Abstract
Signals from the BCR are required for Ag-specific B cell recruitment into the immune response. Binding of Ag to the BCR induces phosphorylation of immune receptor tyrosine-based activation motifs in the cytoplasmic domains of the CD79a and CD79b signaling subunits, which subsequently bind and activate the Syk protein tyrosine kinase. Earlier work with the DT40 chicken B cell leukemia cell line showed that Syk was required to transduce BCR signals to proximal activation events, suggesting that Syk also plays an important role in the activation and differentiation of primary B cells during an immune response. In this study, we show that Syk-deficient primary mouse B cells have a severe defect in BCR-induced activation, proliferation, and survival. Furthermore, we demonstrate that Syk is required for both T-dependent and T-independent Ab responses, and that this requirement is B cell intrinsic. In the absence of Syk, Ag fails to induce differentiation of naive B cells into germinal center B cells and plasma cells. Finally, we show that the survival of existing memory B cells is dependent on Syk. These experiments demonstrate that Syk plays a critical role in multiple aspects of B cell Ab responses.
Highlights
We found that restricted loss of Syk in B cells resulted in a large decrease in Ag-specific IgM and IgG1 in the serum (Fig. 3C), and a large reduction in the numbers of Agspecific IgG1-expressing B cells and germinal center B cells, as well as a reduction in Ab-secreting cells producing Ag-specific IgM or IgG1 (Fig. 3D)
Our results establish that Syk is a key signal transducer that is required for coupling the BCR to all downstream signaling pathways in mature B cells
We have shown previously that survival signals from BAFFR are partially transduced via the BCR to the activation of Syk [13]
Summary
Mice carrying a conditional allele of Syk (Syktm1.1Nns, Sykfl), a constitutive disruption of Syk (Syktm1Tyb, Syk2), an allele of ROSA26 expressing the MerCreMer fusion protein (Gt(ROSA)26Sortm1(cre/Esr1*)Nns, Rosa26MerCreMer, RMCM), disruption of the IgM gene (Ighmtm1Cgn, mMT), and a disruption of Rag (Rag1tm1Mom) have been described previously [10, 13, 14]. Single-cell suspensions of splenocytes were treated with ACK lysis buffer to remove RBCs before staining in PBS, containing LIVE/DEAD fixable nearIR dead cell stain (Life Technologies) and appropriate, pretitered Abs. Abs used, indicating Ag and fluorophore (and clone): CD69-FITC (H1.2F3), CD86-PE (GL-1), streptavidin-PerCP (Becton Dickinson), MHC class II-bio (I-Ab; M5/114.15.2), IgM-PECy7 (II/41), B220-eFluor450 (RA3-6B2; eBioscience), CD19-FITC, B220-PerC, IgM-PE, IgG1-APC, PNA-FITC, CD38-PE/Cy7, CD45.2-ef450, CD45.1-FITC, CD138-PE, IgD-ef450, CD4-FITC, CD8-FITC, CD45.2-PerCP-cy5.5, CD45.1-FITC, B220BV605, CD4-BV650, CD8a-BV650, CD38-bio, Streptavidin-PacO, FAS-PE. To induce primary responses 2.5 3 104 HEL-binding B cells from tamoxifentreated SWHEL/Sykfl/fl or SWHEL/Sykfl/flRMCM mice were transferred into B6.SJL recipient mice together with 5 3 108 HEL-conjugated sheep RBCs (SRBC). To generate a memory pool, splenocytes containing an equivalent of 5 3 105 HEL-binding B cells from SWHEL/Sykfl/+RMCM or SWHEL/ Sykfl/flRMCM mice were transferred into (B6 x B6.SJL)F1 recipient mice together with 1 3 109 HEL33-conjugated SRBC.
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