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Transcriptome analysis of mouse WT and CD22 KO GC B cells

ABSTRACT: Germinal centres (GC) are specialized sites where B cells expand and diversify their antibody genes through somatic hypermutation. GC B-cells are routinely identified through distinct changes on their surface carbohydrates, known as glycans. One striking modification relates to the monosaccharide sialic acid. In mice, this change is mediated through downregulation of an enzyme called CMAH, which results in a GC-specific loss of preferred ligand for CD22, a member of the sialic acid-binding immunoglobulin-type lectins (Siglecs) and an inhibitory co-receptor of the B-cell antigen receptor (BCR). Here, we identified that glycan remodeling, mediated by downregulation of CMAH, is crucial for the GC B-cell response, and production of memory B-cells, plasma cells, and high affinity antibodies. We also demonstrated that the function of these altered glycans is dependent on CD22, highlighting that coordinated loss of preferred ligands acts to modulates the CD22 activity in the GC B-cells. Overall, our study reveals that intrinsic glycan remodeling functions to optimize the B-cell responses in the GC by controlling CD22.

ORGANISM(S): Mus musculus

PROVIDER: GSE196206 | GEO | 2022/02/09

REPOSITORIES: GEO

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Project description:Our goal is to understand how glycosylation on human B cells is regulated during the differentiation and activation. Accumulating evidences have indicated that functions of immune cells are regulated in a glycan-dependent manner. One of well-known examples is B cell regulation by CD22 through its association with sialylated glycans. While recent studies uncovered that B cell activation leads to loss of high affinity glycan ligands for human CD22 (Neu5Ac-a2,6-Gal-B1,4-GlcNAc(6-sulfo)), the gene expression profile of human B cells before and after activation is unknown. Therefore we would like to perform gene expression analysis of human B cells before and after activation. We already have published the glycan profiling and gene expression of mouse B cells before and after activation done in collaboration with the CFG. This study revealed programmed changes in glycosylation relevant to regulation of B cell signaling by CD22. Since the ligands of human CD22 differ in several respects from that of mouse CD22, these experiments are relevant to the evolution of siglec ligands and their conserved functions in B cell biology. In brief, the experimental protocol is as follows: human peripheral B cells are isolated from human peripheral blood of healthy donors and stimulated for 0, 24, 48 and 72 hrs. Human B cells were isolated from human peripheral blood of healthy donors by the negative selection kit (Miltenyi). For the resting B cell samples, isolated B cells were immediately shock-frozen in the liquid N2 and kept at -80˚C until the RNA extraction. For the activated B cell samples, the cells were activated in the RPMI medium (Invitrogen) supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 1 mM non-essential amino acid, 1 mM sodium pyruvate, and 50 M 2-melcaptoethanol, 10 g/mL anti-human IgA, G, M (Jackson Immunoresearch), and 50 nM CpG ODN 2006 (Invivogen) for 3 days. Total RNA was isolated using the TRIzol reagent (Invitrogen).

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Transcriptome analysis of mouse WT and CD22 KO GC B cells

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