To understand how glycosylation on human B cells is regulated during differentiation and activation

ABSTRACT: 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).

ORGANISM(S): Homo sapiens

SUBMITTER: Steven Head 

PROVIDER: E-GEOD-31932 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress