The tyrosine kinase Syk is required for light chain isotype exclusion but dispensable for the negative selection of B cells

Abstract

In this study we set out to test whether Syk was required for negative selection of immature B cells. B cells expressing a B cell antigen receptor (BCR) transgene (3-83, anti-H-2K k ) underwent negative selection independently of Syk in both fetal liver organ culture and radiation chimera models. Furthermore, Syk-independent negative selection was not reversed by transgenic overexpression of Bcl-2. Receptor editing was not apparent in Syk-deficient B cells, presumably as a consequence of the failure of mature edited B cells to develop in the absence of Syk. Interestingly, light chain isotype exclusion by the BCR transgene failed in the absence of Syk. We observed a dramatic reduction in the overall BCR-mediated tyrosine phosphorylation of cellular proteins in Syk-deficient immature B cells. However, the tyrosine phosphorylation of a number of substrates including phospholipase C + 2, although reduced, was not completely abrogated. BCR ligation triggered an increase in calcium flux in the absence of Syk. Thus signaling events that mediate negative selection can still occur in the absence of Syk. This may be due to redundancy with zeta-associated protein 70 (ZAP-70), which we demonstrate to be expressed in immature B cells.

B Cell Receptor Basal Signaling Regulates Antigen-Induced Ig Light Chain Rearrangements

“…B cells deficient in CD19 show strong spontaneous upregulation of Rag, loss of allelic exclusion at L chain loci, and impaired positive selection (27), suggesting that the lack of positive signaling through the BCR is associated with the induction of Rag and failure to progress in development. Similarly, immature B cells lacking the protein tyrosine kinase syk show loss of L chain allelic exclusion (28). Together, these data support the hypothesis that developing B cells require threshold signals from Ig receptors expressed on the cell surface to block further Ig gene rearrangements.…”

“…We hypothesize that the basal signal is transmitted through the following series of interacting proteins: the BCR, Ig␣␤, Syk, Lyn, Btk, and PI3K. Knockouts of syk (28), lyn (45), PI3K p85␣ (66), PI3K p110␦ (67), and btk (Fig. 1) and the inhibition of PI3K with inhibitors (29) result in a phenotype compatible with a role for basal signaling in suppressing Rag induction.…”

“…lyn appears to be involved in positive regulation of BCR signaling in immature B cells by phosphorylating ITAMs of Ig␣ and Ig␤, and lyn-deficient immature B cells show impaired allelic exclusion and developmental progression (28,45). We predicted that if positive signaling is necessary for Rag expression, then it should be impaired in lyn null /HEL-Ig/Rag2-GFP mice.…”

B lymphocytes in humans express ZAP‐70 when activated in vivo

“…Initially, ZAP-70 was detected in B lymphocytes at early maturation stages only, when, in the pro-B to pre-B cell transition, ZAP-70 participates in the transduction of signals delivered by the microenvironment through the B cell antigen receptor (BCR) [2,3].…”

The development of mature B lymphocytes requires the combined function of CD19 and the p110δ subunit of PI3K

“…28 Indeed, L-chain allelic exclusion fails when signaling components of the BCR such as the tyrosine kinases syk and lyn are missing. 43,44 Previously it has been shown that pharmacological inhibition of PI3K can lead to the induction of RAG-gene expression. 45 It remains unclear from our study whether RAG gene expression was extinguished and then reactivated or whether it failed to be shut type I FO cells were difficult to identify in p110δ -/-CD19 -/-double mutant mice; this is most easily appreciated from our analysis of lymph node B cells in the double mutant.…”