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Transcriptomic analysis of B cells from patients with chronic lymphocytic leukemia

ABSTRACT: CD22 (also known as Siglec-2) belongs to the Siglec family of glycan-recognition proteins and negatively regulates B-cell receptor-mediated signaling. CD22 is down-regulated in the B cells of patients with chronic lymphocytic leukemia (CLL). However, the mechanism regulating the transcription of CD22 is incompletely understood. In this study, we defined the minimal promoter of the human CD22 gene and identified the transcription factors that bind to the CD22 promoter by proteomic analysis. This transcriptome dataset (by RNAseq of peripheral blood B cells from 6 patients with CLL) was used (1) to validate the positive correlation between the expression levels of CD22 transcript (assessed by RNAseq) and CD22 protein (assessed by flow cytometry), and (2) to test the correlations between the expression levels of CD22 transcript and those of transcription factors that bind to minimal CD22 promoter.

ORGANISM(S): Homo sapiens

PROVIDER: GSE273421 | GEO | 2025/03/24

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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Transcriptomic analysis of B cells from patients with chronic lymphocytic leukemia

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