To investigate this CD22-dependent inhibition in a more quantitative manner, WT or CD22?/? IgMHEL B cells were co-cultured with different numbers of mHEL-RBCs (2103 C 4107) (inserted graphs on Physique 3B,C). reasoned that introduction of CD22 ligands in RBCs should abolish B cell activation toward its cognate antigen on the surface of RBCs. Accordingly, we employed a glyco-engineering approach wherein synthetic CD22 ligands linked to lipids are inserted into the membrane of RBCs. Indeed, insertion of CD22 ligands into RBC cell surface strongly inhibited B cell activation, cytokine secretion, and proliferation. These results demonstrate that the lack of Siglec ligands on the surface of murine RBCs permits B cell responses to erythrocyte antigens, and shows that Siglec-mediated B cell tolerance is restricted to cell types that express glycan ligands for the B cell Siglecs. and for 35 min at 22 C. Percoll gradients were prepared after osmolality adjustment of Percoll PLUS by adding 9 parts of Percoll PLUS to 1 1 a part of PBS 10x. Mononuclear and polymorphonuclear cells accumulated at the top of the 65% layer and at the interface between the 72% and 65% layers, respectively, and were discarded, RBCs pelleted within the 72% layer. Cells were washed twice with HBSS and >99% of the cells were erythrocytes as determined by a CD45?Ter-119+ staining pattern by flow cytometry (FACS) (Figure 1A). FACS analysis of isolated RBCs with anti-CD41, a marker of mouse platelets, marker) showed that this RBCs did not have bound platelets (>0.03% of CD41+Ter-119+ cells, data not shown). FACS data were obtained on an LSR-II (BD Biosciences) and analyzed using FlowJo software. Open in a separate window Agt Physique 1 FACS analysis reveals MAA-II binding to erythrocytes surface, but not SNA, mCD22-Fc or Siglec-10-FcIsolated mouse red blood cells (RBCs) or total splenocytes were stained with SNA-FITC, biotinylated MAA-II, mCD22-Fc plus anti-human IgG1-APC or Siglec-10-Fc plus anti-human IgG1-APC. RBCs were also stained with TER 119-PE and CD45.2-FITC and splenocytes with CD19-PE. (A) Contour plots of SSC vs FSC and TER 119-PE vs CD45.2-FITC of RBCs. (B) Contour plots of SSC vs FSC and CD19-PE vs SSC-H of splenocytes. (C) Representative histograms of WT RBCs (TER 119+/CD45.2?) and WT B cells (CD19+) are showing the staining profiles for lectins SNA (black line), MAA-II (gray line) or unstained cells (filed gray lines). (D) Representative histograms of WT RBCs and WT B cells of unstained cells (filed gray lines), stained with mCD22-Fc (gray line) or Siglec-10-Fc (black line). (E) RBCs or B cells from WT (black lines) or ST6Gal1?/? (gray lines) mice were stained with mCD22-Fc; filed gray line represent unstained cells. SNA = MAA-II = II. These experiments were independently performed 3 times in triplicate. Probing cells with lectins and Siglecs-Fc Total splenocytes or isolated 1M7 RBCs were re-suspended in phosphate-buffered saline (PBS) with calcium and magnesium (Gibco) and incubated with mouse Fc-block (anti-CD16/32, Biolegend) for 30 min at 4 C, washed and stained for 30 min, 4 C with FITC conjugated lectin (SNA-I) from elderberry bark (Vector Laboratories), biotinylated lectin II (MAA-II) (Vector Laboratories), mCD22-Fc or Siglec-10-Fc. Cells were washed twice with PBS (made up of Ca+2/Mg+) and incubated with Ter-119-PE (Biolegend) and anti-human IgG1-APC (Jackson ImmunoResearch) or streptavidin-FITC. For staining of the B cells, CD19-PE (Biolegend) and anti-human IgG1-APC or 1M7 streptavidin-FITC (Biolegend) were used. Culture of Chinese hamster ovary (CHO) cells expressing mCD22-Fc or Siglec-10-FC Expression of mCD22-Fc or Siglec-10-Fc from stably transfected CHO cells were described previously (22C24). Briefly, CHO cells were maintained in Dulbeccos altered 1M7 Eagles medium/F-12 medium supplemented with 10% fetal bovine serum and 500 g/mL hygromycin B (Roche Applied Science). Supernatants of CHO cells were collected after approximately 10 days of culture, and were used in FACS assays. Insertion of high affinity CD22 ligand into RBCs The preparation of the high affinity CD22 ligand (6BPANeu5Gc-PEG-DSPE) has been described previously (1, 4). 6BPANeu5Gc-PEG-DSPE (BPANeu5Gc) or PEG-DSPE (PEG, as a control) were incubated with RBCs (1107 cells/mL) in HBSS buffer.