Complement receptors regulate differentiation of bone marrow plasma cell precursors expressing transcription factors Blimp-1 and XBP-1

Abstract

Humoral immune responses are thought to be enhanced by complement-mediated recruitment of the CD21-CD19-CD81 coreceptor complex into the B cell antigen receptor (BCR) complex, which lowers the threshold of B cell activation and increases the survival and proliferative capacity of responding B cells. To investigate the role of the CD21-CD35 complement receptors in the generation of B cell memory, we analyzed the response against viral particles derived from the bacteriophage Qbeta in mice deficient in CD21-CD35 (Cr2(-/-)). Despite highly efficient induction of early antibody responses and germinal center (GC) reactions to immunization with Qbeta, Cr2(-/-) mice exhibited impaired antibody persistence paralleled by a strongly reduced development of bone marrow plasma cells. Surprisingly, antigen-specific memory B cells were essentially normal in these mice. In the absence of CD21-mediated costimulation, Qbeta-specific post-GC B cells failed to induce the transcriptional regulators Blimp-1 and XBP-1 driving plasma cell differentiation, and the antiapoptotic protein Bcl-2, which resulted in failure to generate the precursor population of long-lived plasma cells residing in the bone marrow. These results suggest that complement receptors maintain antibody responses by delivery of differentiation and survival signals to precursors of bone marrow plasma cells.

Figure 1.

Maintenance of anti-Qβ antibody titers and generation of BM ASCs are impaired in Cr2^−/− mice. (A) C57BL/6 and Cr2^−/− mice were immunized i.v. with 10 μg Qβ and Qβ-specific serum IgG titers were determined by ELISA. (B) Frequencies of Qβ-specific IgG ASCs in spleen and BM of Cr2^−/− and WT mice were determined by ELISPOT assay. Values are given as the mean ± SEM, with significant differences between means indicated by asterisks (*, P < 0.01; **, P < 0.05). The analysis was repeated twice on days 8, 21, and 100 with a similar result.

Figure 2.

Induction of memory B cells is normal in Cr2^−/− mice. (A) Representative staining of splenocytes from naive and immunized mice to identify Qβ-specific (IgM; IgD; CD4; CD8; CD11b; Gr-1; YO-PRO-1)⁻B220⁺ cells. PNA binding on Qβ-specific isotype-switched B cells is shown. (B) Frequency of Qβ-specific isotype-switched B cells in spleens of Cr2^−/− and WT mice. The analysis was repeated twice on days 8, 21, and 100 with a similar result. (C) Anti-Qβ antibody levels induced in irradiated WT mice 6 d after immunization and adoptive transfer of Qβ-binding (IgM; IgD; CD4; CD8; CD11b; Gr-1; YO-PRO-1)⁻B220⁺ B cells, total (IgM; IgD; CD4; CD8; CD11b; Gr-1; YO-PRO-1)⁻B220⁺ B cells, or B220⁺IgM⁺IgD⁺ B cells. Results are expressed as serum IgG ELISA titers per transferred cells. (D) Qβ-specific serum IgG levels in Cr2^−/− and WT mice 6 mo after primary immunization and 6 d after secondary challenge with Qβ. (E) B cell responses induced in Cr2^−/− and WT mice by injection of 25 μg AP205. Frequencies of AP205-specific IgG ASCs in spleen and BM and of AP205-specific memory B cells in spleen were determined 9 wk after immunization. AP205-specific serum IgG was measured 13 wk after primary immunization and 6 d after antigen recall. All data represent the mean ± SEM, mean values statistically different from WT levels are indicated by asterisks (*, P < 0.01; **, P < 0.05).

Figure 3.

Immunization with Qβ induces efficient GC formation in Cr2^−/− mice. (A) Frequency of Qβ-specific PNA^high B cells in spleens of Cr2^−/− and WT mice. Isotype-switched Qβ-binding PNA^high B cells were identified as shown in Fig. 2 A. Data are expressed as the mean ± SEM, mean values statistically different from WT levels are indicated by asterisks (*, P < 0.01; **, P < 0.05). (B) Immunohistochemical detection of Qβ-specific B cells and PNA-binding cells in serial sections of the spleen of Cr2^−/− and WT mice 12 and 21 d after immunization. Original magnification: ×62.5 (day 12); ×75 (day 21).

Figure 4.

Qβ-specific isotype-switched PNA^low B cells from Cr2^−/− mice exhibit reduced levels of Blimp-1, XBP-1, and Bcl-2 mRNA. Qβ-binding isotype-switched PNA^high and PNA^low B cells, identified as shown in Fig. 2 A, were purified by FACS from three to four pooled spleens 12 d after immunization. Blimp-1, XBP-1, and Bcl-2 mRNA levels were determined by quantitative RT-PCR. (A) Expression of Blimp-1 and XBP-1 in purified cells from Cr2^−/− and WT mice. (B) Blimp-1 and XBP-1 expression in Qβ-specific B cells from chimeric mice having Cr2^−/− B cells and WT FDCs and from control chimeras. 5 × 10⁷ splenocytes from Cr2^−/− or C57BL/6 mice were adoptively transferred into sublethally irradiated C57BL/6-CD45.1 recipients and mRNA levels were determined in CD45.1⁻ B cells from immunized recipient mice. (C) Bcl-2 mRNA levels in sorted cells from Cr2^−/− and WT mice. Expression levels are depicted in relation to β-actin expression. Quantitative RT-PCR of each sample was performed in triplicate. Results are represented as the mean ± SD. One of two similar experiments is shown.

Figure 5.

B220^highPNA^low B cells specific for Qβ are not secreting antibody. (A) Splenocytes from naive and immunized (day 12) WT mice were permeabilized and intracellular expression of Qβ-specific antibodies was detected with Alexa 647-labeled Qβ. (CD4; CD8; CD11b)⁻B220^high and B220^low cells were gated and analyzed for PNA binding. Mean percentages of Qβ-specific plasma cells are indicated. Surface staining was blocked by preincubation with unlabeled Qβ; the specificity of the staining was controlled with Alexa 647-conjugated AP205. (B) Frequency of Qβ-specific B220^lowPNA^low plasma cells in spleen of Cr2^−/− and WT mice on day 12 after immunization. Values are given as the mean ± SEM.

Figure 6.

Qβ-specific isotype-switched PNA^low B cells exhibiting a partial plasma cell phenotype are absent in Cr2^−/− mice. (A) Expression of CD138, VLA-4, LFA-1, and CD44 on Qβ-specific PNA^low and PNA^high B cells from WT mice. B220⁺ splenocytes were purified by magnetic cell sorting; IgM^lowIgD^low B cells binding Qβ and low or high levels of PNA were gated and analyzed for expression of the indicated surface markers. One of three similar experiments is shown. (B) Comparison of CD138, VLA-4, LFA-1, and CD44 expression on Qβ-specific PNA^low and PNA^high isotype-switched B220⁺ splenocytes from Cr2^−/− and WT mice on day 12 after immunization. (C) Phenotype of Qβ-specific B cells in the blood of WT mice on day 12 after immunization. PNA-binding on Qβ-specific (IgM; IgD; CD4; CD8; CD11b; Gr-1; YO-PRO-1)⁻B220⁺ cells was determined. Expression of cytoplasmic Ig in Qβ-specific PNA^low B cells was assessed by analysis of binding of Alexa647-labeled Qβ to permeabilized (CD4; CD8; CD11b)⁻B220^low cells.

Figure 7.

Preplasma memory B cells are induced normally in Cr2^−/− mice. (A) Analysis of B220 and CD138 expression on Qβ-binding (IgD; CD4; CD8; YO-PRO-1)⁻ splenocytes to identify B220⁻CD138⁻ preplasma memory B cells. Mean percentages of B220^-CD138⁻, B220⁺CD138⁻, and CD138⁺ cells in Cr2^−/− and WT mice are indicated. (B) Frequency of Qβ-specific preplasma memory B cells in spleens of Cr2^−/− and WT mice 12 and 21 d after immunization. Results are expressed as the mean ± SEM.

Figure 8.

Isotype-switched Qβ-specific PNA^low B cells are reduced in the absence of GC formation. (A) Frequency of total, PNA^high and PNA^low Qβ-binding isotype-switched B220⁺ cells in spleens of TNFR1^−/− and WT mice 12 d after immunization. (B) Frequency of splenic B220^low plasma cells with cytoplasmic Ig specific for Qβ in immunized TNFR1^−/− and WT mice. (C) Levels of anti-Qβ serum IgG antibodies induced in TNFR1^−/− and WT mice by immunization with Qβ. Mice were boosted on day 55 after primary immunization for analysis of recall responses. All data represent the mean ± SEM, mean values statistically different from WT levels are indicated by asterisks (*, P < 0.01; **, P < 0.05).

Figure 9.

Short-term trapping of Qβ particles is efficient but long-term antigen retention is reduced in Cr2^−/− mice. Histological staining of Qβ antigen on spleen sections from WT and Cr2^−/− mice on days 0, 12, and 21 after immunization with 100 μg Qβ. Original magnification: ×50 (B6, day 0); ×75 (Cr2^−/−, day 0); ×69 (day 12); ×82.5 (day 21).