Human B Cell Lines and Primary B Cells Actively Secrete Granzyme B in Response to IL-2 Family Cytokines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1340-1340
Author(s):  
Bernd Jahrsdoerfer ◽  
Sue E. Blackwell ◽  
Thomas Simmet ◽  
George J. Weiner
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Granzyme B ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Main Function ◽  
Dependent Manner ◽  
Unexpected Findings

Abstract It is widely believed that the main function of B cells is antibody secretion, but not cellular cytotoxicity. Recently we found that human B cells activated with interleukin 21 (IL-21) and antibodies to the B cell receptor (BCR) or immunostimulatory oligonucleotides (CpG ODN) develop a phenotype similar to that of cytotoxic T lymphocytes. B cells treated in such a way start to secrete large amounts of granzyme B (GrB) instead of antibodies and, as in the case of B-chronic lymphocytic leukemia (B-CLL), acquire the capability to induce apoptosis in bystander B-CLL cells in a GrB-dependent manner. Using FACS and ELISpot analyses we could now demonstrate that GrB is actively secreted by B cells in a time-dependent manner and that IL-21 is not the only cytokine that induces GrB in B cells. Also cytokine combinations such as IL-10 and IL-4 as well as IL-10 and IFN-alpha induce GrB in normal B cells and various B cell lines including MEC-1 (CLL), ARH-77 (plasma cell leukemia) and Namalwa (Burkitts lymphoma). We conclude that IL-21 and further cytokines can induce B cells to produce functional granzyme B. Further studies are required to elucidate the interactions with B lymphocytes of cells producing these cytokines such as CD4+ T cells, regulatory T cells, NKT cells and plasmacytoid dendritic cells. Our unexpected findings could have significant implications on our understanding of the role of B cells in immune regulation and for a variety of immune phenomena including auto-, cancer and infectious immunity.

ZAP-70 Is Not Phosphorylated on the Activating Tyrosine Residue (Tyr319) in Chronic Lymphocytic Leukemia and B-Cell Lymphoma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 178-178
Author(s):  
Stefania Gobessi ◽  
Aleksandar Petlickovski ◽  
Luca Laurenti ◽  
Dimitar G. Efremov
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Progressive Disease ◽  
Cell Receptor ◽  
Kinase Domain ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
Cell Receptor Signaling

Abstract The protein tyrosine kinase ZAP-70 is expressed at high levels in leukemic B-cells from chronic lymphocytic leukemia (CLL) patients with progressive disease and short survival. ZAP-70 is a key component of the proximal T-cell receptor signaling pathway and is highly homologous to Syk, an important B-cell receptor signaling (BCR) molecule. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is still not well understood. In T-cells, upon TCR stimulation ZAP-70 becomes phosphorylated on Tyr319 by the Src-like kinase Lck, which results in the release of the ZAP-70 kinase domain from an autoinhibited state to a fully active conformation. The Tyr319 site in ZAP-70 corresponds to the Tyr352 site in Syk, which is phosphorylated in B-cells following BCR stimulation. We therefore investigated the activation status of ZAP-70 and Syk in BCR stimulated CLL B-cells, using phosphorylation of Tyr319 and Tyr352 as markers of their activation. Analysis of 10 ZAP-70-positive CLL samples by immunoblotting with the phospho-ZAP70Tyr319/SykTyr352 antibody revealed that ZAP-70 is not phosphorylated at this site either before or after BCR stimulation, although in control experiments with Jurkat T-cells ZAP-70 became phosphorylated on Tyr319 upon TCR stimulation. Moreover, the Tyr352 site in Syk was phosphorylated following BCR stimulation in 6 of the 10 CLL B-cell samples. To further investigate the reasons for the unexpected lack of ZAP-70 activation in CLL B-cells, we produced stable transfectants of the BJAB lymphoma B-cell line that expressed ZAP-70 at levels similar to those found in CLL cases with progressive disease. In agreement with the CLL B-cell experiments, the Tyr319 site in ZAP-70 was not phosphorylated either before or after BCR stimulation. Since phosphorylation of Tyr319 is Lck-dependent in T-cells, and this kinase is expressed also in CLL B-cells, we ectopically expressed Lck in the ZAP-70-positive BJAB clones. Again, the Tyr319 site was not phosphorylated, indicating that ZAP-70 does not undergo activation of the kinase domain also in this cellular system. In contrast, BCR crosslinking in BJAB cells induced significant phosphorylation of Tyr352 in Syk, which was further enhanced in the clones that coexpressed ZAP-70. Furthermore, analysis of downstream signaling pathways following BCR stimulation showed stronger and prolonged activation of ERK and to a lesser extent Akt in the ZAP-70 positive clones, whereas no difference was observed in terms of activation of PLC-γ 2, JNK and degradation of the NF-kB inhibitor IkB. These data indicate that ZAP-70 does not undergo full activation in B-cells, but can still enhance activation of certain downstream BCR signaling pathways, possibly by affecting the activity of the related PTK Syk.

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.

scholarly journals B-chronic lymphocytic leukemia cells and other B cells can produce granzyme B and gain cytotoxic potential after interleukin-21-based activation

Blood ◽  
2006 ◽  
Vol 108 (8) ◽  
pp. 2712-2719 ◽  
Author(s):  
Bernd Jahrsdörfer ◽  
Sue E. Blackwell ◽  
James E. Wooldridge ◽  
Jian Huang ◽  
Melinda W. Andreski ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Granzyme B ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Barr Virus ◽  
Enhanced Production ◽  
Cpg Oligodeoxynucleotide ◽  
Interleukin 21

AbstractB cells currently are not viewed as being capable of producing granzyme B or being cytotoxic. We found that B-chronic lymphocytic leukemia (B-CLL) cells treated with interleukin-21 (IL-21) produce low levels of granzyme B. The addition of either CpG oligodeoxynucleotide (ODN) or anti-B-cell-receptor antibody (anti-BCR) to IL-21 results in enhanced production of functional granzyme B by B-CLL cells. B-CLL cells treated with IL-21 and CpG ODN undergo apoptosis and are able to induce apoptosis of untreated bystander B-CLL cells. This effect can be inhibited by anti-granzyme B antibody. Benign human B cells, Epstein-Barr virus (EBV)-transformed lymphoblasts, and many standard lymphoma cell lines produce high levels of granzyme B in response to IL-21 and anti-BCR. Our results suggest that the ability to induce production of functional granzyme B by B cells could open new approaches to the therapy of B-CLL and other B-cell malignancies. Our findings also have significant implications for our understanding of the role of B cells for immune regulation and for a variety of immune phenomena, including cancer immunity, autoimmunity, and infectious immunity.

Naïve B Cells Differentiate into Highly Activated Granzyme B-Secreting B Cells in Response to Interleukin 21 and B Cell Receptor Stimulation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2555-2555
Author(s):  
Elisabeth Schwesinger ◽  
Magdalena Hagn ◽  
Thomas Simmet ◽  
Bernd Jahrsdoerfer
Keyword(s):  
B Cells ◽  
B Cell ◽  
Granzyme B ◽  
Self Regulation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
Antigen Uptake ◽  
Interleukin 21

Abstract Human B cells have so far not been known to express the cytotoxic molecule granzyme B (GrB). Recently we found malignant B cells from patients with chronic lymphocytic leukemia, activated with interleukin 21 (IL-21), secrete high amounts of GrB. Here we demonstrate healthy human naïve B cells differentiate into GrB-secreting B cells in response to IL-21 and B cell receptor (BCR) cross-linking. After 72 hours up to 90% of viable B cells were differentiating. GrB-secreting B cells were more resistant to apoptosis than unstimulated B cells and non-viable B cells did not contain GrB, suggesting that the primary function of B-cell-derived GrB is not self-regulation. Differentiation was associated with a CD19+CD27-IgD-CD38-phenotype, increased dextran uptake and strong upregulation of molecules involved in cell adhesion (CD54), antigen-presentation (MHC class II) and co-stimulation (CD86). In summary we describe a novel differentiation pathway of naïve B cells into highly activated GrB-positive B cells in response to IL-21 and BCR stimulation. Our data indicate this differentiation serves potential interactions with target cells including antigen uptake and presentation, but not self-regulation. Our findings may have significant implications for understanding the role of B cells in immunity, and may open novel immunotherapeutic approaches.

Chronic Lymphocytic Leukemia B-Cells Lack or Express Markedly Reduced Levels of the Phosphatase PHLPP, Which Regulates B-Cell Receptor Induced AKT Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2080-2080
Author(s):  
Mirza Suljagic ◽  
Luca Laurenti ◽  
Muhammad Alam ◽  
Pablo G Longo ◽  
Sami N Malek ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Akt Pathway ◽  
Immunoblotting Analysis ◽  
Akt Phosphorylation ◽  
Akt Activation

Abstract The PI3K/AKT pathway plays a central role in regulating cellular growth and survival. This pathway is activated by signals derived from various receptors and is tightly regulated through the action of several phosphatases, including SHIP and PTEN, which hydrolyze the PI3K product PIP3, and the recently identified PHLPP, which directly dephosphorylates AKT. Hyperactivation of the PI3K/AKT pathway has been implicated in the pathogenesis of many types of cancer, including chronic lymphocytic leukemia (CLL) and B-cell lymphoma. In addition, gene expression profiling and real-time RT/PCR analysis have recently shown differential expression of PHLPP mRNA in CLL subsets classified according to the presence of the 13q14 abnormality, with many CLL cases demonstrating absent PHLPP expression altogether. These findings prompted us to compare the levels of PHLPP expression in primary CLL B-cells (n=17) with normal tonsillar B-cells (n=4) and various lymphoma cell lines, including the diffuse large B-cell lymphomas (DLBCL) DHL-4, DHL-6, DHL-8, DHL-10, WSU, Toledo, Ly1, Ly3, Ly7 and Ly18, the Burkitt’s lymphoma BJAB and the prolymphocytic leukemia MEC1. Immunoblotting analysis revealed abundant and uniform expression of PHLPP in normal B-cells and in 7 out of 12 investigated lymphoma cell lines. Higher levels were observed in the BJAB, Ly1 and Ly18 cell lines, whereas PHLPP was undetectable in the DLBCL cell lines WSU and Toledo. Remarkably, PHLPP was either not expressed or was expressed at markedly reduced levels in all of the investigated CLL samples, with levels of expression ranging from 0 to 10% of the levels in normal B-cells. In contrast, the levels of expression of the phosphatase SHIP were relatively similar between CLL and normal B-cells. To determine what are the consequences of reduced PHLPP expression on signaling through AKT in malignant B-lymphocytes, we downregulated PHLPP in BJAB and DHL-4 cells by RNA interference. A significant reduction in the levels of PHLPP was achieved in both cell lines, which amounted to 20–40% of the levels in cells transfected with the control siRNA. Immunoblotting analysis of protein extracts from cells transfected with PHLPP and control siRNA did not show a difference in AKT phosphorylation on Ser473 and Thr308, indicating that a reduction in PHLPP expression is not sufficient to augment basal AKT activity. To determine the effects of PHLPP downregulation on agonist-induced AKT activation, we investigated phosphorylation on Ser473 and Thr308 in BJAB and DHL-4 cells stimulated through the B-cell receptor. In both cell lines downregulation of PHLPP resulted in more than a 50% increase in BCR-induced AKT phosphorylation. In contrast, phosphorylation of other signaling molecules that are also activated by BCR crosslinking, such as PLCγ2 and ERK, appeared unaffected by PHLPP downregulation. These data confirm the functional relevance of PHLPP in AKT regulation in B-lymphoid cells and implicate reduced or absent PHLPP expression in CLL B-cells as a potential determinant of BCR-induced AKT signaling in CLL.

Clinical Significance of Free Circulating CD3 and CD5 in Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4947-4947
Author(s):  
Menna Hodge ◽  
Susan O’Brien ◽  
Adam Abdool ◽  
Michael Keating ◽  
Iman Jilani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Normal Control ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Β2 Microglobulin ◽  
Control Subjects

Abstract </DEL> CD5, a transmembrane protein expressed in T-cells, few B-cells, and chronic lymphocytic leukemia (CLL) B-cells, is the ligand for CD72 and may play a role in B-cell-T-cell communication. CD5 is part of the T-cell receptor (TCR)-CD3 complex in T-cells as well as the B-cell receptor (BCR) complex and serves as substrate for induction of tyrosine kinase activity. Since leukemic cells have high turnover and pour their protein, RNA, and DNA into the circulation, we speculated that free circulating CD3 (cCD3) and CD5 (cCD5) could be detected in the plasma of patients with CLL. We have developed a bead-based sandwich immunoassay to measure cCD3 and cCD5 in the plasma. Using this assay, we assessed the value of cCD5 measurement, alone and after normalization to cCD3 levels, as a tumor marker in CLL. Plasma levels of cCD3 and cCD5 were measured in 85 patients with CLL and 51 normal control subjects. cCD3 and cCD5 levels were significantly higher in patients with CLL (median, 7,465 and 55,806 U/μl, respectively) than in normal control subjects (median, 830 and 1,671 U/μl, respectively). Patients with CLL had significantly higher cCD5:cCD3 ratios (median, 5.28; range, 0–161) than did normal controls (median, 1.70; range, 0–8.06) (P <0.0001). Levels of cCD5, but not cCD3, correlated positively with WBC count, β2-microglobulin level, splenomegaly, and Rai stage (all P <0.01). The cCD5:cCD3 ratio also correlated with Rai stage (P = 0.04) and β2-microglobulin level (P = 0.03). cCD5 levels and the cCD5:cCD3 ratio both correlated with survival (P = 0.03). These findings confirm that free circulating surface markers can be detected in the circulation of patients with CLL, most likely reflect the tumor load, and can be used as tumor markers. The biological and therapeutic relevance of these free circulating proteins should be considered in pharmacokinetic and pharmacodynamic studies.

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

Abstract 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.

scholarly journals DUSP1/6 Inhibition Reduces Tumor Cells and Activates Immune Response in Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2857-2857
Author(s):  
Martina Braun ◽  
Veronika Ecker ◽  
Tanja Neumayer ◽  
Markus Muschen ◽  
Jürgen Ruland ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Immune Cells ◽  
Healthy Donor ◽  
Lymphocytic Leukemia ◽  
Downstream Signaling ◽  
Bcr Signaling

Abstract Chronic lymphocytic leukemia (CLL) is characterized by an expansion of monoclonal, mature B cells that carry auto/polyreactive B cell receptors (BCRs). It is one of the most prevalent B cell malignancies in Western countries. Over the past decade, extensive research has led to significant improvement in understanding its pathogenesis, particularly in regard to the BCR signaling pathway. This has led to the development of novel and promising treatment options, however, none of these approaches are curative and relapses are challenging to treat. Until now allogeneic hematopoietic stem cell transplantation remains the only treatment option for durable remission in poor-risk disease. The therapeutic difficulties result from the heterogeneity, the advanced age of the patients, chemoresistance and relapse of the disease. Here, we test a novel strategy for simultaneously targeting the malignant cells and reverting the CLL-suppressed immune response. Stimulation of the antigen receptors of immune cells readily induces proliferation, differentiation and functional activation. However, strong binding of antigen can also induce negative selection in lymphocytes. CLL B cells constitutively signal through their BCR and thus we set out to investigate whether they rely on the counterbalancing, negative regulation of specific downstream signaling pathways, in particular the mitogen-activated protein kinase (MAPK) pathway. The dual specificity phosphatases 1 and 6 (DUSP1 and DUSP6) dephosphorylate extracellular-signal regulated kinase 1/2 (ERK1/2) and thereby limit ERK1/2 activation. These molecules are frequently downregulated in solid tumors (Khor et al., Int. J. Med.Sci 2013; Okudela et al., Am. J. Pathol. 2009). We therefore set out to analyze the expression levels of DUSP1 and DUSP6 in CLL and found them readily expressed at various levels, comparable to normal B cells. To determine the functional relevance of DUSP1 and DUSP6, we blocked their phosphatase function using the small molecule inhibitor BCI. By treatment with BCI, we induced hyperactivation of the MAPK signaling cascade followed by cell death of the CLL cells. Interestingly, the induction of cell death is specific for CLL cells and does not occur to the same extent in other malignant B cell lymphoma cells or healthy donor-derived B cells. This deleterious effect of BCI was evident in primary patient-derived CLL cells as well as in the CLL-like cell lines MEC-1 and EHEB and in CLL cells derived from the T cell leukemia/lymphoma 1 (TCL1)-driven mouse model. To further investigate the downstream signaling event upon BCI treatment, we conducted a global phosphoproteome analysis. After treatment of primary CLL cells with BCI, the most significant alterations were within the BCR signaling pathway, including hyperphosphorylation of ERK1/2 and followed by a rapid induction of a DNA damage response. These results were validated by immunoblot analysis of human and murine CLL cells and were not detected in BCI-resistant cell lines. Beside the direct effects of BCI on the CLL cells we set out to investigate effects on other immune cells, directly by BCI and secondary via CLL cells treated with BCI. Indeed, we observed changes in immune cell compartment: BCI-treated CLL patient-derived peripheral blood mononuclear cells (PBMCs) resulted in selective enrichment of cytotoxic T cells. Furthermore, BCI treatment of CLL cells fed with Ovalbumin, in co-culture with or without BMDCs and OT-I cytotoxic CD8 T cells (specifically recognizing the SINFEKL peptide), resulted in the induction of immunogenic cell death of CLL cells. This was evidenced by enhanced antigen-specific T cell proliferation and release of the high mobility group box 1 protein (HMGB1). To investigate indirect effects of BCI, we treated CLL cells with BCI at sublethal doses, then washed the cells and co-cultured these primary CLL cells or CLL cell lines with healthy donor-derived PBMCs. The frequencies of CLL-induced myeloid-derived suppressor cells (MDSCs) as well as regulatory T cells (Tregs) were reduced after co-cultivation of PBMCs with BCI-pretreated CLL cells. Taken together, our data indicate that negative feedback inhibition reduces CLL content by induction of immunogenic cell death and activates immune cells to target the CLL-induced dysfunction of the immune system. We therefore propose that inhibition of DUSP1/6 is a promising therapeutic approach for CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pharmacologic Targeting MCL1 with AZD5991 Induces Apoptosis and Mitochondrial Dysfunction in Non-Hodgkin Lymphoma (NHL) Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Tingting Liu ◽  
Fei Xu ◽  
Elana Thieme ◽  
Vi Lam ◽  
Guang Fan ◽  
...  
Keyword(s):  
B Cells ◽  
Mitochondrial Dysfunction ◽  
B Cell ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Synthetic Lethality ◽  
Lymphocytic Leukemia ◽  
Dependent Manner ◽  
Oncology Research

BCL2 family proteins determine cell fate and comprise pro-apoptotic "initiators" (NOXA, BIM, PUMA), anti-apoptotic "guardians" (BCL2, MCL1, BCLX) and pro-apoptotic effectors (BAX/BAK). Venetoclax, a BCL2 inhibitor, received regulatory approval in therapy of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia. However, venetoclax is modestly effective in NHL. MCL1 is a short-lived pro-survival protein that is frequently overexpressed in NHL, leading to increased cell survival and drug resistance. MCL1 transgenic mice develop lymphoma, mostly of the diffuse large B-cell (DLBCL) type. Thus, selective targeting MCL1 represents a promising pharmacologic strategy in NHL. AZD5991 is a small molecule inhibitor highly selective towards MCL1 (Tron et al, 2018) in clinical trials. Here we explored pre-clinical activity of AZ5591 in NHL. Experiments were conducted in activated B-cell (ABC)-like (OCI-LY3, U-2932) and germinal center (GC)-like (OCI-LY18/19, SUDHL4/6/10, VAL) DLBCL cell lines; parental and ibrutinib-resistant Mino mantle cell lymphoma (MCL) cell lines. Peripheral blood mononuclear cells were isolated from patients with CLL and MCL and co-cultured with BAFF- or CD40L-expressing stroma. AZD5991 was obtained from Activ Biochem Ltd. MCL1 expression was detected in 10 tested DLBCL cell lines, highly expressed in 7/10. Meanwhile, BCLX was upregulated in eight, and BCL2 in 5/10 lines. Four cell lines expressed all three proteins. To confirm relevance of this data, we conducted analysis of primary DLBCL lymph nodes (n=30). MCL1 and BCLX were expressed in 50% and 80% of GC-like, and 10% and 25% of non-GC tumors, respectively. Treatment with AZD5991 restricted growth of DLBCL cells in a dose-dependent manner. GC-like cell lines VAL, SU-DHL4 and SU-DHL6 were highly susceptible with IC50~0.2 µM. Interestingly, they expressed relatively low levels of MCL1. Similarly, parental and ibrutinib-resistant Mino MCL cells were susceptible to MCL1 inhibition, with IC50 of 0.1 µM and 0.5 µM, respectively. Meanwhile, SU-DHL10, OCI-LY19 (GC-like), OCI-LY3 and U-2932 (ABC-like) cells were resistant to AZD5591. Consistent with its mechanism of action, immunoprecipitation assays showed that AZD5591 displaced BIM from MCL1 in NHL cells. We then evaluated MCL1 inhibition in primary neoplastic B-cells. CLL and MCL cells from patients were co-cultured with either CD40L- or BAFF-expressing stroma for 24 h. While CD40L predominantly induced BCLX, BAFF upregulated MCL1 in those cells. Consistent with this, BAFF-stimulated cells were highly sensitive to AZD5991, while CD40L-stimulated cells exhibited resistance. Since MCL1 acts in balance with pro-apoptotic effectors BAX and BAK at the mitochondrial membrane, we assessed the physiological effects of MCL1 inhibition on the mitochondrial function. Treatment with AZD5991 induced mitochondrial depolarization and led to a reduction in mitochondrial mass as well as increased generation of reactive oxygen species. This was accompanied by a decrease in cellular maximal respiratory capacity in both DLBCL and parental/ibrutinib-resistant MCL cells. Meanwhile, the rate of glycolysis was not significantly impacted. Interestingly, MCL1 inhibition induced mitophagy in sensitive (VAL) but not resistant cells (OCI-LY3). Next, we evaluated AZD5991 for synthetic lethality in a functional MTS-based screening assay using a panel of 189 small molecule inhibitors that target a variety of distinct signaling pathways activated in cancer (Tyner et al, 2018). AZD5991 demonstrated synergy with other BH3-mimetics. Co-treatment of DLBCL cells with BCL2/X inhibitors AZ4320, venetoclax and navitoclax overcame resistance to AZD5991. In summary, MCL1 inhibition using selective BH3-mimetic AZD5991 restricts cell proliferation and induces apoptosis in a subset of DLBCL, ibrutinib-resistant MCL cells and primary neoplastic B cells. MCL1 inhibition leads to mitochondrial dysfunction and mitophagy. Resistance to MCL1 inhibition may be overcome by concurrent targeting of alternative anti-apoptotic proteins (BCL2/X) in NHL. Disclosures Tyner: Syros: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Aptose: Research Funding; Constellation: Research Funding; AstraZeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Petra: Research Funding; Agios: Research Funding. Danilov:Abbvie: Consultancy; Celgene: Consultancy; Rigel Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Research Funding; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy, Research Funding; Verastem Oncology: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; TG Therapeutics: Consultancy; Nurix: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Karyopharm: Consultancy.

Quantification of Recent Bone Marrow B-Cell and Thymic T-Cell Emigrants in Chemotherapy Naïve Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2353-2353
Author(s):  
Marina Motta ◽  
Claudia Ghidini ◽  
Cinzia Zanotti ◽  
Marco Chiarini ◽  
Luigi Caimi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mutational Status ◽  
Cd4 Lymphocytes

Abstract Abstract 2353 Poster Board II-330 Background Chronic lymphocytic leukemia (CLL) results in accumulation of mature, malignant, monoclonal B cells in blood, lymph nodes, spleen, liver, and bone marrow. Patients with CLL have fundamental defects in both humoral and cell-mediated immunity that significantly impact on its clinical course. It is still not known if these immune abnormalities include a decrease of new B- and T-cell mobilization from their production sites. During early lymphocyte development and differentiation, occurring in bone marrow and in thymus, B- and T-cells undergo rearrangements of B-cell and T-cell receptor genes whereby specific chromosomal sequences are excised to produce episomal DNA products identified respectively as T-cell receptor excisional circles (TRECs) and Kappa-deleting recombination excision circles (KRECs). Being stable circular fragments of DNA, TRECs and KRECs do not replicate during the mitotic process required for cell proliferation and, therefore, they are diluted out by cell divisions and are lost when the cells die. Since T-cells leaving the thymus are 70% TRECs+ and KRECs are present randomly in about 50% of neo-produced normal B-cells, the quantification of TRECs and KRECs allows a good estimation of the thymic and bone marrow output. Aim To investigate the extent of neo-synthesis of normal B and T cells in untreated patients with CLL. Patients and Methods Twelve previously untreated CLL patients were enrolled in this study. M:F ratio was 5:1; median age was 66; 7 patients had mutated IgVH genes, while 5 patients had unmutated IgVH genes; all patients were stage A Binet; FISH analysis including del(13q), del(11q), del(17p) and trisomy 12 detected 13q deletion in 6 patients while the other 6 patients presented no abnormalities. TRECs and KRECs were measured in mononuclear cells isolated from peripheral blood by duplex quantitative Real-Time PCR. This new assay, based on the use of a standard curve prepared with a plasmid containing fragments of TRECs, KRECs and of a reference gene, allows to quantify neo-produced B and T lymphocytes. T-cell subpopulations were determined by flow cytometry as follows: recent thymic emigrants (RTE) as cells with CD4+CD45RA+CCR7+CD31+ phenotype, regulatory T cells (Treg), as CD4+CD25int/highCD127low, RTE-Treg as Treg expressing CCR7+CD31+CD25int/high markers, effector memory (TEM) and central memory (TCM) T cells as lymphocytes displaying CD4+CD45RA-CCR7- and CD4+CD45RA-CCR7+ phenotype. Results The overall number of CD4+ lymphocytes was increased in chemotherapy-naïve patients with CLL (1585/μl vs 953/μl; p=0.02). TRECs were significantly higher in CLL patients compared to age-matched healthy controls (2.9/ml vs 1.4/ml; p=0.04), while the proportion of RTE was lower (24.9% vs 32.4%; p=0.02). No significant differences were observed in the percentage of Treg and RTE-Treg as well as of TCM. On the contrary, the percentage of TEM was higher (22.4% vs 10.4%; p=0.01) in CLL patients. The number of KRECs was lower in CLL patients than in controls (3.8/ml vs 7.4/ml; p=0.004). No correlation between IgVH gene mutational status, Ig levels and KRECs was found. Conclusions The neo-synthesis of normal T, with the exception of Treg, and B cells is reduced in patients with CLL compared to controls, even in a good prognosis, chemotherapy-naïve subset. The increased number of TRECs+ cells and TEM together with low RTE could be ascribed to the increased number of circulating CD4+ lymphocytes which do not appear to be recently mobilized from the thymus (CD31- cells) or to undergo peripheral expansion, but to accumulate in the peripheral blood. The low number of KRECs compared to normal controls could be interpreted as a consequence of the abnormal leukemic B-cell expansion which may impair normal B cell neo-synthesis. The level of KRECs did not seem to correlate to the mutational status of IgVH genes or the Ig level but a larger number of patients is needed to confirm these preliminary data and to establish whether the analysis of TRECs and KRECs may help clarify the complex immune abnormalities in CLL. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document