Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells

Nagy, Zoltán Á.; Hubner, Bernd; Löhning, Corinna; Rauchenberger, Robert; Reiffert, Silke U.; Thomassen-Wolf, Elisabeth; Zahn, Stefan; Leyer, Sigmar; Schier, Eva M.; Zahradnik, Angelika; Brunner, Christoph; Lobenwein, Kurt; Rattel, Benno; Stanglmaier, Michael; Hallek, Michael; Wing, Mark; Anderson, Steve; Dunn, Matt; Kretzschmar, Titus; Tesar, Michael

doi:10.1038/nm736

Cited by 135 publications

(121 citation statements)

References 32 publications

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“…This factor appears to have been overlooked in some previous reports. For example, earlier studies of the effect of L243 (anti-HLA-DR) on Raji cells (identical to the model we used), which claimed that apoptosis occurred, did not mention the dramatic aggregation that occurs (Kostelny et al, 2001;Nagy et al, 2002). One consequence of aggregation, which we documented, is that it may be impossible to disperse the cells in tight aggregates without killing the cells.…”

Section: Discussionmentioning

confidence: 75%

“…Binding of Abs to the cells surface has been frequently reported to induce apoptosis of lymphoma cells, especially if the Ab is crosslinked with a secondary Ab (Truman et al, 1997;Shan et al, 1998;Nagy et al, 2002;Carlo-Stella et al, 2006). In performing such experiments, we noted that Ab binding sometimes induced extensive aggregation of the cells.…”

Section: Cell Aggregation By Absmentioning

confidence: 70%

“…Because of the large aggregates, it was impossible to count the cells with an acceptable level of accuracy, and it was also impossible to test the cells for apoptosis by most of the methods that are usually employed. The aggregates started to develop within 2 h and became increasingly large and dense over the next 24 h. Initial experiments utilised L243 at 30 mg ml À1 , following Nagy et al (2002), but a range of Ab concentrations were tested. Judging microscopically by the extent of aggregation, 3 mg ml À1 had the same effect as 30 mg ml À1 , 30 ng ml À1 produced slight aggregation, and 300 ng ml À1 had an intermediate effect.…”

Section: Cell Aggregation By Absmentioning

confidence: 99%

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Apoptosis assays with lymphoma cell lines: problems and pitfalls

Mattes

2007

Br J Cancer

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Much attention has been focused on the manner in which tumour cells die after treatment with cytotoxic agents. The basic question is whether cells die via apoptosis or via direct damage from the toxic agent. Various assays have been used to make this distinction. However, we show herein that some of the widely used assays for apoptosis do not in fact distinguish between apoptosis and other forms of cell death. More specifically: (1) A sub-G 1 DNA content, identified by propidium iodide staining, does not distinguish between apoptotic and necrotic cells; (2) loss of mitochondrial membrane potential does not distinguish between apoptotic and necrotic cells, unless combined with an assay for an intact cell membrane; (3) subcellular fragments that arise from dead cells or from apoptotic bodies can interfere with some assays for apoptosis such as annexin V staining, as they may be close to the size of intact cells, making it difficult to decide where to set the size threshold; (4) irradiated cells display a large increase in nonspecific Ab binding. This may be partly due to an increase in cell size, but, regardless of the cause, it can lead to a mistaken conclusion that there is an increase in a particular antigen if appropriate control reagents are not tested; and (5) experiments utilising Ab crosslinking have neglected the role of cell aggregation, which can cause multiple problems including death from mechanical stress when cells are handled. Consideration of these factors will improve our ability to determine the mode of cell death.

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Section: Discussionmentioning

confidence: 75%

Section: Cell Aggregation By Absmentioning

confidence: 70%

Section: Cell Aggregation By Absmentioning

confidence: 99%

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Apoptosis assays with lymphoma cell lines: problems and pitfalls

Mattes

2007

Br J Cancer

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“…Interestingly, this can recapitulate a number of events observed during B cell interaction with T cells, including cytoskeleton reorganization, an increase in tyrosine phosphorylation and the calcium concentration, proliferation, differentiation, or apoptosis (1). In addition, high sensitivity of certain B cell lymphomas to cell death following MHC-II cross-linking make anti-MHC-II antibodies potential therapeutic agents (9,37).…”

mentioning

confidence: 91%

SCIMP, a Transmembrane Adaptor Protein Involved in Major Histocompatibility Complex Class II Signaling

Dráber

Vonkova

Štěpánek

et al. 2011

Molecular and Cellular Biology

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“…Considering the great similarity of CSVs containing 1F5 or L243, it is most likely that the CSVs containing L243 also are produced by viable cells. The frequent presence of nuclear fragments in fluorescent CSVs prepared with L243 may be related to the fact that L243 has been reported to induce toxicity, via caspase-independent apoptosis, in most Blymphoma cell lines [10,11]. As noted above, there was no significant amount of cell death in our experiments, but there may have been a low level of cell death, and one dead cell could generate a large number of small nuclear fragments.…”

Section: The Presence Of Nuclear Fragments Within Fluorescent Csvs Prmentioning

confidence: 58%

Characterization of antibody-containing vesicles shed from B-lymphoma cell lines: exposure of annexin V binding sites

Michel¹,

Abu‐Asab²,

Tsokos³

et al. 2006

Leukemia & Lymphoma

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Antibodies (Abs) to CD20 or HLA-DR, after binding to the B-lymphoma cell line RL following an overnight incubation at 37°C, accumulate in the form of shed vesicles, which develop in the center of the cell clusters that are spontaneously formed by this cell line. These vesicles coalesce into fairly stable large structures, which we refer to as conglomerates of shed vesicles (CSVs). In the present study, we have extended our previous investigations into the nature of this material. Electron microscopy revealed a conglomerate of heterogeneous vesicles, which looked like pinched-off cytoplasmic projections. CSVs developed similarly either with or without Ab, demonstrating that CSV production is a spontaneous process that incorporates bound Abs if they are present. Before delivery to CSVs, the Abs capped on the cell surface. CSVs had high expression of annexin V binding sites, which are phagocytic signals that are exposed on damaged cells. For CSVs that were cell bound, which are frequently observed, the annexin V binding sites were only in the CSVs, and not on the surface of the intact cell. Although all CSVs contained both Abs and annexin V binding sites, the precise distribution of these two ligands was generally different. Annexin V binding sites were present on caps as well as on CSVs, and appear as soon as caps are formed. In cells incubated with anti-HLA-DR, CD20 was delivered to the CSVs together with HLA-DR, suggesting an association between these two molecules. CSVs prepared with anti-HLA-DR, but not CSVs prepared with anti-CD20, contained considerable numbers of nuclear fragments, identified by propidium iodide staining.

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Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells

Cited by 135 publications

References 32 publications

Apoptosis assays with lymphoma cell lines: problems and pitfalls

Apoptosis assays with lymphoma cell lines: problems and pitfalls

SCIMP, a Transmembrane Adaptor Protein Involved in Major Histocompatibility Complex Class II Signaling

Characterization of antibody-containing vesicles shed from B-lymphoma cell lines: exposure of annexin V binding sites

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