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. 2009 Mar 10;106(10):3895-900.
doi: 10.1073/pnas.0809736106. Epub 2009 Feb 19.

CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche

Stéphane Chevrier  1 , Céline Genton, Axel Kallies, Alexander Karnowski, Luc A Otten, Bernard Malissen, Marie Malissen, Marina Botto, Lynn M Corcoran, Stephen L Nutt, Hans Acha-Orbea
Affiliations

CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche

Stéphane Chevrier et al. Proc Natl Acad Sci U S A. .

Abstract

Plasma cells represent the end stage of B-cell development and play a key role in providing an efficient antibody response, but they are also involved in numerous pathologies. Here we show that CD93, a receptor expressed during early B-cell development, is reinduced during plasma-cell differentiation. High CD93/CD138 expression was restricted to antibody-secreting cells both in T-dependent and T-independent responses as naive, memory, and germinal-center B cells remained CD93-negative. CD93 was expressed on (pre)plasmablasts/plasma cells, including long-lived plasma cells that showed decreased cell cycle activity, high levels of isotype-switched Ig secretion, and modification of the transcriptional network. T-independent and T-dependent stimuli led to re-expression of CD93 via 2 pathways, either before or after CD138 or Blimp-1 expression. Strikingly, while humoral immune responses initially proceeded normally, CD93-deficient mice were unable to maintain antibody secretion and bone-marrow plasma-cell numbers, demonstrating that CD93 is important for the maintenance of plasma cells in bone marrow niches.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CD93 is expressed on ASC after MMTV and NP-CGG immunizations. (A) Plasmablasts and B cells in the draining lymph nodes of MMTV-immunized and control BALB/c mice. Numbers indicate the percentages of cells. (B) FACS analyses of lymph node cells of C57BL/6 mice at different time points after footpad immunization with 50 μg of NP-CGG in alum. NP-intracellular+, B220int cells (ASC) were analyzed for CD93 and CD138 expression. (C) FACS analysis 40 days after boost immunization of BALB/c mice with ovalbumin (OVA) protein. Mice were fed with BrdU containing water for 20 days before analysis. LLPC in the BM were detected by intracellular staining for OVA-specific antibodies and low BrdU incorporation. (D) Expression of CD93 and surface NP-specific Ab was assessed on GL7+PNA+ B cells 14 days after NP-CGG immunization. (E) Memory B cells, defined as B220+ NP-surface+, present 30 days after boost were analyzed by FACS for the expression of CD93. (F) FACS analyses of splenic cells before and 5 days after i.v. immunization with 30 μg of NP-Ficoll. B220-NP-intracellular+ ASC were analyzed for the expression of CD93 and CD138.
Fig. 2.
Fig. 2.
Characteristics of the 4 B-cell and ASC subpopulations defined by the expression of CD138 and CD93. (A) Six days after MMTV immunization, plasmablasts from draining lymph nodes were sorted by FACS based on expression of B220, CD138, and CD93 expression and recultured for 24 h. Only live cells defined as DAPI– Annexin V– were included in the FACS analysis. (B) MMTV-immunized mice (6 days old) were pulsed i.p. with 300 μg of BrdU and incorporation was assessed in the different populations present in the draining lymph nodes after 2 or 12 h; mean ± SD of 3 independent experiments. **, P < 0.01, CD138 SP compared with DP subset by Student's t test. (C) The 4 subsets of FACS-purified plasmablasts were fixed and stained with anti-IgG2a (brown). Nuclei were counterstained with Mayer's Hematoxylin (blue). Data are representative of 3 independent experiments. (D) FACS-purified populations were analyzed by ELISPOT for IgM- and IgG2a-secreting cells. (E) mRNA levels of Bcl6, Pax5, Blimp-1, Irf-4, and Xbp-1 in the 4 FACS-purified subpopulations were investigated by real-time RT-PCR. Expression was normalized to Pol2A and Pol2G. Data are represented as mean ± SD of triplicate samples. The experiment was performed twice with similar results.
Fig. 3.
Fig. 3.
CD93 is dispensable for ASC differentiation in vitro and for early stages of B-cell responses after TD immunization in vivo. (A) MACS-purified splenic B cells from Blimp-1gfp/+ and CD93−/− Blimp-1gfp/+ mice were activated in vitro with LPS or anti-CD40 + IL-4/IL-5 for 5 days and analyzed by flow cytometry. (B) CD93-deficient and control mice were immunized with MMTV and the frequency of B220–MHCIIintCD138+ ASC was determined in lymph nodes by FACS. Bars are the means ± SD of 3 independent samples. (C) FACS analysis of one representative experiment from (B). (D) CD93–/– and WT mice were immunized with NP-CGG. The percentages of B220+PNA+GL-7+ germinal center cells in the draining lymph node were investigated by flow cytometry before and 7 days after immunization. (E) The frequency of NP-intracellular+B220int ASC was analyzed in the same organs. Data are representative of 3 independent experiments.
Fig. 4.
Fig. 4.
CD93 expression is required for the maintenance of LLPC in the BM. (A) ELISA of total and high affinity NP-specific IgG1 in the serum of CD93-deficient and WT mice after NP-CGG immunization and boost. Data are the mean ± SD of 12 mice. Three additional experiments (n = 9) gave similar results. (B) Mice were immunized with NP-Ficoll and the concentration of IgM and IgG3 NP-specific Ig level was monitored by ELISA. (C) CD93-deficient and control mice were immunized and boosted 30 days later with NP-CGG. The frequency of high affinity and total NP-specific ASC present in the BM was monitored by ELISPOT at day 30. Data are the mean of 3 mice. (D) Transfer of WT or CD93-deficient splenocytes into WT naive recipient 6 days after boost immunization. Level of NP-specific IgG1 monitored by ELISA in the sera at different time points after transfer of WT cells (black circles) or CD93–/– cells (open circles) into WT recipient. (E) Number of NP-specific ASC present in spleen and BM day 1 and 36 after transfer of WT cells (black bar) or CD93-deficient cells (open bar) as detected by ELISPOT. Results were pooled from 3 mice for each condition. (F) Lethally irradiated WT (Ly5.1) mice were reconstituted with a 1:1 mixture of CD45.1 WT and CD45.2 CD93–/– bone marrow. These chimeric mice were immunized and boosted with NP-CGG as described above. Thirty days later the ratio of cells from WT and CD93–/– origin in total BM or in NP-specific cells was investigated. *, P < 0.05, CD93 compared with WT mice by Student's t test. When the normality test failed, a Mann-Whitney Rank Sum Test was performed.
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