Bruton’s tyrosine kinase (Btk) and the adapter protein SLP-65 (Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa) transmit precursor BCR (pre-BCR) signals that are essential for efficient developmental progression of large cycling into small resting pre-B cells. We show that Btk- and SLP-65-deficient pre-B cells have a specific defect in Ig λ L chain germline transcription. In Btk/SLP-65 double-deficient pre-B cells, both κ and λ germline transcripts are severely reduced. Although these observations point to an important role for Btk and SLP-65 in the initiation of L chain gene rearrangement, the possibility remained that these signaling molecules are only required for termination of pre-B cell proliferation or for pre-B cell survival, whereby differentiation and L chain rearrangement is subsequently initiated in a Btk/SLP-65-independent fashion. Because transgenic expression of the antiapoptotic protein Bcl-2 did not rescue the developmental arrest of Btk/SLP-65 double-deficient pre-B cells, we conclude that defective L chain opening in Btk/SLP-65-deficient small resting pre-B cells is not due to their reduced survival. Next, we analyzed transgenic mice expressing the constitutively active Btk mutant E41K. The expression of E41K-Btk in Ig H chain-negative pro-B cells induced 1) surface marker changes that signify cellular differentiation, including down-regulation of surrogate L chain and up-regulation of CD2, CD25, and MHC class II; and 2) premature rearrangement and expression of κ and λ light chains. These findings demonstrate that Btk and SLP-65 transmit signals that induce cellular maturation and Ig L chain rearrangement independently of their role in termination of pre-B cell expansion.

IntroductionB-cell chronic lymphocytic leukemia (B-CLL), 1,2 the most common leukemia in the Western world, is characterized by the accumulation of a monoclonal population of mature B cells that aberrantly express CD5. 3,4 The clinical course of CLL is extremely heterogeneous: whereas some patients survive more than a decade with stable disease, others die of the disease within months despite aggressive treatment. This heterogeneity is associated with variability in the expression pattern of a number of different proteins, and also with the presence of different chromosomal aberrations. 5 Approximately half of the CLL cases have 13q14deletions, apparently involving the linked microRNA molecules miR15 and miR16, which are thought to be negative regulators of the antiapoptotic gene Bcl2. 2 Deletions on chromosome 17p, affecting the p53 protein, are less frequent and are associated with poor prognosis. 6,7 CLLs manifest a unique gene-expression signature that differs from other lymphoid cancers, suggesting a common mechanism of transformation or a homogeneous cell population of origin. 2 CLL has been subdivided into 2 prognostic subsets, based on the presence of somatic mutations of the immunoglobulin (Ig) heavy (H) chain variable (V H ) genes. A total of 50% to 70% of CLL patients have mutated V H genes, probably reflecting antigen-driven postgerminal center (GC) selection, and have a more favorable prognosis than those with unmutated B-cell receptors. CLL Ig V H regions also exhibit unique complementarity determining region 3 (CDR3) features that characterize and differentiate aggressive unmutated from more indolent mutated CLLs. In particular, unmutated poor outcome cases frequently contain long CDR3s with amino acid residues that favor polyreactivity. Interestingly, both unmutated and mutated CLLs are thought to derive from self-reactive B-cell precursors. 8 In this context, it has been found that approximately 3% to 4% of healthy persons older than 40 years of age have a population of monoclonal lymphocytes in their blood with immunophenotypic characteristics of CLL cells. 2 The simian virus 40 (SV40) T antigen is a potent oncogene able to transform many cell types 9,10 and has been implicated in the etiology of various cancers. 11,12 The SV40 T antigen has transforming activity by inactivating p53 and Rb proteins and inducing genomic instability. 13 Several studies point to a causative role of SV40 in the formation of human B-cell malignancies, including non-Hodgkin lymphoma. [14][15][16] Transgenic expression of the SV40 T gene under the control of the IgH enhancer induced hyperproliferation of multilineage hematopoiesis, reminiscent of myelodysplastic syndromes in humans. 17 In this report, we aimed to accomplish sporadic SV40 T gene expression in the B-cell lineage in mice. We introduced the SV40 T gene, without its promoter and in opposite transcriptional orientation between the IgH chain D and J H segments. As antisense transcription takes place across the D-J H region in pro-B cells, 18 it is possible t...

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This information is current as Ig light chain gene rearrangement pre-B cell differentiation and the induction of Bruton's tyrosine kinase and SLP-65 regulate

The adapter protein Slp65 and Bruton's tyrosine kinase (Btk) are key components of the precursor-B (pre-B) cell receptor (pre-BCR) signaling pathway. Slp65-deficient mice spontaneously develop pre-B-cell leukemia, expressing high levels of the pre-BCR on their cell surface. As leukemic Slp65-deficient pre-B cells express the recombination activating genes (Rag)1 and Rag2, and manifest ongoing immunoglobulin (Ig) light-chain rearrangement, it has been hypothesized that deregulated recombinase activity contributes to malignant transformation. In this report, we investigated whether Rag-induced DNA damage is involved in oncogenic transformation of Slp65-deficient B cells. We employed Btk/Slp65 doubledeficient mice carrying an autoreactive 3-83ld BCR transgene. When developing B cells in their bone marrow express this BCR, the V(D)J recombination machinery will be activated, allowing for secondary Ig light-chain gene rearrangements to occur. This phenomenon, called receptor editing, will rescue autoreactive B cells from apoptosis. We observed that 3-83ld transgenic Btk/Slp65 double-deficient mice developed B-cell leukemias expressing both the 3-83ld BCR and the pre-BCR components k5/VpreB. Importantly, such leukemias were found at similar frequencies in mice concomitantly deficient for Rag1 or the non-homologous end-joining factor DNA-PKcs. We therefore conclude that malignant transformation of Btk/Slp65 double-deficient pre-B cells is independent of deregulated V(D)J recombination activity.