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Unlabelled: Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with upregulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of maturation. The aim of our work was to explore CD150 expression both on protein and mRNA levels with the emphasis on CD150 isoforms in malignant B-cell lines at the different stages of maturation in comparison with their normal B cell counterparts., Materials and Methods: Studies were performed on normal tonsillar B-cell subpopulations, B-lymphoblastoid cell lines, malignant B-cell lines of different origin, including pre-B acute lymphoblastic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, and multiple myeloma. Protein CD150 expression was assessed by western blot analysis and the expression level of CD150 isoforms was evaluated using qRT-PCR., Results: Despite the similar CD150 expression both on mRNA and protein levels in normal B-cell subsets and B-lymphoblastoid cell lines, malignant B-cell lines demonstrated substantial heterogeneity in CD150 expression. Only Hodgkin's lymphoma cell lines, Burkitt's lymphoma cell lines BJAB and Raji, and also pre-B cell line BLIN-1 expressed CD150 protein. At the same time total CD150 and mCD150 mRNA was detected in all studied cell lines excluding pre-B cell line REH. The minor sCD150 isoform was found only in Hodgkin's lymphoma cell lines and Burkitt's lymphoma cell line Raji. The nCD150 isoform was broadly expressed in tested B cell lines with exception of REH and Daudi., Conclusion: Malignant B-cell lines at the different stages of maturation only partially resemble their normal counterparts by CD150 expression. In malignant B-cell lines, CD150 expression on mRNA level is much broader than on protein level. CD150 isoforms are differentially expressed in normal and malignant B cells with predominant expression of mCD150 isoform.

Background: X-linked lymphoproliferative disease type 1 (XLP1) belongs to genetically determined primary immunodeficiency syndromes with mutations in SH2D1A/DSHP/SAP gene. The dramatic increase of the risk of B-cell lymphoma development in XLP1 patients is linked not only to SAP deficiency of NK, NKT and T cells, but probably to the impairment of B cell differentiation., Aim: To analyze the receptor-mediated Akt/PKB and ERK1/2 phosphorylation and expression of transcription factors that are involved in B cell maturation in EBV-transformed B-lymphoblastoid cell lines (B-LCLs) from XLP1 patients (XLP B-LCLs) in comparison with conventional B-LCLs., Methods: Studies were performed on EBV-transformed XLP B-LCLs IARC 739, SC-XLP and RP-XLP in comparison with SAP-negative B-LCL T5-1 and SAP-positive B-LCL MP-1. Western blot analysis was used for evaluation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204) phosphorylation in response to ligation of CD150, CD40, and IgM cell surface receptors. The expression levels of transcription factors IRF4, IRF8, BCL6, BLIMP1, SPIB, PU.1 and MITF were assessed using quantitative RT-PCR., Results: It was shown that SAP deficiency in XLP B-LCL did not abrogate CD150-mediated Akt and ERK1/2 phosphorylation. At the same time, ligation of CD150 or IgM affects kinetics and amplitude of ERK1/2 activation. In XLP B-LCL the CD150 signaling with IgM coligation play the dominant role in both Akt and ERK1/2 phosphorylation. We found that significantly reduced IRF4, IRF8 and PU.1 expression levels are the key features of XLP B-LCLs., Conclusion: XLP B-LCLs and conventional B-LCLs have differences in kinetics and amplitude of Akt and ERK1/2 phosphorylation. Analysis of transcription factors profile revealed the distinguishing features of XLP B-LCLs with SAP deficiency that may impair B cell differentiation.Key Words: B-lymphoblastoid cell lines, X-linked lymphoproliferative disease type 1, CD150, CD40, BCR, Akt/PKB, ERK1/2, transcription factors.

Background: Mutations in SH2D1A/DSHP/SAP gene are responsible for the onset of X-linked lymphoproliferative disease type 1 (XLP1) that have increased risk for B-cell lymphoma development. In XLP1 patients SAP deficient NK, NKT and CD8(+) cytotoxic T cells are inefficient in eliminating EBV-infected proliferating B cells that may partially contribute to the lymphoma development. However, little is known about impairment of B cell characteristics in XLP1., Aim: To analyze the cell surface phenotype and functional characteristics of EBV-transformed B-lymphoblastoid cell lines from XLP1 patients (XLP B-LCLs) in comparison with conventional B-lymphoblastoid cell lines (B-LCLs)., Methods: Studies were performed on SAP-negative B-LCLs T5-1, 6.16, RPMI 1788; SAP-positive B-LCL MP-1 and XLP B-LCLs IARC 739, XLP-D, XLP-8005. Cell surface immunophenotyping was performed using flow cytometry analysis. The level of apoptotic cells (Annexin V-binding), cell viability (MTT assay), and cell proliferation (trypan blue exclusion test) were evaluated in response to ligation of CD40, CD95, CD150 and IgM cell surface receptors., Results: A cell surface phenotype and functional features that distinguish XLP B-LCLs from conventional B-LCLs were revealed. XLP B-LCLs showed the upregulated level of CD20, CD38 and CD86 cell surface expression and downregulation of CD40, CD80 and CD150 expression. The major functional differences of XLP B-LCLs from conventional B-LCLs concern the modulation of CD95 apoptosis via CD40 and CD150 receptors and unresponsiveness to proliferative signals triggered by CD40 or colligation of BCR with CD150., Conclusion: The data suggest that the B-LCL from XLP1 patients have an intrinsic defect that affects cell activation, apoptosis, and proliferation.

Aim: To study upstream and downstream events in CD150-mediated Akt signaling pathway in normal human B cells, EBV-transformed lymphoblastoid (LCL) and malignant Hodgkin's lymphoma (HL) B cell lines., Methods: To access protein-protein interaction we applied immunoprecipitation, Western blot analysis and surface plasmon resonance (SPR) technique. A novel modification of SPR technique using reduced glutathione bound to golden surface was proposed. Immunostaining and isolation of cytoplasmic fractions and nuclear extracts were performed to detect proteins' localization in cells. Western blot analysis was performed to follow up the phosphorylation of proteins on specific sites and proteins' expression level., Results: It was shown that CD150 ligation induced Akt activation in normal tonsillar B cells (TBC), SH2D1A positive LCL and HL B cell lines. The p85α subunit of PI3K co-precipitated with CD150 cytoplasmic tail. This direct association depends on tyrosine phosphorylation and is mediated by N terminal SH2 domain of p85α. CD150 initiated phosphorylation of FoxO1 transcription factor in normal B cells as well as in LCL MP-1 and HL cell line L1236. At the same time, CD150 ligation triggered GSK-3β kinase phosphorylation only in immortalized LCL MP-1 and HL cell line L1236., Conclusions: We have demonstrated that CD150 receptor could trigger PI3K-mediated Akt signaling pathway in normal, EBV-transformed and malignant B cells. CD150-mediated phosphorylation of Akt downstream targets GSK-3β and FoxO1 in EBV-transformed and HL cells could be one of the mechanisms to avoid apoptosis and support survival program in these immortalized B cells.

The CD150 receptor is expressed on thymocytes, activated and memory T cells, B cells, platelets, natural killer T cells, and mature dendritic cells, and is also detected on tumor cells of Hodgkin's lymphoma (HL) and diffuse large B-cell lymphoma with an activated B cell phenotype. Here, we report that the level of CD150 expression is elevated during B cell differentiation toward plasma cells. In primary tonsillar B cells and HL cell lines, CD150 signaling regulates the phosphorylation of three types of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and Jun N-terminal kinase 1/2 (JNK1/2). CD150 induced ERK1/2 activation in primary tonsillar B cells and in two HL cell lines. CD150 mediated activation of JNK1/2 p54 and JNK2-gamma kinase isoforms in all CD150(+) B cell lines we tested. CD150 associated with the serine/threonine kinase hematopoetic progenitor kinase 1 (HPK1) regardless of CD150 tyrosine phosphorylation or binding of the SH2D1A adaptor protein to CD150, and HPK1 overexpression enhanced CD150-mediated JNK1/2 phosphorylation. CD150 ligation inhibited cell proliferation of all studied HL cell lines and induced apoptosis in L1236 HL cells that did not depend on JNK activity. As signaling through CD150 modulates MAPK activity in HL tumor cells, CD150 may contribute to regulation of tumor cell maintenance in low-rate proliferating HLs.

The CD150 receptor is expressed on activated T and B lymphocytes, dendritic cells, and monocytes. A TxYxxV/I motif in the CD150 cytoplasmic tail can bind different SH2-containing molecules, including tyrosine and inositol phosphatases, Src family kinases, and adaptor molecules. To analyze CD150-initiated signal transduction pathways, we used DT40 B-cell sublines deficient in these molecules. CD150 ligation on DT40 transfectants induced the extracellular signal-regulated kinase (ERK) pathway, which required SH2-containing inositol phosphatase (SHIP) but not SH2 domain protein 1A (SH2D1A). CD150-mediated Akt phosphorylation required Syk and SH2D1A, was negatively regulated by Lyn and Btk, but was SHIP independent. Lyn directly phosphorylated Y327 in CD150, but the Akt pathway did not depend on CD150 tyrosine phosphorylation and CD150-SHP-2 association. Analysis of CD150 and SH2D1A expression in non-Hodgkin and Hodgkin lymphomas revealed stages of B-cell differentiation where these molecules are expressed alone or coexpressed. Signaling studies in Hodgkin disease cell lines showed that CD150 is linked to the ERK and Akt pathways in neoplastic B cells. Our data support the hypothesis that CD150 and SH2D1A are coexpressed during a narrow window of B-cell maturation and SH2D1A may be involved in regulation of B-cell differentiation via switching of CD150-mediated signaling pathways.

CDw150, a receptor up-regulated on activated T or B lymphocytes, has a key role in regulating B cell proliferation. Patients with X-linked lymphoproliferative disease have mutations in a gene encoding a protein, DSHP/SAP, which interacts with CDw150 and is expressed in B cells. Here we show that CDw150 on B cells associates with two tyrosine-phosphorylated proteins, 59 kDa and 145 kDa in size. The 59-kDa protein was identified as the Src-family kinase Fgr. The 145-kDa protein is the inositol polyphosphate 5'-phosphatase, SH2-containing inositol phosphatase (SHIP). Both Fgr and SHIP interact with phosphorylated tyrosines in CDw150's cytoplasmic tail. Ligation of CDw150 induces the rapid dephosphorylation of both SHIP and CDw150 as well as the association of Lyn and Fgr with SHIP. CD95/Fas-mediated apoptosis is enhanced by signaling via CDw150, and CDw150 ligation can override CD40-induced rescue of CD95-mediated cell death. The ability of CDw150 to regulate cell death does not correlate with serine phosphorylation of the Akt kinase, but does correlate with SHIP tyrosine dephosphorylation. Thus, the CDw150 receptor may function to regulate the fate of activated B cells via SHIP as well as via the DSHP/SAP protein defective in X-linked lymphoproliferative disease patients.