Toll like Receptors (TLRs) in B Cell Chronic Lymphocytic Leukemia (B-CLL).

Abstract Introduction: B-CLL is characterised by the accumulation of long-lived CD5+ B cells. Mature B cells can be activated via TLRs to produce immunoglobulin and proliferate. We have previously reported differential surface expression of CD180, TLR-4 associated receptor, by B-CLL cells in relation to IgVh gene status (Porakishvili N et al., 2004; Blood (ASH annual meeting abstracts) 104: 2807). To further characterise subsets of leukemic cells, we assessed B-CLL cells for TLR mRNA expression. Method: B cells from untreated (n=24) and treated (CHOP n=3, Thalidomide n=1 and chlorambucil and prednisolone n=2) CLL patients, and healthy aged matched controls, (n=14) were isolated by CD19+ selection using iMACs columns (Miltenyi Biotec), total RNA extracted (Qiagen) and primers for TLR2, TLR4, TLR9, CD180 and the adaptor protein MYD88 were used for TaqMan PCR analysis (ABI). GAPDH was used for normalization. Data were expressed as mean delta cycle number (ct) ± standard deviation, giving a value inversely related to the level of expression of the specific gene. Patients were separated into groups according to IgVh gene mutation status or level of Zap 70 expression. Statistical analysis was performed using by Mann-Whitney U test and Spearman rank correlation or Pearson’s correlation coefficient. Results: Overall, there was a heterogeneous pattern of TLR expression in patients and controls with no significant differences between them. Treated patients were characterised by higher mRNA levels for the all TLRs measured (TLR9 p=0.018) and MYD88 (MYD88 p=0.026) compared with untreated patients, despite no significant difference of their GAPDH levels (p>0.05). Significant correlations were seen between mRNA levels for the TLRs within the age matched control group (all p<0.05). Groups of patients were further analysed according to mutated (mutated >2%) or unmutated IgVh genes (unmutated <2%). No significant correlation in the TLR expression levels was found within the mutated patient group (n=8) or Zap 70 low expression group (<0.7 level) (n=11). Preliminary data from patients with an unmutated IgVh gene status (n=4) or according to levels of ZAP 70high expression (n=5) showed correlation of mRNA levels for TLRs and MYD88 expression (all p<0.05). Conclusion: Our data indicates a direct relationship between MYD88 and TLRs transcription levels in healthy control individuals. Lack of significant correlation amongst TLR mRNA levels in CLL patients with mutated IgVh genes and Zap70low expression levels suggests aberrant regulation of TLRs in this group.

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C1GALT1 expression is associated with galactosylation of IgA1 in peripheral B lymphocyte in immunoglobulin A nephropathy

10.21203/rs.2.14899/v2 ◽

2019 ◽

Author(s):

Yue Xing ◽

Lina Li ◽

Yaru Zhang ◽

Fanghao Wang ◽

Dandan He ◽

...

Keyword(s):

B Cells ◽

B Lymphocyte ◽

Meta Analysis ◽

Mrna Level ◽

Immunoglobulin A ◽

Control Group ◽

Expression Levels ◽

Medical Network ◽

Significant Difference ◽

Healthy Control

Abstract Background More and more studies demonstrated that genetic variation at C1GALT1 influences Gd-IgA1 level in IgAN. However, whether the expression of β1, 3-galactosyltransferase (β1, 3Gal-T) was influenced may provide insights into how Gd-IgA1 levels are controlled in IgAN. Methods Thirty IgAN patients diagnosed in Tianjin Medical University General Hospital from April to September 2018 and 30 healthy volunteers whose age and gender matched with patients were enrolled in this study. Total Gd-IgA1 levels in plasma were determined by ELISA and C1GALT1 levels were determined by RT-PCR. Four databases (PubMed, EMBASE, CNKI, WanFang Medical Network) were searched to identify eligible studies that evaluated a difference in the expression of C1GALT1 in IgAN patients compared with total controls (non-IgAN and health controls). The C1GALT1C1 expression levels, which was indispensable to β1, 3Gal-T of IgA1, was also been compared. Results Gd-IgA1 levels were remarkable higher in IgAN patients compared with healthy control. The expression levels of C1GALT1 gene were remarkably down-regulated in IgAN patients compared with healthy control. And the mRNA level of C1GALT1 was inversely correlated to Gd-IgA1 levels. In meta-analysis, six articles including 316 participants that analyzed the expression of β1, 3Gal-T were met inclusion criteria. There was no significant difference in the expression of C1GALT1 between IgAN patients compared with controls. And we found patients with IgAN had lower levels of C1GALT1 gene expression in the B cells compared to controls. The C1GALT1C1 levels in the IgAN patients were not different from the levels in the control group, which were unchanged no matter according to different ethnic population, different control group and different cell source. Two studies including 46 persons compared enzymatic activity of β1, 3Gal-T in B cells, and the result showed the β1, 3Gal-T activity was decreased in B cells. Conclusions We found expression levels of C1GALT1 were remarkably downregulated in IgAN patients and negatively correlated with higher levels of Gd-IgA1. Subsequent meta-analysis validated the low expression and activity of β1, 3Gal-T in B cells in patients with IgAN. However, there was no apparent disparity in the aspect of C1GALT1C1 expression between IgAN and control groups.

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TCL1 Expression in Chronic Lymphocytic Leukemia Correlates with the Intensity of 11q Deletions and ZAP-70.

Blood ◽

10.1182/blood.v110.11.2068.2068 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2068-2068

Author(s):

Laura Rassenti ◽

L. Huynh ◽

G.W. Basak ◽

E.M. Ghia ◽

D. Van Dyke ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Chromosomal Abnormalities ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Expression Levels ◽

Significant Difference ◽

The Relationship ◽

11Q Deletion

Abstract The TCL1 (T-cell leukemia/lymphoma1) oncogene is a coactivator of the AKT oncoprotein, an essential molecule in the transduction of antiapoptotic signals in T and B cells. Eμ-TCL transgenic mice with B cells with high TCL1 expression develop the aggressive phenotype of chronic lymphocytic leukemia (CLL). Studies in human CLL have found that expression of TCL1 correlates with high expression of ZAP-70 and use of unmutated IgVH genes. The expression of TCL1 may be regulated in part by microRNA, miR-29 and miR-181, which map to chromosome 11(11q). Because aberrations at 11q have been associated with poor prognosis in CLL, we interrogated the relationship between deletions at 11q and expression of TCL1 and ZAP-70. We used a direct immunophenotyping method to investigate the relative co-expression levels of TCL1 and ZAP-70 within CLL cells and examined the relationship between such levels and the proportion of leukemia cells within the CLL population bearing 11q deletions, as detected by FISH analysis. Direct staining of intracellular TCL1 protein was performed by using the monoclonal anti-TCL1 antibody (clone 1–21) labeled with Alexa647 in combination with the established ZAP-70 protocol (NEJM2004; 351:893) together with mAb directed against CD5 and B cell surface antigens. Negative staining levels were set using isotype control antibodies. FISH was performed on interphase nuclei by using uniform and cross-validated procedures at all CRC sites using the CLL-panel from Vysis. Chromosomal abnormalities were detected in 76% (520) of the 680 CLL samples analyzed. Sixteen percent of the patients had leukemia cells with monoalleleic deletions at 11q. We performed flow cytometry for intracellular co-expression TCL1 and ZAP-70 on cryopreserved samples obtained from 25 CLL patient samples with varying proportions of cells with the 11q deletion (10% to 98% abnormal cells with 11q deletion, mean 70%) and 30 CLL samples lacking any chromosomal abnormalities. We detected significantly higher levels of TCL1 in CLL cells that expressed ZAP-70 and/or unmutated IgVH genes. The ZAP-70pos cases (39/55) had a median percent of TCL1pos cells of 34% compared to 15% for the ZAP-70neg cases. The cases using unmutated IgVH genes (43/55) had a median percent of TCL1pos cells of 31%, which was greater than the 19% median observed for cases that used mutated IgVH genes. Multiparameter analyses revealed that the ZAP-70 positive fraction of each CLL clone had significantly higher levels of TCL1 than did the ZAP-70 negative cells (mean=45% versus 29%, respectively, p=0.002). We observed a significant difference between the expression levels of TCL1 for CLL cells that had deletions in 11q relative to that of CLL cells lacking any chromosomal abnormalities (mean=41% versus 18%, respectively p=0.0002). In addition, we observed a relationship between the levels of TCL1 expressed in leukemia cell populations and the relative proportion of leukemia cells with deletions at 11q. This study reveals a relationship between the levels of TCL1 expression in CLL leukemia-cell expression of ZAP-70, and the relative proportions of leukemia cells having deletions at 11q. Studies are in progress to define whether these relationships can be explained by altered expression of microRNA that map to 11q, which might also account for the noted adverse prognosis of CLL that has deletions at 11q.

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C1GALT1 expression is associated with galactosylation of IgA1 in peripheral B lymphocyte in immunoglobulin A nephropathy

10.21203/rs.2.14899/v1 ◽

2019 ◽

Author(s):

Yue Xing ◽

Lina Li ◽

Yaru Zhang ◽

Fanghao Wang ◽

Dandan He ◽

...

Keyword(s):

B Cells ◽

B Lymphocyte ◽

Meta Analysis ◽

Mrna Level ◽

Immunoglobulin A ◽

Control Group ◽

Expression Levels ◽

Medical Network ◽

Significant Difference ◽

Healthy Control

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

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-03910-x ◽

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.

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Expression of AIM2 in rheumatoid arthritis and its role on fibroblast-like synoviocyte

10.21203/rs.2.23420/v1 ◽

2020 ◽

Author(s):

fujuan qiu ◽

Chen Yong ◽

Qiu Fujuan ◽

Zhao Xiaofeng ◽

Xiao Changhong

Keyword(s):

Rheumatoid Arthritis ◽

Mtt Assay ◽

Control Group ◽

Expression Levels ◽

Potential Target ◽

Caspase 1 ◽

Significant Difference ◽

And Migration ◽

Fibroblast Like Synoviocytes ◽

Proliferation And Migration

Abstract Background To determine whether any differences of AIM2 inflammasome expression levels between rheumatoid arthritis (RA) and osteoarthritis (OA) and investigate the effects of AIM2 when transferred into RA fibroblast-like synoviocytes (RA-FLS).Methods Serum AIM2 levels between OA and RA patients were compared by ELISA. Different expression levels of AIM2, ASC, Caspase-1 and IL-1β between RA and OA synovium were semi-quantified by RT-qPCR and immunohistochemical (IHC) staining. IHC staining were recorded by H scores, and determine the correlation with ESR and CRP levels of RA patients. SiRNA AIM2 was transferred to RA-FLS and observe its effects on proliferation and migration by MTT assay and transwell test respectively.Results In RA sera, no significant difference was observed between OA and RA patients. However, in affected knee synovium, AIM2, ASC, Caspase-1 and IL-1β were expressed higher in RA than that of OA. Plus, H score of AIM2, ASC, and IL-1β were positively correlated to ESR and CRP levels in RA patients. After transferred AIM2 siRNA to FLS and incubation for 48 hours, the proliferation of FLS were significantly inhibited, and the apoptosis rate were significantly increased compared to FLS in control group. However, no effect on migration was detected.Conclusions AIM2 participated in the proliferation of FLS, and might be a potential target for therapy.

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CD6 Ligation Modulates the Bcl-2/Bax Ratio and Protects Chronic Lymphocytic Leukemia B Cells From Apoptosis Induced by Anti-IgM

Blood ◽

10.1182/blood.v89.8.2833 ◽

1997 ◽

Vol 89 (8) ◽

pp. 2833-2841 ◽

Cited By ~ 68

Author(s):

Lyda M. Osorio ◽

Angelina De Santiago ◽

Miguel Aguilar-SantelisesHå ◽

kan Mellstedt ◽

Mikael Jondal

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

T Cell Activation ◽

Cell Activation ◽

Scavenger Receptor ◽

Interleukin 2 ◽

Lymphocytic Leukemia ◽

Small Subset ◽

Mrna Levels ◽

Membrane Glycoproteins

Abstract CD6 and CD5 belong to a scavenger-receptor cysteine-rich (SRCR) super family of membrane glycoproteins that are expressed on chronic lymphocytic leukemia B (B-CLL) cells, normal T cells, and a small subset of normal B cells. CD6 configures in the membrane in relation to the cellular activation level and can act as a coreceptor for T-cell activation. We have examined a group of progressive and nonprogressive B-CLL cells. Most B-CLL cells were positive for CD6 and the expression of CD6 was increased after activation with Staphylococcus aureus Cowan I plus interleukin-2 or 12-O-tetradecanoylphorbol 13-acetate, although anti-CD6 antibodies did not increase proliferative responses to these stimuli. However, anti-CD6 stimulation was found to protect against anti-IgM–induced apoptosis in B-CLL. baxα upregulation and bcl-2 downregulation were found in anti-IgM– and glucocorticoid (GCC)-induced apoptotic cells, respectively. Furthermore, CD6 cross-linking downregulated baxα mRNA levels in anti-IgM–treated cells, resulting in an increased bcl-2/baxα ratio. CD6 activation also prevented bcl-2 mRNA downregulation and apoptosis induced by GCC in one of six GCC-sensitive patients. These data suggest that an interaction between CD6 and its ligand might contribute to B-CLL survival through the modulation of the Bcl-2/Bax ratio.

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Correlation between expression levels of IL-37, GM-CSF, and CRP in peripheral blood and atherosclerosis and plaque stability

European Journal of Inflammation ◽

10.1177/2058739219844068 ◽

2019 ◽

Vol 17 ◽

pp. 205873921984406

Author(s):

Tao Zheng ◽

Qingyun Zhou ◽

Zhe Chen ◽

Qinning Wang

Keyword(s):

Peripheral Blood ◽

Density Lipoprotein ◽

Inflammatory Factors ◽

Control Group ◽

Case Group ◽

Expression Levels ◽

Gm Csf ◽

Significant Difference ◽

Group A ◽

Group B

The study aimed to study the correlation between expression levels of interleukin-37 (IL-37), granulocyte macrophage colony-stimulating factor (GM-CSF), and C-reactive protein (CRP) in peripheral blood and the status of atherosclerosis (AS) and plaque stability and to confirm the clinical significance of these inflammatory factors in the pathogenesis of AS. A total of 64 AS patients (case group) were selected and divided into unstable plaque group (group A, 28 cases) and stable plaque group (group B, 36 cases) according to the color ultrasonography results of arterial vessels. At the same time, 30 healthy subjects were classified into the control group. General information of the enrolled subjects was collected, including levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), CRP, and homocysteine (Hcy). The expression levels of IL-37 and GM-CSF in the serum of peripheral blood samples collected from these subjects were measured by enzyme-linked immunosorbent assay (ELISA). There was no significant difference between the case group and the control group in the levels of TC, TG, HDL, and LDL ( P > 0.05). However, the expression level of Hcy in the case group was significantly higher than that in the control group ( P < 0.05). Compared with the control group, the expression levels of IL-37, GM-CSF, and CRP in the case group were significantly increased ( P < 0.05). In addition, compared with group B, the expression level of GM-CSF in group A was significantly increased ( P < 0.05), while no significant difference was detected between group A and group B in the expression levels of IL-37 and CRP ( P > 0.05). In conclusion, inflammatory factors IL-37, GM-CSF, CRP, and Hcy were all involved in the pathogenesis of AS, and the increased levels of GM-CSF were closely related to the progress of unstable plaques. These results may aid the early diagnosis/treatment of AS.

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Lymphocyte aggregation in response to adrenergic stimulation

Blood ◽

10.1182/blood.v71.5.1470.1470 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1470-1474 ◽

Cited By ~ 5

Author(s):

DE Hammerschmidt ◽

C Jeanneret ◽

M Husak ◽

M Lobell ◽

HS Jacob

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Binding Studies ◽

Beta Adrenergic ◽

Cell Counts ◽

Significant Difference ◽

Induced Aggregation

Abstract A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.

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Chronic Lymphocytic Leukemia (CLL) Cells Require Microenvironmental Stimuli to Resist Apoptosis through Activation of STAT3 Mediated by VEGF.

Blood ◽

10.1182/blood.v112.11.1069.1069 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1069-1069

Author(s):

Iris Gehrke ◽

Julian Paesler ◽

Rajesh Kumar Gandhirajan ◽

Regina Razavi ◽

Alexandra Filipovich ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

Lymphocytic Leukemia ◽

Vegf Receptor ◽

Mrna Levels ◽

Activated State ◽

Survival Effect

Abstract B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, but incompetent B-cells due to a decrease of apoptosis rather than an increase in proliferation. Vascular endothelial growth factor (VEGF) has been suggested to play an important role in this so called apoptotic block. However, so far little is understood whether VEGF is acting mainly as a microenvironmental stimulus and/or whether CLL cells themselves contribute to the enhanced apoptotic resistance by maintaining an autocrine VEGF loop. Moreover, it is unknown by which mechanisms VEGF prevents apoptosis and whether this can be circumvented by inhibition of VEGF signaling. By quantitative real time PCR we found no significant difference in mRNA VEGF levels in B-cells from CLL patients and healthy donors after isolation from blood. In contrast, ELISA revealed clearly increased levels of secreted VEGF in plasma of CLL patients and in the supernatant under culture conditions compared to healthy individuals. In addition, we found the VEGF receptor 2 (VEGFR2), which is existent in CLL and healthy B-cells, in a phosphorylated, hence activated state, to a significantly higher extent in CLL cells as assessed by intracellular phospho flow cytometry. In conclusion, despite its expression in healthy B-cells VEGF does not seem to be secreted and therefore, no VEGF receptor phosphorylation takes place. Whereas CLL cells exhibit a long life span in vivo, they die rapidly in vitro, suggesting major survival factors being existent in the CLL cells microenvironment. We found levels of secreted VEGF in supernatant decreasing with time in culture, going along with decreasing levels of phosphorylated VEGFR2 and increasing cell death as assessed by Annexin V-FITC/PI staining. This further supports the role of VEGF in CLL cell survival. Coculturing primary CLL cells with the bone marrow stromal derived cell line HS5 dramatically increased VEGF transcription and secretion and improved cell survival. Hence, VEGF expression in CLL cells is not only mediated by autocrine, but also paracrine stimuli involving bone marrow stromal. Knocking down VEGF in HS5 cells and subsequent coculture with CLL cells might prove the major role of VEGF in this survival supporting coculture setting. Besides coculturing also supplement of culture medium with recombinant human VEGF (rhVEGF) increased survival, but to a lesser extent than coculture, indicating a direct cell-cell interaction as advantageous. Furthermore, we found a downregulation of anti apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia 1 (MCL1) and BclXL upon VEGF stimulation. Also cyclinD1 was upregulated as seen by immunoblotting. We further tried to discover the underlying mechanism of how VEGF mediates its pro survival effect and found STAT3 to become phosphorylated on tyrosine 705 upon VEGF stimulation. In CLL STAT3 is known to be constitutively phosphorylated on serine 727. This phosphorylation is not sufficient to induce target gene expression though. We could show that Y705 phosphorylation of STAT3 is responsible for upregulation of anti apoptotic BCLXL and cyclinD1. A PCR array detecting mRNA levels of 84 transcription factors in untreated and VEGF stimulated CLL cells shall provide more information about mechanistical details how VEGF mediates it pro survival effect. Since VEGF seems to be a major player in CLL cell survival it might be a suitable target to overcome the apoptotic block. In first experiments we found an induction of apoptosis after neutralization of VEGF or inhibition of the VEGF receptor. This additionally highlights the severe importance of VEGF in the apoptotic block in CLL cells. Therefore, VEGF might serve as an excellent therapeutic target in CLL.

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CD72 Expression Patterns in ZAP70 Positive and Negative Chronic Lymphocytic Leukemia: Potential Regulatory Role in BCR Signalling

Blood ◽

10.1182/blood.v112.11.4159.4159 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4159-4159

Author(s):

Francisco P. Careta ◽

Rodrigo A. Panepucci ◽

Daniel M Matos ◽

Rodrigo Proto-Siqueira ◽

Wilson A. Silva-Junior ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Expression Patterns ◽

Surrogate Marker ◽

Lymphocytic Leukemia ◽

Negative Regulator ◽

Beta Catenin ◽

Cell Surface Glycoprotein ◽

Expression Levels

Abstract Introduction: Absence of mutations in IgVH genes or higher number of ZAP70+ cells (as a surrogate marker) in chronic lymphocytic leukemia (CLL) B-cells defines a patient group with a poorer clinical course. These features relate to the role of BCR signalling in the proliferation and survival of CLL B-cells, and establish a link between these markers and the biology of CLL prognostic subgroups. The identification of additional players in this context may help to better understand the molecular basis of this disease and contribute to develop new therapeutic approaches. A search for genes potentially related to BCR signalling, when comparing mutated and unmutated CLL cases using serial analysis of gene expression, revealed a 4-fold increase of CD72 tags in unmutated samples, a specific B cell surface glycoprotein known to transmit both positive and negative signals in BCR signalling. Objective: This finding lead us to explore the potential role of CD72 on BCR signalling in distinct CLL prognostic subgroups, as defined by ZAP70 expression. Methods: Percentage of ZAP70+ and CD72+ cells were evaluated by flow cytometry on gated CD19+CD5+ cells in 25 CLL samples. Positive cases for ZAP70 and CD72 were defined using a cut-off of 35% and 40% positive cells, respectively. Real time PCR was used to quantify the expression levels of 3 genes related to proliferation and survival, RELB, Beta-Catenin (CTNNB1) and AKT1, on 16 CD19+ enriched (purity > 90%) CLL samples. Results: Samples were classified as 11 ZAP70+ and 14 ZAP70−. Median percentage of CD72+ cells in ZAP70+ was significantly higher than for ZAP70− cases (82% compared to 39%, respectively, P=0.0029). Furthermore, percentages of CD72 and ZAP70 were positively correlated (r=0.5930 and P=0.0009). Interestingly, ZAP70+ cases were restricted to CD72+ cases (n=11, CD72+ZAP70+ [+/+]), whereas six CD72+ cases were ZAP70− (ZAP70−CD72+ [−/+]). Finally, there were 8 cases CD72−ZAP70− [−/−]. No differences among these 3 groups were observed in regard to laboratory parameters (white blood cells, total lymphocytes, lymphocyte percentage, haemoglobin, haematocrit and platelet number). Despite the reduced number of samples analysed (6 +/+, 6 −/− and 4 −/+), transcripts for RELB (P<0.05), CTNNB1 (P<0.05), and AKT1(P=0.057) were expressed at higher levels in ZAP70+CD72+ than in ZAP70−CD72+ samples. Additionally, the transcripts were expressed at higher levels in ZAP70−CD72− than in ZAP70−CD72+ samples, and this difference was statistically significant (P<0.05) for CTNB1 and AKT1, but not for RELB (P=0.054). Conclusion: Our data indicate that higher percentages of ZAP70+ cells are associated with higher expression levels of transcripts related to proliferation and survival of CLL B-cells. In the absence of ZAP70 expression, CD72 may act as a negative regulator of the BCR pathway, as indicated by the lowest levels of transcripts on ZAP70−CD72+ cases.

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