Retinoic acid and α-galactosylceramide regulate the expression of costimulatory receptors and transcription factors responsible for B cell activation and differentiation

Abstract

Mature naïve B cells possess a number of BCR coreceptors and other antigen receptors, including the MHC class I-like molecule CD1d, but little is known of the response of B cells to stimulation by the CD1d ligand, α-galactosylceramide (αGalCer). Previously, we showed that all-trans-retinoic acid (RA) increases the expression of CD1d and the magnitude of CD1d-mediated antibody production in vivo. Potential mechanisms could include changes in the expression of costimulatory molecules and transcription factors that regulate plasma cell formation. In the present study, we have used isolated purified B cells and in vivo studies to demonstrate that αGalCer and RA initiate a regulated expression of several genes essential for B cell activation and differentiation, such as Pax-5, Blimp-1, IRF-4 and activation-induced cytidine deaminase (Aid). Moreover, whereas αGalCer mainly increased the expression of Pax-5, CD40 and CD86 that are critical for B cell activation, RA predominantly increased CD138⁺ and Fas⁺-PNA⁺ B cells, which represent more advanced B cell differentiation. It is also noteworthy that αGalCer enriched a CD19hi subset of B cells, which represent B cells with more differentiated phenotype and higher potential for antibody production. In vivo, treatment with αGalCer enriched the CD19hi population, which, after sorting, produced more anti-TT IgG by ELISPOT assay. Together, our data demonstrate that RA and αGalCer can regulate B cell activation and differentiation at multiple levels in a complementary manner, facilitating the progress of B cells towards antibody secreting cells.

Keywords: AID; CD138; CD19; CD40; CD86; GC; IRF; Pax-5; RA; TT; activation induced deaminase; all-trans-retinoic acid; germinal center; interferon regulatory factor; tetanus toxoid; α-galactosylceramide; αGalCer.

Fig. 1

RA and αGalCer differentially regulate the expression of genes that control B cell proliferation and differentiation. Spleen B cells were isolated and cultured in 24-well plates (10⁶ cells/1 ml medium) in the presence and absence of RA (20 nM) and/or αGalCer (100 ng/ml) for 2 days. Cells were then subjected to real-time qPCR analysis to determine the expression Pax-5 (A), Blimp-1 (B), IRF-4 (C) and Aid (D) genes. The graphs represent two independent experiments each in duplicate. *, P < 0.05.

Fig. 2

αGalCer increases Pax-5 protein in spleen B cells. Purified B cells (5 × 10⁵ cells/0.5 ml in 48-well plates) were cultured for 4 days in the presence and absence of RA (20 nM) and/or αGalCer (100 ng/ml) or anti-μ (1 μg/ml), and subjected to flow cytometric analysis to detect CD19 and Pax-5 molecules. Live B cells were gated on CD19⁺ cells to evaluate the Pax-5 levels. A. Representative histograms showing the expression level of Pax-5 in B cells. The horizontal line indicates the gate for Pax-5⁺ cells. B and C, αGalCer increased Pax-5 expression levels as shown by increased percent of positive cells and MFI (Mean fluorescence intensity). *, P < 0.05 compared to control. The data shown are representative of at least three independent experiments each performed in triplicate.

Fig. 3

αGalCer and anti-μ increased the co-stimulatory molecules CD40 and CD86 on B cells. Splenocytes were cultured in the presence and absence of RA (20 nM), anti-μ (1 μg/ml) and/or αGalCer (100 ng/ml) at 5 × 10⁵ cells/0.5 ml in 48-well plates for a total of 4 days. Cells were harvested each day and subjected to flow cytometric analysis. Cells were first gated on CD19 and IgD positive cells, and then analyzed for CD40 and CD86 positive cells. A and B, Representative histograms showing the CD40 and CD86 expression on CD19⁺-IgD⁺ B cells after 2 days of culture and treatment. The negative control is shown in filled grey from cells stained with isotype control antibody, cell treated with medium only by the dashed line, and cells treated with αGalCer by the solid line. C. CD40 and CD86 double positive B cells are shown in the upper right quadrant, a representative graph from cells treated with αGalCer and anti-μ for 2 days. D. CD40 and CD86 double positive cells were increased after stimulation. The data shown are representative of three independent experiments, each performed in triplicate. Data were analyzed using a two-way ANOVA followed by Bonferroni post hoc test. Groups with different letters differed significantly, a>b>c>d, P < 0.05.

Fig. 4

RA and αGalCer regulate B cell differentiation in vivo. Spleens from the immunized animals with and without RA and/or αGalCer treatment (refer to Material and Methods) were collected and subjected flow cytometry analysis and immunofluorescent staining. A. Spleen weight is increased after αGalCer treatment. B. Serum TT-specific IgG levels determined by ELISA. C. Immunofluorescent staining of mouse spleens showing the spleen follicle with marginal zone (MZ) and germinal center (GC) identified by IgM (Green) and Ki-67 (Red). D. Comparison of CD40-CD86 positive cells after treatments. Splenocytes were first gated on CD19⁺ cells, and then analyzed for their CD40-CD86 staining. E. RA and αGalCer increased CD19hi-IgMhi positive B cells. The in vivo experiment was performed more than three times with n=3 to 5 each time.

Fig. 5

Characteristics of the CD19⁺ B cells and the correlation of CD40, CD86 and Pax-5 expression in CD19⁺ B cell subsets. Spleen B cells were cultured in the presence and absence of RA and/or αGalCer for 4 days. B cells were stained with CD19, CD1d, IgG1, or IgD, respectively. A group of cells were also labeled with CFSE on day 0 to monitor the B cell division. From A to E, Differential characteristics of CD19lo (red) and CD19hi (blue) B cells. A sample of B cells cultured for 4 days in the presence of RA (20 nM) was presented. A. Gating of CD19lo and CD19hi cells. B. Cell distribution on forward and side scatter. C. CFSE dye dilution on day 4 (B cells of day 0 are in gray). sIgG1 (D) and CD1d (E) expressions were shown, filled gray showed negative staining using the isotype control antibodies. F. αGalCer and RA increase CD19hi expression. The A gate was set to analyze the CD19hi B cell population. G and H. αGalCer and anti-μ differentially regulated CD40⁺Pax-5hi, and CD86⁺Pax-5hi cells in CD19hi and CD19lo cells. B cells cultured for 24 hours were analyzed for their CD40, CD86, Pax-5 and CD19 expression. Live cells were first gated on CD19hi and CD19lo groups and then analyzed for CD40 and Pax-5 or CD86 and Pax-5. Data are representative of three or more independent experiments, n=3 per experiment. Groups with different letters differed significantly, a>b>c>d, P < 0.05.

Fig. 6

RA and αGalCer differentially regulate plasmacyte differentiation. B cells were cultured in the presence and absence of RA, αGalCer and/or anti-μ for 4 days and costained with antibodies. Cells were gated on their CD19 expression and then analyzed for CD138 or Fas-PNA staining. A. RA increased CD138⁺ B cells. B. CD19hi population contained a higher percentage of Fas⁺-PNA⁺ B cells, and RA increased Fas⁺-PNA⁺ on both CD19hi and CD19lo populations. Groups with different letters differed significantly, a>b>c>d, P < 0.05.

Fig. 7

αGalCer increases the anti-TT IgG secreting B cells in CD19hi B cells. Splenocytes from TT-immunized mice with and without αGalCer were collected and stained for CD19, then sorted into CD19hi and CD19lo B cells for ELISPOT assay. A. Representative histograms showing the CD19+ cell population before and after sorting. B. ELISPOT assay showing that CD19hi B cells in αGalCer treated mice had higher number of antibody-secreting cells. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc test, n=3. Groups with different letters differed significantly, a>b, P < 0.05.