Modulation of Igβ is essential for the B cell selection in germinal center

Abstract

The positive and negative selection of antigen-reactive B cells take place in the germinal center (GC) during an immune responses. However, the precise molecular mechanisms underlying these selection machineries, including the involvement of antigen receptor signaling molecules, remain to be elucidated. We found that expression levels of Igα and Igβ, which are the essential components of B cell antigen-receptor complex, were differentially regulated in GC B cells and that the expression of Igβ was more prominently down-regulated in a portion of GC B cells. The suppression of Igβ down-regulation reduced the number of GL7(+)GC B cells and the affinity maturation in T-dependent responses was markedly impaired. In addition, the disease phenotypes in autoimmune-prone mice were ameliorated by blocking of Igβ down-regulation. These results suggest that Igβ down-regulation is involved in the normal positive selection in GC and the accumulation of autoreactive B cells in autoimmune-prone mice.

Figure 1

Down-regulated Igβ expression in the germinal center. (a) Frozen sections of the spleen from B6 mice, 10 days after the immunization with NP-CGG precipitated in alum, were stained with PNA (green) and antibodies to Igβ (red) and CD38 (blue). (b) Naïve B cells (shaded), B220⁺CD38⁻ cells (blue line) and B220⁺GL7⁺cells (red line) were analyzed for the expression level of Igβ. (c) Quantitative RT-PCR of Igβ mRNA in sorted naïve B cells (B220⁺IgM⁺CD38⁺) and GC B cells (B220⁺CD38⁻) from B6 mice 10 days after the immunization with NP-CGG. Cells were stained with propidium iodide to exclude dead cells and apoptotic cells. Data were normalized to the expression levels of the β-actin transcript. (d) Surface IgM in mouse splenic B cells was cross-linked with 10 ug/ml anti-IgM F(ab’)₂ to induce endocytosis for indicated periods of time and remaining levels of cell surface IgM were analyzed (left). Expression levels of Igβ mRNA were analyzed by RT-qPCR in B cells whose surface IgM was down-modulated by cross-linking for the indicated periods (right).

Figure 2

Classification of GC B cells by expression level of Igβ. (a) Flow cytometric analysis of splenocytes from immunized B6 mice. B220⁺B cells (left) cells were further gated to B220⁺CD38⁺cells (top right) and B220⁺CD38⁻ cells (bottom right) and analyzed for the expression levels of Igβ and GL7. The numbers indicate the percentage of cells in each square, and the number in the parenthesis indicate the percentage of cells in each square within the CD38⁻GL7⁻ cells. (b) Suppressed IgD expression in B220⁺CD38⁻GL7⁻ and B220⁺CD38⁻GL7⁺B cells. (c) Expression of cell surface Igα in GC B cells. Cell surface expression levels of Igα and GL7 were examined in B220⁺CD38⁺naive B cells (top) and B220⁺CD38⁻ GC B cells (bottom) by staining with anti-GL7 mAb in combination with rabbit polyclonal antibody raised against extracellular portion of Igα. Numbers indicate the percentages of the cells within the gates. (d) Transcription levels of Igβ, Bcl6 and AID in GC subsets (left) and surface Fas expression in B220⁺CD38⁻GL7⁻ and B220⁺CD38⁻GL7⁺GC subsets (right). RT-qPCR were carried out in sorted B220⁺CD38⁻GL7⁻IgβHi cells (Igβ-Hi), B220⁺CD38⁻GL7⁻IgβInt cells (Igβ-Int), B220⁺CD38⁻GL7⁻IgβLo cells (Igβ-Lo) and B220⁺CD38⁻GL7⁺cells (GL7⁺) 10 days after the immunization. (e) Cell cycle analysis of GC B cells. Splenocytes from immunized B6 mice were stained with Hoechst 33342 dye in combination with antibodies to Igβ and indicated surface markers, followed by FACS analysis. Naive B cells (top), B220⁺CD38⁻GL7⁻ (bottom left) and B220⁺CD38⁻GL7⁺B cells (bottom right) were analyzed.

Figure 3

Class switching events in GC B cells (a) Expression of IgG1 and Igβ in B220⁺IgM⁺CD38⁺naive B cells (top) and B220⁺CD38⁻ GC B cells (bottom). (b) Expression levels of germline transcript and surface expression of IgM and IgG1 in the GC B cell subsets. Expression of IgM germline transcript (GLT-μ), IgG1 germline transcript (GLT-γ1) and IgG1 postswitch transcript were assessed by RT-qPCR in each population, respectively (top). Cell surface expression of IgM and IgG1 in each GC subset was examined (bottom). (c) Frequency of somatic hypermutation in VH186.2 gene of GC B cell subsets. Number of nucleic acid mutations (left) and amino acid mutations (right) are shown.

Figure 4

Forced expression of Igβ in B cells impaired the antibody production and affinity maturation. (a) Expression of Igβ in B220⁺CD38⁻GL7⁻ splenocytes from immunized Igβ-Tg (Igβ-Tg; line) and wild-type litter mates (wild type; shaded). (b) Expression of CD38 and GL7 in B220⁺spleen cells from immunized wild type litter mates (top left) and Igβ-Tg mice (top right). Cell cycle analysis of CD38⁻GL7⁻ GC B cells from wild type litter mates (bottom left) and Igβ-Tg mice (bottom right). Numbers indicate the percentages of cells in each gate. (c) Serum anti-NP titers in each isotype after the immunization. Igβ-Tg (open circle) and wild type litter mates (closed circle) were immunized with NP-CGG on day 0 (1°) and boosted 42 d later (2°). Sera were collected at indicated day and assayed by ELISA as described in Methods section. Error bars indicate SEM for each group. Each group consists of 5 mice. Representative data from 3 repeated experiments is shown. (d) Affinity maturation of anti-NP IgG1 antibody in NP-CGG immunized Igβ-Tg (closed bar) and wild type litter mates (open bar). Error bars indicate SEM for each group. (e) Decreased frequency of somatic hypermutation in immunized Igβ-Tg mice. CD38⁻GL7⁺GC B cells were isolated from Igβ-Tg and wild type litter mates 21 days after the immunization. Total RNA was extracted and reverse transcribed to obtain cDNA samples. VH186.2 gene was then amplified and sequenced for the mutation analysis.

Figure 5

Impaired GC formation by inhibition of IL-21-mediated Igβ down-regulation. (a) Expression of Igβ in activated splenic B cells supplemented with IL-21. Splenocytes depleted of T cells were cultured with IL4 (10 ng/ml), and anti-CD40 antibody (1 μg/ml) supplemented with indicated concentrations of IL-21 for 6 days. Expression levels of Igβ and GL7 were analyzed by flow cytomeric analysis. The numbers under the square gate indicate the percentages of Igβ down-regulated population in the gate. Representative data from 3 experiments is shown. (b) Average percentages and SEM of Igβ low cells from repeated experiments as in (a) were shown. (c) Flow cytomtric analysis of Igβ expression in B220⁺CD38⁻GL7⁻ splenocytes from immunized wild type (shaded) and IL-21R-deficient (line) mice. (d) Flow cytometric analysis of the splenic B cells (B220⁺) from wild type litter mates (left) and IL-21R-deficient mice (right) 10 days after the immunization with NP-CGG. The numbers indicate the percentage of CD38⁻GL7⁺ cells within the gate. (e) Cell cycle analysis of B220⁺CD38⁻GL7⁻ GC B cells from immunized IL-21R-deficient mice. Splenocytes from wild type litter mates (left) and IL21R-deficient mice (right) 10 days after the immunization were stained with Hoechst 33342 dye in combination with antibodies to Igβ.

Figure 6

Amelioration of auto-reactive antibody production and proteinuria by suppression of Igβ down-regulation in BXSB-Yaa autoimmune prone mice. Igβ-Tg mice were back-crossed to BXSB-Yaa for 9 generation and obtained male mice were subjected to further analyses. Serum titers in NZB and wild type C57BL/6 mice were also measured concomitantly as positive band negative controls, respectively. (a) Percentages of Igβ-Hi and Igβ-Int B cell population in B220⁺CD38⁻GL7⁻ GC B cell subset from immunized C57BL/6 (open bars), BXSB-Yaa background wt (dark shaded bars) and Igβ-Tg (light shaded bars) mice are shown. (b) Serum titers of anti-dsDNA antibodies were measured in BXSB-Yaa background wt and Igβ-Tg mice at indicated ages. Each symbol indicate the serum levels for individual mouse. Thick lines indicate the average values and thin lines indicate SEM values. (c) Protineuria was measured at 20 weeks of age. Each circle indicate the serum levels for individual mouse. (d) Survival curve of BXSB-Yaa background wt and Igβ-Tg mice.