Thomas Wossning

Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling

B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.

Amplification of B Cell Antigen Receptor Signaling by a Syk/ITAM Positive Feedback Loop

We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first tyrosine whereas Syk phosphorylates both tyrosines of the BCR immunoreceptor tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive allosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein tyrosine phosphatases, the activity of which is regulated by H(2)O(2) and the redox equilibrium inside the cell.

N-linked glycosylation selectively regulates autonomous precursor BCR function

Developing B cells express distinct classes of B cell antigen receptors (BCRs) that differ in their heavy chain (HC). Although only μHC is expressed in early stages, δHC-containing BCRs dominate on the surface of mature B cells. The reason for the tightly regulated expression of these receptors is poorly understood. Here we show that μHC was specifically required for precursor BCR (pre-BCR) function and that δHC was unable to form a functional pre-BCR. A conserved asparagine (N)-linked glycosylation site at position 46 (N46) in the first conserved domain of μHC was absolutely required for pre-BCR function, and swapping that domain with δHC resulted in a functional δHC-containing pre-BCR. When tested in the context of the BCR, μHC with a mutant N46 showed normal function, which indicated that N46-glycosylation is specifically required for pre-BCR function. Our results suggest an unexpected mode of pre-BCR function, in which binding of the surrogate light chain to N46 mediates autonomous crosslinking and, concomitantly, receptor formation.

Identification of a Pre-BCR Lacking Surrogate Light Chain

SLP-65−/− pre-B cells show a high proliferation rate in vitro. We have shown previously that λ5 expression and consequently a conventional pre-B cell receptor (pre-BCR) are essential for this proliferation. Here, we show that pre-B cells express a novel receptor complex that contains a μ heavy chain (μHC) but lacks any surrogate (SL) or conventional light chain (LC). This SL-deficient pre-BCR (SL−pre-BCR) requires Ig-α for expression on the cell surface. Anti-μ treatment of pre-B cells expressing the SL−pre-BCR induces tyrosine phosphorylation of substrate proteins and a strong calcium (Ca2+) release. Further, the expression of the SL−pre-BCR is associated with a high differentiation rate toward κLC-positive cells. Given that B cell development is only partially blocked and allelic exclusion is unaffected in SL-deficient mice, we propose that the SL−pre-BCR is involved in these processes and therefore shares important functions with the conventional pre-BCR.

FoxO1 induces Ikaros splicing to promote immunoglobulin gene recombination

During murine B cell development, PI3 kinase inhibits Ig gene rearrangement by suppressing FoxO1, which mediates Ikaros mRNA splicing; Ikaros is needed for Ig gene recombination.

Responsiveness of B cells is regulated by the hinge region of IgD

Mature B cells express immunoglobulin M (IgM)- and IgD-isotype B cell antigen receptors, but the importance of IgD for B cell function has been unclear. By using a cellular in vitro system and corresponding mouse models, we found that antigens with low valence activated IgM receptors but failed to trigger IgD signaling, whereas polyvalent antigens activated both receptor types. Investigations of the molecular mechanism showed that deletion of the IgD-specific hinge region rendered IgD responsive to monovalent antigen, whereas transferring the hinge to IgM resulted in responsiveness only to polyvalent antigen. Our data suggest that the increased IgD/IgM ratio on conventional B-2 cells is important for preferential immune responses to antigens in immune complexes, and that the increased IgM expression on B-1 cells is essential for B-1 cell homeostasis and function.

The BTG2-PRMT1 module limits pre-B cell expansion by regulating the CDK4-Cyclin-D3 complex

Developing pre-B cells in the bone marrow alternate between proliferation and differentiation phases. We found that protein arginine methyl transferase 1 (PRMT1) and B cell translocation gene 2 (BTG2) are critical components of the pre-B cell differentiation program. The BTG2-PRMT1 module induced a cell-cycle arrest of pre-B cells that was accompanied by re-expression of Rag1 and Rag2 and the onset of immunoglobulin light chain gene rearrangements. We found that PRMT1 methylated cyclin-dependent kinase 4 (CDK4), thereby preventing the formation of a CDK4-Cyclin-D3 complex and cell cycle progression. Moreover, BTG2 in concert with PRMT1 efficiently blocked the proliferation of BCR-ABL1-transformed pre-B cells in vitro and in vivo. Our results identify a key molecular mechanism by which the BTG2-PRMT1 module regulates pre-B cell differentiation and inhibits pre-B cell leukemogenesis.

Deregulated Syk inhibits differentiation and induces growth factor–independent proliferation of pre–B cells

Wossning et al. 2006. J. Exp. Med. doi:10.1084/jem.20060967[OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20060967%26rft_id%253Dinfo%253Apmid%252F17130299%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%