High-Level Expression of the T Cell Chemokines CCL3 and CCL4 by Chronic Lymphocytic Leukemia B Cells in Nurselike Cell Co-Cultures and in Response to BCR Stimulation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 342-342
Author(s):  
Jan A. Burger ◽  
Matthias Niedermeier ◽  
Andrea Bürkle ◽  
Elena Hartmann ◽  
William G. Wierda ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Serum Levels ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
High Level Expression ◽  
High Level

Abstract Despite their apparent longevity in vivo, isolated Chronic Lymphocytic Leukemia (CLL) B cells generally undergo spontaneous apoptosis in vitro when cultured under conditions that support the growth of human B cell lines. This suggests that interactions between CLL cells and a distinct tissue microenvironment in the marrow and the lymphatic tissues, where CLL cells are in close contact with accessory cells (mesenchymal stromal cells, and CD68+ “nurselike cells”/NLC), are critical for the progression of the disease. NLC can be detected in secondary lymphoid tissues from CLL patients and appear to be an integral part of the CLL microenvironment, comparable to lymphoma-associated macrophages in follicular lymphoma. The molecules involved in CLL-NLC cross talk are only partially understood. Therefore, we examined the gene expression profile of purified CLL B cells using Affymetrix U133 Plus 2.0 Arrays to define distinct expression profiles induced in purified CLL B cells by co-culture with NLC. When compared to freshly isolated blood CLL B cells, we found that CLL B cells co-cultured for 14 days with NLC displayed high level expression of two T cell chemoattractants, macrophage inflammatory protein-1a and b (MIP-1 a, MIP-1b, also called CCL3 and CCL4). CCL3 and CCL4 expression levels correlated with ZAP-70 expression by the CLL cells, suggesting that B cell receptor signaling is involved in inducing the expression of these chemokines. Supernatants from CLL-NLC co-cultures harvested 7 and 14 days after initiation of the cultures revealed high CCL3 and CCL4 protein levels (up to >30 ng/ml) by ELISA, predominantly in the same CLL cases that displayed high CCL3, CCL4 and ZAP-70 expression by expression profiling. Moreover, serum samples from CLL patients were tested for CCL3 and CCL4 protein expression by ELISA. These studies demonstrated higher CCL3 and CCL4 serum levels in CLL patients when compared to healthy volunteers. CCL3 and CCL4 serum levels were found to be higher in CLL patients that were CD38+, displayed non-mutated IgVH genes, and b2 microglobulin levels that are > 4 mg/L. In vitro, B cell receptor (BCR) triggering of CLL cells, using anti-IgM antibodies, induced a rapid and robust induction of CCL3 and CCL4 protein production by CLL B cells. In contrast, CD40 triggering did not induce expression of these chemokines. These studies reveal a novel mechanism of cross-talk between CLL B cells and their microenvironment, namely the induction of two T cell chemokines, CCL3 and CCL4, by CLL-NLC interaction. Thus, we provide the first evidence that neoplastic B cells are an important source of T cell chemokines, that can induce recruitment of T cells of helper/effector phenotype to sites of cognate T cell-CLL interactions. Besides inducing the outgrowth of NLC, this is another mechanism how CLL cells actively create a microenvironment that favors their growth and survival. Figure Figure

scholarly journals High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation

Blood ◽  
2009 ◽  
Vol 113 (13) ◽  
pp. 3050-3058 ◽  
Author(s):  
Jan A. Burger ◽  
Maite P. Quiroga ◽  
Elena Hartmann ◽  
Andrea Bürkle ◽  
William G. Wierda ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cross Talk ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
High Level Expression ◽  
Accessory Cells ◽  
High Level

Abstract In lymphatic tissues, chronic lymphocytic leukemia (CLL) cells are interspersed with CD68+ nurselike cells (NLCs), T cells, and other stromal cells that constitute the leukemia microenvironment. However, the mechanism regulating colocalization of CLL and these accessory cells are largely unknown. To dissect the molecular cross talk between CLL and NLCs, we profiled the gene expression of CD19-purified CLL cells before and after coculture with NLCs. NLC coculture induced high-level expression of B-cell maturation antigen and 2 chemoattractants (CCL3, CCL4) by CLL cells. CCL3/CCL4 induction in NLC cocultures correlated with ZAP-70 expression by CLL cells. High CCL3/CCL4 protein levels were found in CLL cocultures with NLCs, and CCL3/CCL4 induction was abrogated by R406, a Syk inhibitor, suggesting that NLCs induce these chemokines via B-cell receptor (BCR) activation. BCR triggering also caused robust CCL3/CCL4 protein secretion by CLL cells. High CCL3 and CCL4 plasma levels in CLL patients suggest that this pathway plays a role in vivo. These studies reveal a novel mechanism of cross talk between CLL cells and their microenvironment, namely, the secretion of 2 T-cell chemokines in response to NLC coculture and BCR stimulation. Through these chemokines, CLL cells can recruit accessory cells and thereby actively create a supportive microenvironment.

scholarly journals Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells

2021 ◽  
Author(s):  
Sarah Wilmore ◽  
Karly-Rai Rogers-Broadway ◽  
Joe Taylor ◽  
Elizabeth Lemm ◽  
Rachel Fell ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mrna Translation ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Mrna Stabilization

AbstractSignaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4389-4395 ◽  
Author(s):  
Freda K. Stevenson ◽  
Federico Caligaris-Cappio
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Of Origin ◽  
Regulatory B Cell ◽  
V Genes

Abstract The finding that chronic lymphocytic leukemia (CLL) consists of 2 clinical subsets, distinguished by the incidence of somatic mutations in the immunoglobulin (Ig) variable region (V) genes, has clearly linked prognosis to biology. Antigen encounter by the cell of origin is indicated in both subsets by selective but distinct expression of V genes, with evidence for continuing stimulation after transformation. The key to distinctive tumor behavior likely relates to the differential ability of the B-cell receptor (BCR) to respond. Both subsets may be undergoing low-level signaling in vivo, although analysis of blood cells limits knowledge of critical events in the tissue microenvironment. Analysis of signal competence in vitro reveals that unmutated CLL generally continues to respond, whereas mutated CLL is anergized. Differential responsiveness may reflect the increased ability of post-germinal center B cells to be triggered by antigen, leading to long-term anergy. This could minimize cell division in mutated CLL and account for prognostic differences. Unifying features of CLL include low responsiveness, expression of CD25, and production of immunosuppressive cytokines. These properties are reminiscent of regulatory T cells and suggest that the cell of origin of CLL might be a regulatory B cell. Continuing regulatory activity, mediated via autoantigen, could suppress Ig production and lead to disease-associated hypogammaglobulinemia. (Blood. 2004;103:4389-4395)

scholarly journals Defective T cell-mediated, isotype-specific immunoglobulin regulation in B cell chronic lymphocytic leukemia

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 1012-1020 ◽  
Author(s):  
JS Moore ◽  
MB Prystowsky ◽  
RG Hoover ◽  
EC Besa ◽  
PC Nowell
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Activity ◽  
Lymphocytic Leukemia ◽  
Specific Immunoglobulin ◽  
Cell Chronic Lymphocytic Leukemia

The consistent occurrence of T cell abnormalities in patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that the non- neoplastic host T cells may be involved in the pathogenesis of this B cell neoplasm. Because potential defects of immunoglobulin regulation are evident in B-CLL patients, we investigated one aspect of this by studying the T cell-mediated immunoglobulin isotype-specific immunoregulatory circuit in B-CLL. The existence of class-specific immunoglobulin regulatory mechanisms mediated by Fc receptor-bearing T cells (FcR + T) through soluble immunoglobulin binding factors (IgBFs) has been well established in many experimental systems. IgBFs can both suppress and enhance B cell activity in an isotype-specific manner. We investigated the apparently abnormal IgA regulation in a B-CLL patient (CLL249) whose B cells secrete primarily IgA in vitro. Enumeration of FcR + T cells showed a disproportionate increase in IgA FcR + T cells in the peripheral blood of this patient. Our studies showed that the neoplastic B cells were not intrinsically unresponsive to the suppressing component of IgABF produced from normal T cells, but rather the IgABF produced by the CLL249 host T cells was defective. CLL249 IgABF was unable to suppress IgA secretion by host or normal B cells and enhanced the in vitro proliferation of the host B cells. Size fractionation of both normal and CLL249 IgABF by gel-filtration high- performance liquid chromatography (HPLC) demonstrated differences in the ultraviolet-absorbing components of IgABF obtained from normal T cells v that from our patient with defective IgA regulation. Such T cell dysfunction may not be restricted to IgA regulation, since we have found similar expansion of isotype-specific FcR + T cells associated with expansion of the corresponding B cell clone in other patients with B-CLL. These data suggest that this T cell-mediated regulatory circuit could be significantly involved in the pathogenesis of B-CLL.

scholarly journals Growth- and differentiation-associated expression of bcl-2 in B-chronic lymphocytic leukemia cells

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2981-2989 ◽  
Author(s):  
M Schena ◽  
LG Larsson ◽  
D Gottardi ◽  
G Gaidano ◽  
M Carlsson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Messenger Rna ◽  
Phorbol Esters ◽  
Lymphocytic Leukemia ◽  
Mantle Zone ◽  
Cell Stage ◽  
High Level

Abstract The bcl-2 gene is translocated into the Ig loci in about 80% of human follicular lymphomas and in 10% of B-type chronic lymphocytic leukemias (B-CLL), resulting in a high level of expression. We have compared the expression of bcl-2 transcripts and protein in B-CLL cells in their normal equivalent CD5+ B cells and in normal B-cell populations representative of different in vivo and in vitro stages of activation and proliferation. We report here that bcl-2 was expressed in 11 of 11 cases of CD5+ B-CLL clones, contrasting with the absent expression in normal CD5+ B cells. Activation of 173 and 183 B-CLL cells by phorbol esters (12-O-tetradecanoylphorbol-13-acetate [TPA]) to IgM secretion without concomitant DNA synthesis resulted in a rapid but transient downregulation of bcl-2 expression. In contrast, the reduction of bcl-2 at both the messenger RNA and protein levels was sustained after mitogenic stimulation, suggesting that bcl-2 expression and proliferation are inversely related in these cells. This notion was further supported by immunocytochemical analysis showing that bcl-2 was primarily expressed in small resting lymphocytes and in cells differentiating to the plasma cell stage, but less expressed in Ki67- positive proliferating B blasts. Moreover, it was also supported by the low level of bcl-2 in exponentially growing Epstein-Barr virus-carrying lymphoblastoid and B-CLL cell lines. The regulation of bcl-2 expression in B-CLL resembled that of normal tonsillar follicular B cells, in which a high level of expression was found in resting mantle zone B cells but not in the proliferating germinal center B cells. Based on these findings and the role of bcl-2 in maintaining B-cell memory, we propose that the phenotype of B-CLL cells corresponds to a mantle zone memory-type B cell.

The Akt/Mcl-1 Pathway Plays a Prominent Role in Mediating Pro-Survival Signals Derived from the B-Cell Receptor in Chronic Lymphocytic Leukemia B-Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 341-341
Author(s):  
Pablo G. Longo ◽  
Luca Laurenti ◽  
Stefania Gobessi ◽  
Simona Sica ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Igm Antibodies ◽  
B Cell Survival ◽  
Sustained Activation ◽  
Constitutively Active

Abstract Studies of the immunoglobulin variable region gene repertoire have provided compelling evidence that antigen-stimulation through the B-cell receptor (BCR) plays a crucial role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). In addition, previous studies from our lab have shown that CLL B-cells become more resistant to spontaneous and chemotherapy-induced apoptosis following sustained engagement of the BCR with immobilized anti-IgM antibodies, which mimic stimulation with membrane-bound antigens. Investigation of downstream signaling pathways revealed that sustained BCR engagement induces prolonged activation of the PI3K/Akt and MEK/ERK pathways, which are key regulators of survival and proliferation in various cell types. To further define the role of sustained activation of the Akt and ERK kinases in regulating CLL growth and survival, we transfected constitutively active mutants of Akt (myr.Akt) and MEK2 in primary leukemic cells and evaluated changes in the expression of relevant apoptosis- and cell-cycle regulatory proteins. Introduction of constitutively active MEK2 resulted in activation of ERK, but did not induce significant changes in the levels of most investigated proteins (Bcl-2, Bcl-xL, Bim, Bax or Mcl-1). The only exception was the inhibitor of apoptosis protein XIAP, which showed increased expression in most but not all experiments. In contrast, transfection of myr.Akt showed a consistent increase in the levels of the antiapoptotic protein Mcl-1, which ranged from 1.5 to more than 4-fold higher levels with respect to cells transfected with control vectors. Increased expression of Mcl-1 was observed in all experiments and paralleled the rise in Mcl-1 that occurred following stimulation of CLL B-cells with immobilized anti-IgM antibodies. The increase in Mcl-1 protein levels was entirely due to post-transcriptional mechanisms, since quantification by real-time PCR did not show an increase in Mcl-1 mRNA levels. Constitutively active Akt also upregulated Bcl-xL and XIAP, although this increase was lower than the increase in Mcl-1. In addition, CLL cells transfected with myr.Akt showed induction of cyclin D3 and an increase in cell size and viability, indicating that sustained activation of Akt is required for both leukemic cell survival and cell cycle progression. To determine the relative importance of Mcl-1, Bcl-xL and XIAP in CLL B-cell survival, we downregulated expression of these proteins in primary CLL B-cells by RNA interference. Surprisingly, downregulation of Bcl-xL and XIAP had no effect on CLL B-cell survival. In contrast, silencing of Mcl-1 induced rapid and potent apoptosis in all investigated cases and abrogated the prosurvival effect of stimulation with immobilized anti-IgM antibodies. Together, these data provide direct evidence that pro-survival BCR signaling in CLL B-cells is mediated, at least in part, through the Akt/Mcl-1 pathway. In addition, they suggest that Mcl-1 could be an attractive candidate for targeting, either with small molecule inhibitors or with pharmacological agents that interfere with BCR signals propagated by the Akt kinase.

The Bruton Tyrosine Kinase Inhibitor, PCI-32765, Inhibits Activation and Proliferation of Human Chronic Lymphocytic Leukemia Cells in the NSG Xenograph Mouse Model of the Tissue Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 596-596 ◽  
Author(s):  
Sarah E. M. Herman ◽  
Xiameng Sun ◽  
Joseph J. Buggy ◽  
Georg Aue ◽  
Patricia Perez-Galan ◽  
...  
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Cell Receptor ◽  
Gene Signature ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Tissue Microenvironment

Abstract Abstract 596FN2 PCI-32765, a specific inhibitor of Bruton's tyrosine kinase (Btk), can disrupt several signaling pathways involved in tumor microenvironment interactions. In vitro, PCI-32765 has been demonstrated to induce apoptosis, to varying degrees, in tumor cells and prevent CpG-ODN induced proliferation of cultured chronic lymphocytic leukemia (CLL) cells (Herman et al, Blood 2011). PCI-32765 has been shown to be well tolerated in CLL with preliminary clinical trial data showing that >85% (34/39) of patients remained on therapy at a median follow-up of four months. In addition, a significant shrinkage of lymph nodes has been observed in the majority of patients displaying lymphadenopathy. As with other B-cell receptor (BCR) directed therapies, PCI-32765 results in an initial increase in the absolute lymphocyte count. These observations are not explainable by the available in vitro data, demonstrating the need for in vivo investigation. In order to study the effect of PCI-32765 in vivo we chose to use the recently established NOD scid gamma null (NSG) - human CLL xenograft model with some modifications (Bagnara et al., Blood 2011). NSG mice were conditioned with 25 mg/kg busulfan 24 hours before injection of 1 × 108 CLL peripheral blood mononuclear cells previously labeled with 1μM CFSE. We first demonstrated that xenografted CLL cells isolated from the mouse spleen acquire an activated phenotype and proliferate, mimicking the phenotype of CLL cells isolated from human lymph nodes (Sun et al., abstract submitted). Next we sought to use this model to investigate the effect ot PCI-32765 on CLL cell activation and proliferation. Mice received PCI-32765 or vehicle in their drinking water at 0.16 mg/ml dissolved in 1% HP-beta-CD starting at the time of busulfan treatment. Mice were bled weekly and sacrificed between 3 and 4 weeks post xenografting. We found that PCI-32765 treatment resulted in a significant reduction in proliferation (defined as CFSE low cells) compared to mice that received vehicle water; this was observed in all three biological compartments: peripheral blood (84.5% decrease, p=0.007), spleen (72.4% decrease, p=0.012) and bone marrow (92.5% decrease, p=0.049). In comparison, PCI-32765 treatment did not result in a significant reduction in T-cell proliferation in any of the compartments (p>0.4). Although peripheral blood CLL counts were comparable between treated and untreated mice, we found that there were substantially more CLL cells in the spleens of the vehicle treated mice than in those of the PCI-32765 treated mice. In contrast, no differences in T-cell number or localization were observed between treated and untreated mice. Lastly, we sought to determine whether activation of CLL cells in the microenvironment could be blocked by PCI-32765. As we have previously shown, CLL cells in the human lymph node display a gene signature indicating B-cell receptor (BCR) and NF-kB activation compared to CLL cells in the peripheral blood (Herishanu et al., Blood 2011). We used quantitative RT-PCR (pre-designed Taqman Gene Expression assays) to measure expression of representative BCR and NF-kB target genes. PCI-32765 significantly reduced expression of EGR1 (p=0.049), EGR3 (p=0.023) and GFI1 (p=0.023) (BCR signature) and CCL3 (p=0.013) and CCND2 (p=0.046) (NF-kB signature) compared to vehicle treated mice. In addition, we also observed decreases in the proliferation gene signature (CDT1, PCNA and RRM2) (signature score, p=0.035) in the CLL cells from mice treated with PCI-32765; consistent with the assessed CFSE proliferation measurements. Taken together, our results show that PCI-32765 inhibits CLL activation and proliferation in the tissue microenvironment in vivo without affecting T-cell proliferation. These results demonstrate that targeting Btk is sufficient to block key interactions between tumor cells and the microenvironment and thus warrants the use of PCI-32765 as a targeted agent in CLL. Disclosures: Buggy: Pharmacyclics, Inc.: Employment.

Anergic Response to B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia: The Differing Roles of IgD and IgM in the Microenvironment and Peripheral Blood.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2889-2889
Author(s):  
Tom Butler ◽  
Alexander Montoya ◽  
Andrew James Clear ◽  
Rita Coutinho ◽  
David C Taussig ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
Cell Receptor Signaling

Abstract Abstract 2889 Chronic Lymphocytic Leukemia (CLL) cells depend on B cell receptor signaling as well as other microenvironmental survival signals (1). Drugs targeting the BCR signaling pathways are showing exciting results in CLL clinical trials. A peculiarity of CLL is that IgD signaling is generally preserved, whilst IgM signaling is decreased and it has been suggested that this pattern mimics anergic B-cells, and might be consistent with chronic autoantigen exposure. We examined the differing roles of IgM and IgD signaling in CLL using a theoretical framework of anergy. Peripheral blood (PB) CLL cells exhibited higher IgD expression, as compared to IgM (n=204, p<0.0001), but this did not have prognostic impact. When we examined IgM and IgD expression in LN biopsies compared to paired PB (n=10) expression, IgM expression was lower in LN (p=0.002) whilst IgD expression was unchanged. Although the number of these paired samples is small, cases with lower LN IgM levels had poorer prognosis, and we are investigating this further with a larger cohort. We hypothesize that reduced LN IgM expression reflects antigen engagement and an anergic response in the microenvironment. We sought to replicate Mockridge et al' s model of reversible anergy (2) by monitoring the dynamic changes in IgM/D expression after in vitro incubation. Most (18/20) PB CLL samples underwent calcium (Ca) flux after IgD crosslinking, whereas only 13/20 cases underwent IgM Ca flux, and the level of Ca flux was less than with IgD, a well recognized anergic pattern. Incubation for 24h in vitro led to partial restoration of IgM Ca flux and some improvement in IgD Ca flux. This was impaired by treatment with anti-IgD or IgM F(ab)2 fragments, mimicking antigen exposure, and in keeping with a model of CLL cells engaging autoantigen in vivo. Further support for the pro-survival role of the BCR in CLL was demonstrated by the finding that both IgD and IgM ligation was associated with reduced apoptosis in vitro, with a significant decrease in apoptosis with IgD ligation as compared to IgM. To examine the mechanistic differences of signaling via IgM and IgD further, we used high-throughput mass-spectrometry based phosphoproteomics. This allows analysis of multiple active signaling pathways without a priori knowledge of which pathways to investigate. 6 CLL samples were compared to 5 tonsil controls. 4,575 unique phosphopeptides were identified using MASCOT proteomics software and quantified using a label-free technique based on extracted ion currents. 174 phosphoproteins (p<0.001, fold change up to >4000-fold) were over-expressed in CLL relative to healthy B-cells. These included components of RNA processing complexes, cytoskeletal regulators and MAPK signaling pathway components. Kinase prediction based on phosphoprotein substrates confirmed activation of kinases known to be active in CLL (such as AKT1, ERK1/2, CK2), but several novel kinases (such as CaMK1, CRIK, ROCK1 and BCKDK) were also active in CLL relative to healthy controls. Evaluation of differentially expressed phosphoproteins after BCR ligation included components of the spliceosome, regulators of the cytoskeleton, as well as known BCR signaling components. BCR-induced kinase activities included mTOR, CDK family members, MAPKs, BCKDK and others. There was much overlap between kinases active after IgM and IgD ligation, but also marked differences in CLL and tonsil BCR signaling. CONCLUSIONS Anergic IgM signaling is contrasted with IgD as a dynamic and plastic process that appears different in the LN and PB compartments in CLL. Mass-spectrometry based phosphoproteomics offers a powerful tool for interrogating intracellular signaling, with networks of phosphorylation characterizing the topology of pathways. BCR signaling in healthy B-cells has not previously been studied using this approach and comparisons with CLL highlight known pathways as well as suggesting novel treatment targets. The ultimate goal is to identify kinases active in CLL that will provide rational and effective drug combinations. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2327-2335 ◽  
Author(s):  
A. Alfarano ◽  
S. Indraccolo ◽  
P. Circosta ◽  
S. Minuzzo ◽  
A. Vallario ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Alternatively Spliced

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

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