Role of β2 integrins in the prevention of apoptosis induction in chronic lymphocytic leukemia B cells

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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Different Expression of FcgammaRIIb in Chronic Lymphocytic Leukemia and Human Normal B Lymphocytes

Blood ◽

10.1182/blood.v112.11.3134.3134 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3134-3134

Author(s):

Carol Moreno ◽

Rajendra Damle ◽

Sonia Jansa ◽

Gerardo Ferrer ◽

Pau Abrisqueta ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

B Lymphocytes ◽

Surface Membrane ◽

Memory B Cells ◽

Lymphocytic Leukemia ◽

Cd38 Expression ◽

B Cell Subsets

Abstract The Fcgamma receptors (FcγRs) are a family of molecules that modulate immune responses. FcγRIIb is an inhibitory FcγR that bears immunoreceptor tyrosine-based inhibitory motifs which transduce inhibitory signals on coligation with the surface membrane Ig of the B-cell antigen receptor (BCR). The role of FcγRIIb in controlling B cell activation through inhibition of BCR signaling has been extensively studied in animal models. Nevertheless, data on FcγRIIb are scant in human normal and neoplastic B cells, this being due to the lack of a specific antibody for human FcγRIIb. Consequently, there is little information on this receptor in chronic lymphocytic leukemia (CLL). Considering the activated nature of CLL cells and the central role of the BCR in the biology of the disease, studies of FcγRs are warranted. We used a novel specific mAb directly conjugated with Alexa 488 fluorophore that solely reacts with the human FcγRIIb (MacroGenics, Inc.) to investigate the receptors expression on CLL and normal human B cells. The study population included 84 patients with CLL and 24 age- and sex-matched controls. FcγRIIb expression was assessed as the mean fluorescence intensity (MFI) of surface membrane staining. In CLL cells, FcγRIIb was measured on CD19+CD5+ cells in combination with CD38, CD49d or CD69. Normal B cells were immunostained for CD19, CD5, IgD and CD38 expression and B cell subsets: naïve (IgD+CD38−), activated (IgD+CD38+) and memory B cells (IgD−CD38−) were studied for their relative expression of FcγRIIb. FcγRIIb expression was found significantly higher in naïve B cells compared to activated and memory B cells [median MFI: 17420 (11960–21180) vs. 11.140 (7899–16970) and 11.830 (6984–17100); p<0.001]. Significant differences were also observed between CD5− and CD5+ normal B cells. In contrast, FcγRIIb expression was lower in CLL cells than in CD5+ and CD5− normal B lymphocytes [median MFI: 6901(1034–42600), 10180 (5856–14820) and 12120 (7776–16040); p<0.05)]. Interestingly, FcγRIIb expression was variable within individual CLL clones, this being higher in CD38+ and CD49d+ cells than in CD38− and CD49d− cells (p<0.05). Furthermore, the highest density of FcγRIIb was observed on those cells which coexpressed CD38 and CD49d. In contrast, no significant differences were observed between FcγRIIb and the expression of the activation antigen CD69. Although CD69 and CD38 expression was significantly higher on unmutated IGHV cases, no correlation was found between FcγRIIb levels and IGHV mutational status. Similarly, there was no correlation between FcγRIIb and other poor prognostic variables such as ZAP-70 (≥20%), CD38 (≥ 30%) or high risk cytogenetics. Nevertheless, cases with ≥ 30% CD49d+ cells had higher FcγRIIb expression than those with <30% CD49d+ cells (p=0.006). The findings presented in this study suggest a hierarchy of FcγRIIb expression in normal B-cells, CLL cells and their subpopulations: circulating normal CD5− B cells > circulating normal CD5+ B cells > circulating CD5+ CLL B cells. In addition, although FcγRIIb is present on all normal B cell subsets its expression is higher in naïve B cells. Furthermore, in CLL FcγRIIb density is greater in CD38+ and CD49d+ cells within the clone. Although CD49d and FcγRIIb on CLL clones is linked in a direct manner, there is no relationship with FcγRIIb density and IGHV mutations, ZAP-70, CD38 and unfavorable cytogenetic markers. Finally, the relationship between FcγRIIb expression on CLL cells and functional responses to BCR and other receptor-mediated signals deserve further investigation.

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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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CD1d Expression Is Higher In Chronic Lymphocytic Leukemia Patients With Unfavorable Prognosis

Blood ◽

10.1182/blood.v122.21.5278.5278 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5278-5278

Author(s):

Agnieszka Bojarska-Junak ◽

Iwona Hus ◽

Anna Dmoszynska ◽

Jacek Rolinski

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Conflicts Of Interest ◽

Staining Intensity ◽

Lymphocytic Leukemia ◽

Disease Stage ◽

Inkt Cells ◽

Cd38 Expression

Abstract Chronic lymphocytic leukemia (CLL) is characterized by a very heterogeneous clinical course, which is slow and indolent in most of the patients, however some patient experience rapid disease progression and anticancer therapy is required shortly after the diagnosis. Many issues in CLL development and progression are still unclear. The functional consequences of CD1d expression on tumour cells are not well understood. However, increasing evidence suggests that they may affect iNKT cells.The role of CD1d expression in CLL immunopathogenesis remains undefined. In this study, we investigated the potential role of CD1d in CLL by analyzing the level of CD1d expression on leukemic B cells in peripheral blood of120 patients and assessed its correlation with prognostic markers such as ZAP-70 and CD38 expression, Rai stages and unfavourable cytogenetic changes.Measuring CD1d expression by flow cytometry and qRT-PCR, we showed lower CD1d molecule and CD1d mRNA expression in B cells of CLL patients than of healthy controls. The frequency of CD1d+/CD19+ cells, CD1d staining intensity and CD1d transcript levels increased with the disease stage. CD1d expression was positively associated with ZAP-70 and CD38 expressions as well as with unfavourable cytogenetic changes (17p deletion, 11q deletio),. We established the relationship between high CD1d expression and shorter time to treatment and overall survival. The percentage of CD1d+/CD19+cells inversely correlated with the percentage of iNKT cells. iNKT cells ζ-chain expression was downregulated in the high-CD1d group.These results suggest that high CD1d expression is associated with poor prognosis of CLL and might be involved in disease progression. Disclosures: No relevant conflicts of interest to declare.

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The prognostic role of CXCR3 expression by chronic lymphocytic leukemia B cells

Haematologica ◽

10.3324/haematol.10649 ◽

2007 ◽

Vol 92 (3) ◽

pp. 349-356 ◽

Cited By ~ 27

Author(s):

E. Ocana ◽

L. Delgado-Perez ◽

A. Campos-Caro ◽

J. Munoz ◽

A. Paz ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Prognostic Role ◽

Cxcr3 Expression

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Targeting PAK1 Overcomes Resistance to Ibrutinib in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2021-150715 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1546-1546

Author(s):

Zijuan Wu ◽

LEI Fan ◽

Luqiao Wang ◽

Hanning Tang ◽

Yi Miao ◽

...

Keyword(s):

Drug Resistance ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

High Throughput ◽

Combination Index ◽

Lymphocytic Leukemia ◽

Chromatin Conformation ◽

Rna Seq ◽

Ibrutinib Resistance

Abstract Objective: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance. Methods: An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining. Results: Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G). To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I). IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI<1) of this combinatorial therapy (Figure 1K). Conclusions: In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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The Role of Alpha 2 Macroglobulin in Chronic Activation of the Complement System in Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2020-141198 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-17

Author(s):

Naseba Naseraldeen ◽

Regina Michelis ◽

Masad Barhoum ◽

Judith Chezar ◽

Tamar Tadmor ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Complement System ◽

Serum Levels ◽

Lymphocytic Leukemia ◽

Classical Complement Pathway ◽

A Company ◽

The Complement System

The immunotherapy treatments offered for Chronic lymphocytic leukemia (CLL) activate various cellular and biochemical mechanisms, including the complement system. Recently it was shown that the classical complement pathway (CP) in CLL patients is persistently activated at a low level through (Michelis et al. PlosOne, 2019), and that the mechanism of chronic CP activation involves the formation of IgG-hexamers (Michelis et al. PlosOne, 2020). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur only after antigen binding. The study investigated the formation of IgG-hexamers in CLL patients and normal controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and NC were used for separation of IgG-hexamers. The obtained IgG-hexamers were measure and used for assessment of CP activity. For evaluation of the presence of IgG-hexamers on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG-hexamers were higher in patients compared to NC. Also, they activated the complement system to a higher extent in patients than in NC. Alpha 2 macroglobulin (A2M) was recognized as the antigen causing the hexamerization of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the surface of the cells. The exact course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens. Disclosures Tadmor: Neopharm: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Medison: Consultancy, Speakers Bureau; 6. Novartis Israel Ltd., a company wholly owned by Novartis Pharma AG: Consultancy, Speakers Bureau. Aviv:Abbvie: Honoraria.

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Significantly Shorter Telomeres in T-Cells of ZAP-70+/CD38+ Patients with Chronic Lymphocytic Leukemia (CLL): An Important Role of T-Cells in This Subgroup of CLL?.

Blood ◽

10.1182/blood.v110.11.1120.1120 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1120-1120

Author(s):

Alexander Roeth ◽

Dirk de Beer ◽

Holger Nueckel ◽

Ludger Sellmann ◽

Ulrich Duehrsen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Telomere Length ◽

Memory T Cells ◽

Lymphocytic Leukemia ◽

Healthy Individuals ◽

Cell Numbers

Abstract BACKGROUND: In contrast to other B-cell neoplasias, chronic lymphocytic leukemia (CLL) is not only characterized by a clonal expansion of specific B-cells, but also by an increase in non-leukemic T-cells, most likely involved in sustaining the growth of the leukemic B-cell clone. Based on ZAP-70, CD38 and the IgVH mutation status, two prognostic groups of CLL patients can be identified. Our aim was to characterize the replicative histories of the B- and T-cells in the two groups of CLL patients compared to healthy individuals. PATIENTS and METHODS: Blood samples from 73 patients with CLL (ZAP-70−/CD38−: n = 29, ZAP-70+/CD38+: n = 30, ZAP-70/CD38 discordant: n = 14) were analyzed. The quantity and characteristics of the lymphocyte subsets was assessed by a cell counter and by immunophenotypic analysis. The replicative histories of naive and memory T-cells as well as B-cells was determined by measurements of telomere length in peripheral blood leukocytes of CLL patients and healthy individuals by automated multicolor flow-FISH. RESULTS: As expected, the average telomere length of the clonal B-cells was short. The telomere length was, however, significantly shorter for the ZAP-70+/CD38+ patient samples (2.46 ± 1.08 kb) than for the ZAP-70−/CD38− patient samples (5.06 ± 1.76 kb, p < 6.7 x 10−9). Interestingly, also the naive and memory T-cells from ZAP-70+/CD38+ CLL patients exhibited significantly shorter average telomere lengths (mean ± std: 4.85 ± 1.58 kb; 4.39 ± 1.09 kb) than T-cells from ZAP-70−/CD38− CLL patients (6.64 ± 1.72 kb, p < 2.2 x 10−4; 6.22 ± 1.5 kb, p < 7.4 x 10−6). These results are in line with the observed higher absolute T-cell numbers in the ZAP-70+/CD38+ CLL patients compared to ZAP-70−/CD38− CLL patients. Moreover, the average telomere loss in relation to time from primary diagnosis to sample date was higher for naive T-cells than memory T-cells in ZAP-70+/CD38+ patients (7.8 vs. 5.8 bp/month). When we compared the telomere length to age-related percentiles calculated from over 400 healthy individuals aged 0–102 years practically all telomere length values of the naive and memory T-cells from the ZAP-70+/CD38+ CLL patients fell below the 50th percentile, whereas the values of naive and memory T-cells from the ZAP-70−/CD38− CLL patients were within the normal distribution. CONCLUSIONS: We can confirm significantly shorter telomere length values for the B-cells of the ZAP-70+/CD38+ CLL patients. In addition, we can also demonstrate significantly shorter telomeres in T-cells of ZAP-70+/CD38+ CLL patients, which are below the 50th percentile compared to controls, and a higher telomere loss over time for naive T-cells of ZAP-70+/CD38+ CLL patients. As telomere length shortens approximately 50 to 100 bp per cell division the observed decrease in telomere length of the T-cells in ZAP-70+/CD38+ CLL patients equals to approximately 18 to 36 population doublings. This is by far more than expected by the slightly higher T-cell numbers in the peripheral blood. Our observations imply an extensive expansion of the T-cell compartment in ZAP-70+/CD38+ CLL patients and suggest an important role of T-cells in this subgroup of CLL patients.

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MiR-181b in Chronic Lymphocytic Leukemia B Cells Is Regulated By Cellular Interaction with CD4+ T Cells and Increases the CTL Toxicity Versus the Leukemic Clone

Blood ◽

10.1182/blood.v126.23.4134.4134 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4134-4134

Author(s):

Mirco di Marco ◽

Serena Veschi ◽

Rosa Visone ◽

Giuseppe Leone ◽

Paola Lanuti ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd4 T Cells ◽

Lymphocytic Leukemia ◽

Healthy Donors ◽

Leukemic Clone

Abstract Clinical progression of chronic lymphocytic leukemia (CLL) is characterized by gradual reduction of the ratio T/B cells, along with immune cell dysfunction due, at least in part, to T cell defects, such as decreased expression of CD40L and reduced signaling via the TCR CD3. This compromise the ability of T cells to respond and to eliminate leukemic cell from CLL patients. Enhanced activation of either allogenic or autologous T cells can drive the death of CLL cells in vitro and in human subjects. Changes in microRNAs expression also characterize clinical progression of CLL with a strong decrease of miR-181b/a and miR-130a associated with the more aggressive phase of the disease. The miR-181b targets anti-apoptotic proteins, such as BCL-2 and MCL1 and its expression correlates with those protein levels in CLL. In this study we demonstrate that the expression of those microRNAs in CLL-B cells, are regulated by T cells. We co-cultured allogenic pure CLL-B cells with either activated (CD2, CD3 and CD28 antibodies, used to mimic antigen-presenting cells) or not activated CD4+ T cells from healthy donors. We observed a significant increase of mir-181b/a and miR-130a expression in CLL B-cells after co-culture with activated CD4+ T cells in 8 out of 11 cases. A significant increase of these miRs was also determined in purified CLL B-cells after 4 days activation of peripheral blood mononuclear cells (PBMCs) from CLL patients, even if in minor rate. By the use of specific antibodies, co-culture with Hela CD40 expressing cells and transwell experiments, we established that this effect is a T/B contact-dependent signaling mediated through CD40L-CD40 interaction. We determine that increased expression of the 3 miRs occurs at the transcriptional level. Since the expression of miR-181b showed the most significant variation in previous experiments it was selected for further analyses. We next investigated the in vivo role of the miR-181b in highly immunodeficient mice. The CLL cell line, MEC-01, infected with either the LV-miR-181b_coGFP or the LV-CTRL_coGFP was intravenously inoculated in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were sacrificed after 4 weeks and assayed for percentage of GFP+ cells in bone marrow and spleen compartments. The miR-181b did not show any specific effect into the leukemic clone. However when the same cells were inoculated in an environment hosting mature T cells, miR-181b consistently influences the death of leukemic cells (Fig 1B), suggesting that T cells are required to potentiate the apoptotic role of this miRNA. To explain what we observed in vivo, we mixed in vitro MEC-01 infected with either the LV-miR-181b or the LV-CTRL and CD8+ T cells from healthy donors. After few hours of contact T cells showed stronger cytotoxic effect on MEC-01 carrying miR-181b as compared to the control. Mixed lymphocyte reaction CD40L-activated CLL and T cells is used to generate effector CTLs. Therefore we grew T cell with CD40L-activated MEC-01 in which the expression of miR-181b was either shut down by lentiviral vector or unchanged as control. After one week, we monitored by cytofluorimetry the CD38 surface marker on T cells since its expression has been associated with more active CTLs and, by ELISA, the release of IL-10, the inhibitor of the potent inducer of CTLs INF-g. We demonstrate that activated MEC-01 with higher expression of miR-181b leads to an increase of the cell number expressing CD38 and this was accompanied by a reduced release of IL-10 from B cells through down-regulation of c-FOS, which we show to be target of the miR-181b and to promote the transcription of the IL-10. In conclusion, our data suggest a role of the miR-181b in the immune response against CLL-B cells. We show that an efficient activation of CD4+ T cells through CD3-complex pathway and a right CD40L-CD40 interaction lead to a significant increase of the some miRNAs deregulated over the progression of chronic lymphocytic leukemia, namely miR-181b. This miRNA potentiates the cytotoxicity of T cells favoring the killing of the leukemic clone. Disclosures No relevant conflicts of interest to declare.

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High Expression of Haematopoietic Cell Specyfic Lyn Substrate-1 (HS1) Predicts Survival of B-Cell Chronic Lymphocytic Leukemia Patients

Blood ◽

10.1182/blood.v118.21.2853.2853 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2853-2853

Author(s):

Aleksandra Butrym ◽

Miroslaw Majewski ◽

Justyna Dzietczenia ◽

Tomasz Wrobel ◽

Kazimierz Kuliczkowski ◽

...

Keyword(s):

Prognostic Factors ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Patient Survival ◽

Clinical Course ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Mutational Status ◽

Cell Chronic Lymphocytic Leukemia

Abstract Abstract 2853 B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults in western countries. It is characterized by B lymphocyte accumulation in peripheral blood, bone marrow, lymph nodes and other lymphatic organs. Leukemic cells derive most commonly from B lymphocytes, rarely from T or NK cells. B-CLL is known from its heterogeneous clinical course from indolent to very aggressive. In spite of many known prognostic factors (such as immunoglobulin heavy chain gene mutational status – IgVH, expression of ZAP70 and CD38), is still difficult to classify a single patient to particular risk group and to predict CLL clinical course. That is why new prognostic factors are still needed. HS1 (hematopoietic cell specific Lyn substrate-1) is an intracellular protein, which expression occurs mainly in hematopoietic cells. HS1 plays an important role in regulating T cell immune synapse and affects many functions of NK cells, including the lysis of target cells, adhesion, chemotaxis and clustering of actin in the lytic synapse. The role of HS1 in B cells is poorly understood. This protein was identified in B cells as the primary receptor substrate for phosphorylation by BCR after antigenic stimulation. Other studies have confirmed the role of HS1 in the process of clonal expansion and deletion induced by antigen-receptor interaction in B cells and T. HS1 is rapidly phosphorylated in B cells in the vicinity of tyrosine residues and is a substrate for tyrosine kinases: the Src family and Syk, including Lyn, FGR, Fyn and Lck. It has been shown that HS1 interacts with the cell cytoskeleton in both: normal and leukemic B cells. HS1 protein is an important regulator of motility, migration and adhesion of leukemic cells and is involved in cytoskeleton rearrangement. HS1 can have impact on homing and migration of CLL cells. It can indirectly promote disease progression and influence patient survival. The aim of this study was to evaluate HS1 expression in CLL patients in connection with other known prognostic factors and patient survival. Material and methods: 92 untreated CLL patients (45 women and 47 men), aged between 42 and 88 years (median age 67 years), were included into the study. Diagnosis was made basing on typical clinical, hematological and immunophenotypical picture. The control group was consist of 28 healthy matched people (11 men and 17 women), aged between 36 and 79 years (median age 59 years). HS1 protein expression was determined by western blot. Comparative semi-quantitative indication of the degree of saturation of the bands analyzed by densitometry using the gel documentation system Gel-Doc (Bio-Rad) and a computer program to analyze the 1-D Quantity One (Bio-Rad). Assuming conventional units [AU - arbitrary units], depending on the saturation band, patients were divided into four groups with the expression of HS1 protein expressed in value from 0 to 3. Lack of expression was expressed as 0 [AU], and expression of the strongest, with the highest saturation band measured as 3 [AU]. Mutational status of IgVH, as well as CD38 and ZAP70 expression were also analyzed. Results: HS1 expression was significantly higher in CLL patients comparing to controls. Positive correlation was shown between HS1 and: age (p=0.0454), Rai stage (p=0.0412), leukocytosis (p=0.0129) and β2-microglobulin (p=0.0342). There was negative correlation between HS1 and hemoglobin level (p=0.0464) and platelet count (p=0.0310). Patients with lymphocyte doubling time shorter or equal to 6 months had higher expression of HS1. Expression of HS1 significantly influenced survival of CLL patients. Patients with higher HS1 expression had shorter survival than those with lower HS1 expression (p=0.0329). Conclusions: 1. Higher HS1 expression is observed in more advanced CLL stages. 2. Expression of HS1 in CLL cells is matched with shorter patient survival The relationship between expression of HS1 and survival of patients with B-CLL. Disclosures: No relevant conflicts of interest to declare.

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