scholarly journals Apoptosis induced by polyclonal antilymphocyte globulins in human B- cell lines

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1051-1059 ◽  
Author(s):  
N Bonnefoy-Berard ◽  
L Genestier ◽  
M Flacher ◽  
JP Rouault ◽  
G Lizard ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Immunosuppressive Agents ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Lymphoma Cell ◽  
Dna Breaks ◽  
Nuclear Condensation ◽  
Susceptible Cell ◽  
Barr Virus

Antilymphocyte and antithymocyte globulins (ALG) are currently used as immunosuppressive agents in clinical transplantation and for the treatment of severe aplastic anemia. ALG contain a mixture of antibodies that recognize T- and B-cell-specific antigens but mostly nonlineage-specific molecules. We reported previously that ALG could inhibit the proliferation of activated B cells and B cell lines (Bonnefoy-Berard et al, Blood 79:2164, 1992). We show here that ALG induce apoptosis of several human hematopoietic cell lines, as shown by nuclear condensation and fragmentation in fluorescence and electronic microscopy and by double-strand DNA breaks shown by DNA electrophoresis. Apoptosis was achieved without elevation of intracellular Ca2+ and requirement for mRNA and protein synthesis. Most of the B-cell lines tested (Epstein-Barr virus [EBV]-transformed lymphoblastoid cell lines, EBV-negative and groups I/III EBV-positive Burkitt's lymphoma cell lines, as well as other B-lymphoma cell lines) were susceptible to ALG-induced cytotoxicity. Myelomonocytic and T-cell lines were much less susceptible than B-cell lines. Susceptibility to ALG-induced cytotoxicity was not correlated with intracellular Bcl-2 level. Most cell lines that express high levels of Fas/Apo-1 antigen were susceptible to ALG. However, several lines of evidence support the conclusion that, in addition to Fas/Apo-1, other cell surface molecules can mediate ALG-induced apoptosis. The cytotoxic activity could be fully removed by adsorption on susceptible cell lines but not on a resistant cell line, indicating that it was mediated by antibodies specific for surface antigens expressed only on susceptible cell lines. Apoptosis was triggered by ALG F(ab')2 fragments as well as by intact ALG. This cytotoxic property of ALG may account for their antiproliferative effect and might contribute to some extent to the relatively lower risk of posttransplant lymphoproliferative disorders previously reported in ALG-treated patients.

scholarly journals Apoptosis induced by polyclonal antilymphocyte globulins in human B- cell lines

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1051-1059 ◽  
Author(s):  
N Bonnefoy-Berard ◽  
L Genestier ◽  
M Flacher ◽  
JP Rouault ◽  
G Lizard ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Immunosuppressive Agents ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Lymphoma Cell ◽  
Dna Breaks ◽  
Nuclear Condensation ◽  
Susceptible Cell ◽  
Barr Virus

Abstract Antilymphocyte and antithymocyte globulins (ALG) are currently used as immunosuppressive agents in clinical transplantation and for the treatment of severe aplastic anemia. ALG contain a mixture of antibodies that recognize T- and B-cell-specific antigens but mostly nonlineage-specific molecules. We reported previously that ALG could inhibit the proliferation of activated B cells and B cell lines (Bonnefoy-Berard et al, Blood 79:2164, 1992). We show here that ALG induce apoptosis of several human hematopoietic cell lines, as shown by nuclear condensation and fragmentation in fluorescence and electronic microscopy and by double-strand DNA breaks shown by DNA electrophoresis. Apoptosis was achieved without elevation of intracellular Ca2+ and requirement for mRNA and protein synthesis. Most of the B-cell lines tested (Epstein-Barr virus [EBV]-transformed lymphoblastoid cell lines, EBV-negative and groups I/III EBV-positive Burkitt's lymphoma cell lines, as well as other B-lymphoma cell lines) were susceptible to ALG-induced cytotoxicity. Myelomonocytic and T-cell lines were much less susceptible than B-cell lines. Susceptibility to ALG-induced cytotoxicity was not correlated with intracellular Bcl-2 level. Most cell lines that express high levels of Fas/Apo-1 antigen were susceptible to ALG. However, several lines of evidence support the conclusion that, in addition to Fas/Apo-1, other cell surface molecules can mediate ALG-induced apoptosis. The cytotoxic activity could be fully removed by adsorption on susceptible cell lines but not on a resistant cell line, indicating that it was mediated by antibodies specific for surface antigens expressed only on susceptible cell lines. Apoptosis was triggered by ALG F(ab')2 fragments as well as by intact ALG. This cytotoxic property of ALG may account for their antiproliferative effect and might contribute to some extent to the relatively lower risk of posttransplant lymphoproliferative disorders previously reported in ALG-treated patients.

Identification of a Diagnostic Gene Signature for SGN-40, Anti-CD40 Monoclonal Antibody, in Pre-Clinical NHL Models and the Role of FAS in SGN-40 Mediated Apoptosis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1593-1593
Author(s):  
Xiaoyan Shi ◽  
Bart Burington ◽  
Tom Januario ◽  
Jeffrey T Lau ◽  
Shang-Fan Yu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Gene Signature ◽  
Resistant Cell ◽  
Lymphoma Cell ◽  
Hodgkin's Lymphoma ◽  
Sensitive Cell ◽  
Non Hodgkin's Lymphoma

Abstract SGN-40, an anti-CD40 monoclonal antibody, is a humanized IgG1 antibody that binds to CD40, mediates effector cell functions (ADCC/ADCP) and activates downstream signaling pathways. SGN-40 has shown activity in a phase I single agent multi-dose trial in non Hodgkin’s lymphoma, with greatest activity in diffuse large B-cell lymphoma (Advani et al. 2008, ICML). Previous in vitro studies implicated down-regulation of the germinal center expressed protein Bcl-6, upregulation of p53 family member TAp63a, and FAS death receptor induction as potential mechanisms leading to lymphoma cell death (Lewis et al. 2007, AACR, ASH). In order to further define the apoptosis signaling mechanism, we assessed the ability of SGN-40 to inhibit proliferation and promote apoptosis across a large panel of non-Hodgkin’s lymphoma cell lines. SGN-40 reduced cell viability in 58% (18/31) of cell lines tested. To identify the genes that may be promoting apoptosis and/or inhibiting proliferation, we compared gene expression levels before and after SGN-40 exposure in both sensitive and resistant cell lines, as well as in normal B-cells. SGN-40 strikingly and specifically upregulated FAS on the cell surface of sensitive cell lines. Furthermore, the addition of soluble FAS-Fc dampened SGN-40-induced apoptosis in a subset of sensitive cell lines, suggesting a dependence on a FAS-FASL interaction. These data imply that FAS-dependent apoptosis may directly contribute to the anti-tumor effect of SGN-40. Our data also demonstrate that SGN-40 sensitivity is dependent on the point in B-cell development at which the NHL cell lines were transformed. Sensitive cell lines had a gene signature characteristic of minimal activation of CD40 signaling prior to SGN-40 exposure, whereas resistant cell lines had a signature consistent with prior constitutive signaling downstream of CD40. Thus, SGN-40 appears to elicit its apoptotic properties through activation of CD40 signaling in NHL cell lines not previously exposed to CD40L signaling in the germinal center environment at the time of lymphocyte transformation (GCB lymphomas). In order to develop a clinically feasible assay from FFPE tissue, we developed a 14-gene signature by Stepwise Linear Modeling, utilizing genes from the CD40 pathway activation and GCB gene sets; the classifier gave >96% accuracy (30/31) on the ‘training’ set of cell lines and 75% (3/4) accuracy on a ‘test’ set of xenografts. Overall, our data provides unique insights into SGN-40 mechanisms of action, provides a testable hypothesis of the clinical mechanism of action, and a potential diagnostic test to identify patients more likely to benefit from SGN-40. Efforts are currently underway to test the clinical relevance of these findings.

Comparative Phosphoproteomics Study Between Obinutuzumab (GA101) Vs. Rituximab (RTX) Against RTX Sensitive/Resistant Burkitt Lymphoma (BL): Differentially Phosphorylated B Cell Receptor, Fc-Gamma Receptor, Phagocytosis and Natural Killer Cell-Mediated Cytotoxicity Signaling Pathway Proteins

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1550-1550
Author(s):  
Aradhana Tiwari ◽  
Delphine CM Rolland ◽  
Mona Elmacken ◽  
Janet Ayello ◽  
Carmella van de Ven ◽  
...  
Keyword(s):  
Cell Death ◽  
Natural Killer Cell ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Line ◽  
Cell Lines ◽  
Killer Cell ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Bcr Signaling

Abstract Background: Burkitt Lymphoma (BL) is the most common form of NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007, Cairo et al. JCO, 2012). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Goldman/Cairo et al. BJH, 2014). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab vs. RTX, a Type I CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathways are differentially altered following obinutuzumab vs RTX against RTX-sensitive/resistant BL. Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji/Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides and subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL, in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database (McDonnell and Lim et al, Blood, 2013). Silencing of PLCG2 in Raji and Raji4RH cell lines was carried out according to the manufacturer's instructions (Dharmacon, PA, USA). Results: Four hundred and eighteen out of total 661 proteins in Raji and 377 out of total 534 proteins in Raji4RH were differentially phosphorylated (>1.5 fold) after obinutuzumab treatment. Of these proteins, 46 were expressed at significantly higher levels in obinutuzumab vs. RTX in Raji (Figure 1). However, Raji4RH, the RTX resistant cell line did not show significant increase in phosphorylation of protein following in obinutuzumab vs. RTX. Proteins differentially phosphorylated in response to obinutuzumab vs. RTX are involved in the BCR (PLCG2, BTK, GSK3B and RAF-1), FC gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF-1), and Natural Killer cell-mediated cytotoxicity (MAPK1, RAF-1, PLCG2 and MAPK3) signaling pathways in Raji vs. Raji4RH (Figure 2). Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B), validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, revealed up-regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line. Silencing one of the BCR signaling pathway protein, PLCG2 significantly increased cell proliferation and decreased cell death after obinutuzumab vs. RTX treatment in Raji (P=0.0001 & 0.004) however, there was no change in Raji 4RH RTX resistant cell line. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, phagocytosis and cytotoxicity signaling pathways in BL. Knockdown of PLCG2 significantly enhanced BL proliferation and reduced cell death after obinutuzumab vs. RTX treatment. These results offer insights into alternate therapeutic strategies in the treatment of RTX resistant BL. Disclosures Klein: Roche: Employment.

B Cell Lymphomas Escape Bone Morphogenetic Protein (BMP) Induced Growth Inhibition: Downregulation of BMP Receptors and Upregulation of Inhibitory Smads.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2605-2605 ◽  
Author(s):  
Kanutte Huse ◽  
Maren Bakkebo ◽  
Morten Oksvold ◽  
Erlend B. Smeland ◽  
June H. Myklebust
Keyword(s):  
B Cells ◽  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Resistant Cell ◽  
Lymphoma Cell ◽  
Receptor Expression ◽  
Type I ◽  
B Cell Lymphomas

Abstract Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily and mediate their effects mainly through the Smad signaling pathway. TGF-β is one of the most potent negative regulators in hematopoietic cells, and many cancers develop reduced sensitivity towards TGF-β induced growth inhibition by several mechanisms, including functional loss of TGF-β receptors and Smad proteins. We have previously shown that BMP-6 inhibits the growth of normal peripheral blood B cells. As high BMP-6 mRNA expression is associated with poor outcome in diffuse large B cell lymphoma (DLBCL; Rosenwald et al, N Engl J Med 2002), we hypothesized that reduced sensitivity towards BMP-induced growth inhibition might contribute to lymphomagenesis. In the current study, 10 B lymphoma cell lines (representing Burkitt, DLBCL and FL) and tumor material from lymphoma patients were investigated to unravel the role of BMPs in lymphomas. We found that 5 – 7 out of 10 lymphoma cell lines were resistant towards BMP-2, -4, -6 and -7 induced growth inhibition. In comparison, only 3 of the cell lines were resistant towards TGF-β. Analysis of BMP receptor expression by FACS analysis showed that all lymphoma cell lines and the malignant B cells from primary lymphoma biopsies expressed the BMPR type I Alk-2, whereas the expression of Alk-3 and Alk-6 was variable. Interestingly, the expression of BMPRII was low or undetectable in BMP-6 resistant cell lines, whereas it was highly expressed in 3 out of 4 sensitive cell lines. Also, malignant B cells from lymphoma biopsies showed reduced levels of BMPRII, suggesting that downregulation of BMPRII is a mechanism for evading BMP induced growth inhibition. Interestingly, upregulation of Smad6 or Smad7 was seen in 3 of the BMP-6 resistant cell lines and might represent another way of escaping the inhibitory effects of BMP. The lymphoma cell lines were investigated for endogenous production of BMPs by real-time RT-PCR, and 2 out of 10 cell lines had detectable BMP-6 mRNA, whereas 7 cell lines expressed BMP-7 mRNA. Analysis of purified malignant B cells or normal tumor infiltrating T cells from patient biopsies, confirmed the expression of BMP-6 and BMP-7 in the malignant B cells. Altogether, the data suggest that escape from BMP induced growth inhibition might contribute to increased tumor growth in B cell lymphomas.

Evaluation of MLN4924, an investigational NEDD8-activating enzyme (NAE) inhibitor, as a novel targeted agent with single-agent antitumor activity in mantle cell lymphoma (MCL) and Hodgkin lymphoma preclinical models.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e18539-e18539
Author(s):  
Richa Dawar ◽  
Matthew John Barth ◽  
Cory Mavis ◽  
Jospeh J. Skitzki ◽  
Myron Stefan Czuczman ◽  
...  
Keyword(s):  
B Cell ◽  
Hodgkin Lymphoma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Cell Lymphoma ◽  
Resistant Cell ◽  
Chemotherapeutic Agents ◽  
Lymphoma Cell ◽  
Anti Tumor Activity

e18539 Background: MLN4924 is a novel, potent, and selective inhibitor of NAE, an essential component of the ubiquitin-proteosome system (UPS). We have previously demonstrated that the UPS plays a pivotal role in the development of rituximab and chemotherapy resistance in B-cell lymphomas. There is a scientific need to target the UPS more efficiently in an attempt to reverse acquired resistance to biological and chemotherapeutic agents in relapsed/refractory aggressive lymphoma. To this end, we studied the anti-tumor activity of MLN4924 in rituximab-chemotherapy sensitive and resistant pre-clinical models. Methods: A panel of Burkitt (BL), diffuse large B-cell (DLBCL), MCL and HL lymphoma cell lines and primary tumor cells isolated from patients with non-Hodgkin lymphoma (NHL) (N=10) were exposed to escalating doses of MLN4924 alone or in combination with a panel of chemotherapeutic agents for up to 72 hrs. Cell viability was determined by alamar Blue reduction or CellTiter-glo assay. Apoptosis was determined by Western blotting. Cell cycle analysis was performed by flow cytometry. Results: MLN4924 demonstrated activity in all cell lines in a time-and dose-dependent manner, including the rituximab/chemotherapy-resistant cell lines. The most potent activity was noted in MCL and HL cell lines (IC50 doses were tenfold lower as compared to DLBCL or BL cell lines). A variable degree of anti-tumor activity was observed in primary tumor cells isolated from NHL patients. Induction of apoptosis was observed in rituximab-resistant cell lines. MLN4924 exhibited synergistic anti-tumor activity when combined with bortezomib, bendamustine, and cytarabine in MCL cell lines. Conclusions: MLN4924 exhibits potent in vitro cytotoxic activity against a variety of human B-cell lymphoma cell lines and primary tumor cells isolated from NHL patients. Significant activity was observed in MCL cell lines. Experiments investigating the in vivo activity of MLN4924 are ongoing. MLN4924 is a highly promising agent for the treatment of relapsed/refractory MCL or HL.

Expression and Function of CD28 on Epstein-Barr Virus-Positive B Cell Lines and AIDS-Associated Non-Hodgkin’s Lymphoma Cell Lines

Tumor Biology ◽  
2003 ◽  
Vol 24 (2) ◽  
pp. 82-93 ◽  
Author(s):  
Daniel Widney ◽  
W. John Boscardin ◽  
Arjang Kasravi ◽  
Otoniel Martínez-Maza
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Lymphoma Cell ◽  
Barr Virus ◽  
Epstein Barr ◽  
And Function

scholarly journals The XIAP Inhibitor BMT-062789 Displays Anti-Tumor Activity in Cell Line Models of De Novo and Rituximab Relapse/Refractory Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5375-5375 ◽  
Author(s):  
Kyle L. Runckel ◽  
Cory Mavis ◽  
Juan J Gu ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
De Novo ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Lymphoma Cell ◽  
Lymphoma Cell Line ◽  
Raji Cells ◽  
Ic50 Values ◽  
Anti Tumor Activity

Abstract The addition of rituximab to front line therapy for aggressive lymphomas has improved clinical outcomes, but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for rituximab associated chemotherapy cross-resistance our group developed several rituximab resistance cell lines (Raji 4RH and RL 4RH), which also display significant resistance to a wide range of chemotherapy agents. These rituximab resistant cell lines (RRCL)s exhibit multiple deregulations in the BCL-2 and inhibitor of apoptosis (IAP) protein families, including loss of the pro-apoptotic proteins Bax and Bak. We previously demonstrated that the X linked inhibitor of apoptosis protein (XIAP) is critically required for chemotherapy resistance in the RRCLs, and that an shRNA knockdown of XIAP increased chemotherapy response in both in vitro and in vivo models of rituximab resistant lymphoma. BMT-062789 is a heterodimeric mimetic of the second mitochondrial activator of caspases (SMAC) developed by Bristol-Myers Squibb, which can inhibit both the caspase 9 and caspase 3/7 binding domains of XIAP. BMT-062789 demonstrated dose and time dependent single agent anti-tumor effect (as measured by the Cell TiterGlo luminescent viability assay) in a panel of lymphoma cell lines, with IC50 values of less than 5uM for all cell lines tested except the rituximab resistant cell line Raji 4RH. The Burkitt's lymphoma cell line Daudi and diffuse large B-cell lymphoma cell line U2932 were particularly sensitive to BMT-062789 with IC50 values of 0.91uM and 0.76uM respectively. To investigate if the observed anti-tumor effect of BMT-062789 was due to increased apoptosis we exposed rituximab sensitive (Raji, RL) and rituximab resistant (Raji 4RH, RL 4RH) cells to escalating doses of BMT-062789 with or without the addition of 20uM etoposide for 48 hours. The induction of apoptosis was measured by flow cytometry with an Annexin-V:PE-Cy7 conjugate and Sytox blue (a DNA stain). 2uM BMT-062789 alone triggered apoptosis in 75% of Raji cells and 65% of RL cells. It is also worth adding that 2uM BMT-062789 induced higher rates of apoptosis than 20uM etoposide alone in both cell lines. The combination of 2uM BMT-062789 and 20uM etoposide together triggered apoptosis in 80% of Raji cells and 85% of RL cells, indicating that BMT-062789 may be able to augment the anti-tumor activity of conventional chemotherapy. More importantly, the combination of BMT-062789 and etoposide was also able to induce apoptosis in the rituximab resistant cell line models Raji 4RH and RL 4RH. 3uM BMT-062789 in combination with 20uM etoposide triggered apoptosis in 90% of Raji 4RH cells and 55% of the RL 4RH cells, which is a substantial improvement compared to 15% apoptosis with 20uM etoposide alone in each cell line. Additional studies are in progress to evaluate the anti-tumor effect of BMT-062789 in ex vivo samples from lymphoma patients with de novo and relapse/refractory disease. In summary, the novel heterodimeric XIAP inhibitor BMT-062789 has anti-tumor effect at low micromolar concentrations in lymphoma cell line models, including models of rituximab resistant disease. These results support earlier studies by our group indicating that XIAP is critical for survival in models of rituximab resistant lymphoma, and establish that XIAP inhibitors may have potential clinical value for the treatment of both de novo, and rituximab relapse/refractory lymphomas. Disclosures No relevant conflicts of interest to declare.

scholarly journals Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Lisa Grossman ◽  
Chris Chang ◽  
Joanne Dai ◽  
Pavel A. Nikitin ◽  
Dereje D. Jima ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Cellular Aggregation ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out. Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

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