Neutrophils express CD52 and exhibit complement-mediated lysis in the presence of alemtuzumab

Blood ◽  
2009 ◽  
Vol 114 (14) ◽  
pp. 3052-3055 ◽  
Author(s):  
Lyn R. Ambrose ◽  
Anne-Sophie Morel ◽  
Anthony N. Warrens
Keyword(s):  
Monoclonal Antibody ◽  
Adverse Event ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Disease States ◽  
Dose Dependent

Neutropenia is a recognized adverse event in patients treated with the humanized anti-CD52 monoclonal antibody alemtuzumab. However, as it is widely believed that neutrophils do not express CD52, the etiology of alemtuzumab-associated neutropenia is unclear. We have found that neutrophils express both mRNA coding for CD52 and the protein itself on the cell surface. We confirmed cell-surface expression using 3 different anti-CD52 antibodies, and note that neutrophils express lower levels of CD52 than lymphocytes and eosinophils. Further, incubation of alemtuzumab with neutrophils results in dose-dependent, complement-mediated lysis in the presence of both heterologous and autologous complement. These data offer an explanation for the etiology of alemtuzumab-associated neutropenia. In a climate of increased use of alemtuzumab in leukemia and other disease states, as well as in transplantation, these data highlight the need for increased vigilance of emerging neutropenia in patients treated with alemtuzumab.

scholarly journals Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

2008 ◽  
Vol 295 (1) ◽  
pp. G16-G26 ◽  
Author(s):  
Mubeen Jafri ◽  
Bryan Donnelly ◽  
Steven Allen ◽  
Alex Bondoc ◽  
Monica McNeal ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Biliary Atresia ◽  
Cell Lines ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Newborn Mice ◽  
A Cell

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as α2β1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express α2β1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether α2β1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the α2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the α2β1-integrin. Newborn mice were pretreated with a monoclonal antibody against the α2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed α2β1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-α2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the α2β1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

scholarly journals Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas

2009 ◽  
Vol 343 (1) ◽  
pp. 28-41 ◽  
Author(s):  
Paul W. Price ◽  
Elizabeth C. McKinney ◽  
Youliang Wang ◽  
Loren E. Sasser ◽  
Muthugapatti K. Kandasamy ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Membrane Immunoglobulin

Preclinical In Vitro and In Vivo Evidence Support a Therapeutic Role for the CD70 Directed Monoclonal Antibody (SGN-70) in Waldenström’s Macroglobulinemia (WM).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2490-2490 ◽  
Author(s):  
Evdoxia Hatjiharissi ◽  
Allen W. Ho ◽  
Lian Xu ◽  
Kelly E. O’Connor ◽  
Zachary R. Hunter ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Disease Progression ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Induction Of Apoptosis ◽  
Direct Induction

Abstract Introduction: WM is a B-cell disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells (LPC), along with excess mast cells (MC) which support the growth and survival of BM LPC through multiple TNF-family ligands including CD40L, APRIL and BLyS/BAFF. Importantly, BM LPC stimulate cell surface expression of TNF-family ligands through release of sCD27 which induces CD70 on MC. We therefore have sought the development of agents which could target CD27-CD70 interactions. As such, we examined the therapeutic potential of directly targeting CD70 using the fully humanized monoclonal antibody SGN-70 (Seattle Genetics, Inc., Bothell WA). Methods-Results: As part of these studies, we used flow cytometric analysis to evaluate the expression of CD70 on primary WM patient BM LPC and MC, as well as 2 WM cell lines (BCWM.1 and WM-WSU). These studies demonstrated cell surface expression of CD70 on BM LPC and MC from 20/26 (77%) and 10/11 (90%) WM patients, respectively. We next assessed the ability of the SGN-70 antibody to eradicate primary WM LPC (n=5) and WM cell lines by assessing for direct induction of apoptosis, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) as well as induction of TNF family ligands on primary WM MC and the LAD2 MC line. Following incubation of WM LPC with SGN-70 (0.01–20 μg/ml), no direct induction of apoptosis or CDC activity was observed. However, SGN-70 mediated significant dose-dependent ADCC against WM LPC and MC at concentrations of 0.1–20 ug/ml. Importantly, SGN-70 blocked sCD27-induced expression of CD40L and APRIL on primary WM MC and LAD2 MC. To further evaluate the therapeutic potential of SGN-70 in an in vivo model, SCID-hu mice bearing BCWM.1 WM cells were treated with SGN-70 (1 mg/kg, i.p., qOD) Serum human IgM and sCD27 levels were measured by ELISA to monitor for tumor engraftment and disease progression. SGN-70 initiated 6 weeks following tumor engraftment blocked tumor growth in 12/12 treated mice, whereas all 5 untreated mice demonstrated disease progression. The results of these studies provide the framework for clinical trials to examine the therapeutic potential of the SGN-70 monoclonal antibody in WM.

scholarly journals CD44-Mediated Adhesiveness of Human Hematopoietic Progenitors to Hyaluronan Is Modulated by Cytokines

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 1905-1914 ◽  
Author(s):  
Stéphane Legras ◽  
Jean-Pierre Lévesque ◽  
Rachida Charrad ◽  
Kohji Morimoto ◽  
Caroline Le Bousse ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Adhesive Interaction ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Marrow Stroma ◽  
Adhesive Interactions

Abstract Adhesive interactions between CD34+ hematopoietic progenitor cells (HPC) and bone marrow stroma are crucial for normal hematopoiesis, yet their molecular bases are still poorly elucidated. We have investigated whether cell surface proteoglycan CD44 can mediate adhesion of human CD34+ HPC to immobilized hyaluronan (HA), an abundant glycosaminoglycan of the bone marrow extracellular matrix. Our data show that, although CD34+ cells strongly express CD44, only 13.3% ± 1.1% spontaneously adheres to HA. Short-term methylcellulose assay showed that HA-adherent CD34+ cells comprised granulo-monocytic and erythroid committed progenitors (19.6% ± 2.5% and 7.3% ± 1.0% of the input, respectively). More primitive progenitors, such as pre–colony-forming units, also adhered to HA. Moreover, we found that CD44-mediated adhesion of CD34+ cells to HA could be enhanced by phorbol 12-myristate 13-acetate (PMA), the function-activating anti-CD44 monoclonal antibody H90, and cytokines such as granulocyte-monocyte colony-stimulating factor, interleukin-3 (IL-3), and stem cell factor. Enhancement through PMA required several hours, was protein-synthesis–dependent, and was associated with an increase of CD44 cell surface expression, whereas stimulation of adhesion by H90 monoclonal antibody and cytokines was very rapid and without alteration of CD44 expression. H90-induced activation occurred at 4°C and lasted for at least 2 hours, whereas activation by cytokines required incubation at 37°C and was transient. These data, which show for the first time that CD34+ HPC can directly adhere to HA via CD44, point out that this adhesive interaction to HA is a process that may also be physiologically regulated by cytokines.

Anti-N-Terminal CD20 Monoclonal Antibody and L26 Dual Staining Identifies An Irreversible Resistant Mutant Genes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4145-4145
Author(s):  
Yuji Mishima ◽  
Yasuhito Terui ◽  
Yuko Mishima ◽  
Kiyohiko Hatake
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Binding Site ◽  
Immunohistochemical Analysis ◽  
Surface Expression ◽  
Terminal Deletion ◽  
Cell Surface Expression ◽  
Clinical Specimens ◽  
Deletion Mutations ◽  
Cytoplasmic Region

Abstract Abstract 4145 [Introduction] Recently, we reported that gene mutations of CD20 were involved in resistance to rituximab therapy, and we proposed that C-terminal deletion mutations of CD20 might be related to relapse/resistance after rituximab therapy. Many of these cases were diagnosed as CD20 negative by the immunohistochemical analysis using the L26 monoclonal antibody used routinely in most clinical laboratories. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. So, we could not distinguish whether protein expression of CD20 extremely decreased or whether the epitope of the antibody was lost by these mutations. To make this clear, we determined the binding site of L26 antibody on CD20 protein in the present study. In addition, we developed new antibodies that recognize amino acid sequence close to the amino terminal of CD20 molecule. Then we investigated clinical specimens with these antibodies together with L26 to elucidate characteristics of CD20 molecules having C-terminal mutations. [Methods] To determine the binding site of L26 antibody on CD20, we made a series of constructs of the CD20 molecules with deletion mutations in the C-terminal cytoplasmic domain and introduced them into retrovirus vectors. A CD20 negative multiple myeloma cell line, KMS12PE cells were then transformed, and we established six kinds of sub-lines with the various C-terminal deletion mutations of CD20 and used them for epitope-mapping. On the other hand, we screened the CD20 gene sequence of the clinical specimen of rituximab-resistant patients and identified several cases with the mutation in the C-terminal cytoplasm region. The immunochemistry using L26 and newly developed antibodies, as well as membrane expression of CD20 molecules using the rituximab were analyzed. [Results] The epitope analysis of L26 antibody using a series of CD20 deletion mutations revealed that L26 recognizes near the C-terminus of CD20 cytoplasmic region. These results showed that most of CD20 molecules with the C-terminal deletion mutation and frame-shift mutation could not be recognized by L26. The immunohistochemical analysis performed for clinical specimens revealed that the cells that were stained by antibodies recognizing N-terminal region of CD20 but not by L26 were indeed included in some rituximab-resistant cases. DNA sequencing analysis revealed that all these cases had mutated CD20 genes in its C-terminal cytoplasmic region. In addition, a cell-surface expression analysis using flowcytometry demonstrated that the cells having these mutations has reduced cell surface expression of CD20 compared with those of normal CD20. [Discussion] In this study, we determined the recognition site of L26 and demonstrated that L26 couldn't recognize CD20 with the resistant mutations. In contrast, newly developed antibodies against N-terminal region of CD20 could stain even these CD20 molecules. These results suggest that combination use of these antibodies and L26 enables to detect the onset of irreversible rituximab-resistant clones with the CD20 mutations. Disclosures: Hatake: Chugai Pharmaceutical Co., Ltd: Honoraria, Research Funding.

scholarly journals CD3- large granular lymphocytes recognize a heat-inducible immunogenic determinant associated with the 72-kD heat shock protein on human sarcoma cells

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1374-1382 ◽  
Author(s):  
G Multhoff ◽  
C Botzler ◽  
M Wiesnet ◽  
G Eissner ◽  
R Issels
Keyword(s):  
Monoclonal Antibody ◽  
Heat Shock ◽  
Cell Surface ◽  
Mhc Class I ◽  
Surface Expression ◽  
Class I ◽  
Cell Surface Expression ◽  
Effector Cells ◽  
Heat Treated ◽  
Sarcoma Cells

Traditionally, heat shock proteins (HSPs) are believed to be located intracellularly, where they perform a variety of chaperoning functions. Recently, evidence has accumulated that some tumor cells express HSPs on the cell surface. The present study confirms this finding and correlates HSP72 cell surface expression, induced by nonlethal heat shock, with an increased sensitivity to interleukin-2-stimulated CD3- natural killer (NK) cells. After nonlethal heat shock, a monoclonal antibody directed against the major heat-inducible 72-kD HSP (HSP72) stains the cell surface of sarcoma cells (ie, Ewing's sarcoma cells or osteosarcoma cells) but not that of normal cells (ie, peripheral blood lymphocytes, fibroblasts, phytohemagglutin-stimulated blasts, B- lymphoblastoid cell lines) or of mammary carcinoma cell line MX-1 carcinoma cells. In this study, we show for the first time a correlation of HSP72 cell surface expression with an increased susceptibility to lysis by NK effector cells. This finding is supported by the following points: (1) HLA-disparate effector cells show similar, elevated lysis of HSP72+ heat-treated sarcoma cells; (2) CD(3-) NK cells, but not CD3+ cytotoxic T lymphocytes, are responsible for the recognition of heat-shocked sarcoma cells; (3) by antibody-blocking studies, an immunogenic HSP72 determinant, which is expressed selectively on the cell surface of heat-treated sarcoma cells could be correlated with NK recognition; (4) the reported phenomenon is independent of a heat-induced, transient downregulation of major histocompatibility complex (MHC) class-I expression; and (5) blocking of MHC class-I-restricted recognition, using either MHC class-I- specific monoclonal antibody W6/32 on the target cells or alpha/beta T- cell receptor monoclonal antibody WT31 on effector cells, also has no inhibitory effect on the lysis of HSP72+ tumor cells. Finally, our in vitro data might have further clinical implications with respect to HSP72 as a stress-inducible, sarcoma-specific NK recognition structure.

Increased Expression of u-PA and u-PAR on Monocytes by LDL and Lp(a) Lipoproteins – Consequences for Plasmin Generation and Monocyte Adhesion

1999 ◽  
Vol 81 (04) ◽  
pp. 594-560 ◽  
Author(s):  
Florence Ganné ◽  
Marc Vasse ◽  
Jean-Louis Beaudeu ◽  
Jacqueline Peynet ◽  
Arnaud François ◽  
...  
Keyword(s):  
Fold Increase ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Atheromatous Plaque ◽  
Monocyte Adhesion ◽  
Blood Monocytes ◽  
Proteolytic Cascade ◽  
Ecm Degradation ◽  
Dose Dependent Increase ◽  
Dose Dependent

SummaryMonocyte-derived foam cells figure prominently in rupture-prone regions of atherosclerotic plaque. As urokinase/urokinase-receptor (u-PA/u-PAR) is the trigger of a proteolytic cascade responsible for ECM degradation, we have examined the effect of atherogenic lipoproteins on monocyte surface expression of u-PAR and u-PA. Peripheral blood monocytes, isolated from 10 healthy volunteers, were incubated with 10 to 200 µg/ml of native or oxidised (ox-) atherogenous lipoproteins for 18 h and cell surface expression of u-PA and u-PAR was analysed by flow cytometry. Both LDL and Lp(a) induced a dose-dependent increase in u-PA (1.6-fold increase with 200 μg/ml of ox-LDL) and u-PAR [1.7-fold increase with 200 μg/ml of ox-Lp(a)]. There is a great variability of the response among the donors, some of them remaining non-responders (absence of increase of u-PA or u-PAR) even at 200 μg/ml of lipoproteins. In positive responders, enhanced u-PA/u-PAR is associated with a significant increase of plasmin generation (1.9-fold increase with 200 μg/ml of ox-LDL), as determined by an amidolytic assay. Furthermore, monocyte adhesion to vitronectin and fibrinogen was significantly enhanced by the lipoproteins [respectively 2-fold and 1.7-fold increase with 200 μg/ml of ox-Lp(a)], due to the increase of u-PAR and ICAM-1, which are receptors for vitronectin and fibrinogen. These data suggest that atherogenous lipoproteins could contribute to the development of atheromatous plaque by increasing monocyte adhesion and trigger plaque weakening by inducing ECM degradation.

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