+ cells. Numbers in each histogram represent the mean fluorescence intensities of the respective cell populations in relative units. Representative data from three independent experiments are shown.
- FIG. 6.
Blockage of CD134 or CD154 inhibits Ig-SC development and Ig production. (A) Splenic cell suspensions (unlabeled or labeled with CFSE) were depleted of CD138+ cells and cultured for 7 days with 2 μg/ml ConA plus 1 μg/ml CT in the presence of αCD134L, αCD154, αCD134 plus αCD154, or an appropriate isotype control Ab. Frequencies of cells expressing the B220low, CD138+ phenotype were determined and are given as percentages. (B) Levels of IgA, IgM, and IgG secreted into culture supernatants were determined by ELISA. (C) Frequencies of dividing CD19+ cells in cultures treated with αCD154 or control Ab were analyzed by FACS at day 4 of culture and are given as percentages (means ± standard deviations). Representative data from three independent experiments are shown.
and , significant differences at P values of ≤0.01 and ≤0.001, respectively, compared to the αCD134L and αCD154 controls. - FIG. 7.
Modulation of cytokine production by CT, LT-IIa, and LT-IIb. Splenic cell suspensions were cultured with 2 μg/ml of ConA for 4 days in the presence or absence of 1 μg/ml of CT, LT-IIa, or LT-IIb, at which time the levels of IL-6, IL-10, and IFN-γ secreted into the culture supernatants were measured by a multiplex cytokine assay.
, , and , significant differences at P values of ≤0.05, ≤0.01, and ≤0.001, respectively, compared to cells stimulated with ConA alone. - FIG. 8.
Inhibition of IFN-γ production by CT favors Ig-SC development and Ig production. Splenic cell suspensions were depleted of CD138+ cells and cultured for 7 days with 2 μg/ml ConA plus 1 μg/ml CT in the presence or absence of 10 ng/ml of IFN-γ or 10 ng/ml of IL-4. (A) Levels of IgA, IgM, and IgG secreted into culture supernatants were determined by ELISA.
, significant differences at a P value of 0.001 compared to CT or CT + IL-4. (B) Frequencies of IFN-γ-producing CD4+ or CD8+ T cells in cultures treated with ConA plus CT or ConA alone were determined by FACS at day 2 of culture and are given as percentages (means ± standard deviations). (C) Purified preparations of CD8+ or CD8− cells, preincubated in the presence or absence of CT, were mixed together as described in the text and stimulated for 7 days with ConA, and the frequencies of cells expressing CD8 and CD138 were determined by FACS and are given as percentages. Representative data from three independent experiments are shown. (D) Levels of IgA, IgG, and IgM secreted into culture supernatants were determined by ELISA. (E) Levels of IFN-γ secreted into culture supernatants were measured by a Luminex cytokine assay. and , significant differences at P values of ≤0.01 and ≤0.001, respectively, compared to CD8+-untreated and CD8−-untreated preparations.
