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B cell response to Anti-IgM and CD40 treatment

ABSTRACT: Temporal analysis (60, 180, 360 min) of B cells treated with Anti-IgM alone, CD40 alone or Anti-IgM and CD40 (all in triplicates). Keywords: other

ORGANISM(S): Mus musculus

PROVIDER: GSE1019 | GEO | 2004/02/13

SECONDARY ACCESSION(S): PRJNA90255

REPOSITORIES: GEO

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2010-06-09 | E-GEOD-1014 | biostudies-arrayexpress

B cell response to ELC and lipopolysaccharide treatment

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A single-round of antigen receptor signaling programs naÃ¯ve B cells to receive T cell help

Project description:To simulate transient B cell activation that is the likely initiator of T-dependent responses, we examined the molecular and functional consequences of a single-round of immunoglobulin M (IgM) signaling. This form of activation triggered early cytosolic signaling and transcription factor NF-kB activation indistinguishably from conventional continuous IgM cross-linking, but did not induce G1 progression. However, single-round IgM signaling changed the expression of chemokine and chemokine receptor genes implicated in initiating T-dependent responses, as well as accentuated responsiveness to CD40 signaling. Several features of single-round IgM signaling in vitro were recapitulated in B cells after short-term exposure to antigen in vivo. We propose that transient BCR signals prime B cells to receive T cell help by increasing the probability of B-T encounter and creating a cellular environment that is hyper-responsive to CD40 signaling. Primary B lymphocytes were isolated using Auto-MACS (Miltenyi Biotec) by negative selection. B cell purity was 90-95% based on flow cytometric analysis with CD19 staining. Purified B cells (2x10^6/ml) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 55nM beta-mercaptoethanol, 2mM L-glutamine and 100IU penicillin and 100ug/ml streptomycin at 37degrees C. For pulsed anti-IgM treatment experiments, B cells were incubated with 10ug/ml goat anti-mouse IgM F(abâ)2 (Jackson ImmunoResearch Laboratories) at 4 degrees C for 30 min. Unbound anti-IgM was removed from the medium by washing and centrifuging the cells at 4 degrees C. The cells were resuspended in chilled complete medium and shifted to 37 degrees C by placing in an incubator or in water-bath. For continuous anti-IgM treatment experiments, B cells were stimulated with 10ug/ml anti-IgM at 4 degrees C for 30 min, then incubated at 37 degrees C.

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