Dendritic cell-dependent inhibition of B cell proliferation requires CD22

Abstract

Recent studies have shown that dendritic cells (DCs) regulate B cell functions. In this study, we report that bone marrow (BM)-derived immature DCs, but not mature DCs, can inhibit BCR-induced proliferation of B cells in a contact-dependent manner. This inhibition is overcome by treatment with BAFF and is dependent on the BCR coreceptor CD22; however, it is not dependent on expression of the CD22 glycan ligand(s) produced by ST6Gal-I sialyltransferase. We found that a second CD22 ligand (CD22L) is expressed on CD11c(+) splenic and BM-derived DCs, which does not contain ST6Gal-I-generated sialic acids and which, unlike the B cell-associated CD22L, is resistant to neuraminidase treatment and sodium metaperiodate oxidation. Examination of splenic and BM B cell subsets in CD22 and ST6Gal-I knockout mice revealed that ST6Gal-I-generated B cell CD22L plays a role in splenic B cell development, whereas the maintenance of long-lived mature BM B cells depends only on CD22 and not on alpha2,6-sialic acids produced by ST6Gal-I. We propose that the two distinct CD22L have different functions. The alpha2,6-sialic acid-containing glycoprotein is important for splenic B cell subset development, whereas the DC-associated ST6Gal-I-independent CD22L may be required for the maintenance of long-lived mature B cells in the BM.

FIGURE 1

iDCs and splenic DCs, but not mDCs, inhibit BCR-induced proliferation of B cells. Purified CFSE-labeled B cells from WT mice were cocultured with either WT BM-derived iDCs (A), DCs purified ex vivo from spleens (B), or BM-derived mDCs (C) and the indicated graded doses of anti-IgM. CFSE dilution of the B cells was examined 72 h postincubation by flow cytometry. For the BM-derived iDCs, two different B cell:DC ratios were examined (4:1 and 16:1). The histograms shown in A and C are from cultures where the B cell:DC ratio was 4:1. Results from both ratios are shown in graph format. For the splenic DCs (B), four different B cell:DC ratios were used; the percentage of dividing cells in the cultures are indicated in the graph. These results with the BM-derived iDCs are representative of at least eight independent experiments and results with the splenic DCs are representative of three independent experiments. D, B cells and iDCs were cultured in the presence of the indicated doses of BAFF and 1 μg/ml anti-IgM at different B cell:DC ratios (32:1, 24:1, and 16:1). CFSE dilution was examined after 48 and 72 h of BCR cross-linking and the percentage of dividing cells in the cultures are shown. The results from the B cell:DC ratios of 24:1 are shown. These results are representative of three independent experiments.

FIGURE 2

iDC-mediated inhibition of WT B cells is contact dependent. Purified CFSE-labeled B cells from WT mice were cocultured with WT BM-derived iDCs and the indicated concentrations of anti-IgM (“contact”). In some of the cocultures, the DCs and B cells were physically separated using Transwells with a pore size of 0.4 μm. CFSE dilution of the B cells was examined 72 h postincubation by flow cytometry. Two different ratios of B cells:DCs were examined (4:1 and 16:1). These results are representative of six independent experiments.

FIGURE 3

iDC-mediated inhibition of B cell proliferation requires CD22, but not ST6Gal-I sialyltransferase. A, Purified CFSE-labeled B cells from CD22-deficient mice were cocultured together (“contact”) or in Transwells with WT BM-derived iDCs and graded doses of anti-IgM (“contact”). B, Purified CFSE-labeled B cells from WT mice were cocultured with ST6Gal-I-deficient BM-derived iDCs and graded doses of anti-IgM. The same method described in Fig. 2 was used. These results are representative of at least three independent experiments.

FIGURE 4

Soluble CD22 binds to WT and ST6GalI KO DCs. A-C, The binding of purified human IgG, Fc fragment (shaded), CD22 Rg1-7 (bold solid line), or CD22 Rg3-7 (dotted thin line) to the surface of WT and ST6Gal-I-deficient BM-derived iDCs (A), B cells (B), and WT splenic DCs (C) was quantified by flow cytometry. D, The binding of human IgG, Fc fragment (shaded), and CD22 Rg1-7 on WT (thick line) and ST6Gal-I KO (thin line) BM cells was examined by flow cytometry. The cells were stained with purified control or fusion proteins along with anti-human IgG-PE secondary and other surface markers. In the histograms shown, the cells were gated on CD11c⁺ cells (A), CD19⁺ cells (B), CD11c⁺B220^-CD8α⁺ (C), or CD11c⁺B220^-CD8α^- cells, and CD11c⁺ or CD11c^- cells (D). The results shown are representative of three independent experiments.

FIGURE 5

Cleavage of sialic acids does not disrupt CD22 binding on DCs. A, The binding of the purified chimeric CD22 fusion proteins to the surface of WT DCs and B cells following cleavage of α2,6-linked sialic acids (A) or to BM cells following sodium metaperiodate oxidation (B) was quantified by flow cytometry. A, Arthrobacter ureafaciens neuraminidase-treated cells were stained with the indicated fusion proteins or control human IgG (Fc fragment) along with anti-human IgG-PE secondary and anti-CD19-FITC (for the splenocytes) or anti-CD11c-FITC (for the iDCs). In the histograms shown, the cells were gated on CD19⁺ (B cells) or CD11c⁺ (iDCs). The neuraminidase-treated human IgG control was similar to the untreated sample shown. B, BM cells treated by sodium metaperiodate oxidation were stained with anti-CD11c-FITC and control or CD22 fusion proteins as indicated. In the histograms shown, the cells were gated on CD11c⁺ cells. The mean fluorescence intensity of the histograms for the experiment shown above (n = 4 mice) are as follow: untreated CD22 Rg1-7, 198 ± 4.2; untreated CD22 Rg3-7, 37 ± 3.5; treated CD22 Rg1-7, 243 ± 35; and treated CD22 Rg3-7, 50 ± 5.4. The results shown are representative of four experiments.

FIGURE 6

CD22 and ST6Gal-I sialyltransferase are required for development of MZ and T2 precursor B cells, but only CD22 is required for the maintenance of B cells in the BM. A, Total BM was collected from WT and ST6Gal-I KO mice and analyzed for surface expression of B220 and IgM by flow cytometry. The percentage of B220⁺IgM^high cells is shown. B, Total splenocytes were isolated from WT, CD22, and ST6Gal-I KO mice. The splenocytes were analyzed for surface expression of IgM, IgD, and CD21 by flow cytometric analysis. The cell numbers or percentage of each B cell subset is shown in the graphs. Data represent mean ± SD. n, More than three mice per genotype. C, CD22 expression in the B cell subsets. WT splenocytes were stained with anti-IgM, anti-IgD, anti-CD21, and anti-CD22 and analyzed by flow cytometry. The graph shows the average mean fluorescence intensity of CD22 expression for each B cell subset. n = 9 mice. *, p < 0.05; and **, p < 0.01.