A Comparative Study of the JAM Test and⁵¹Cr‐Release Assay to Assess the Cytotoxicity of Dendritic Cells on Hematopoietic Tumor Cells

Abstract

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [3H] thymidine (3H-TdR) uptake by tumor cells; 2) to induce cytolysis on 51Cr-labeled tumor cells; 3) and to induce DNA fragmentation on 3H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of 3H-TdR. However no cytolysis was verified by 51Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between 51Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released 51Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h 51Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.

Hepatocytes as cytotoxic effector cells can induce cell death by CD95 ligand-mediated pathway

“…40,41 The validity of the finding that primary woodchuck and human hepatocytes, as well as WCM-260 and HepG2 cells, eliminated CD95-bearing cells was ascertained using P815 pre-incubated with Jo2 MAb. This antibody is uniquely noncytolytic to P815 cells, most likely due to a defect in the cell CD95-activated signal transduction, but blocks recognition of cell surface CD95 by CD95L.…”

“…In preliminary experiments, it was established using woodchuck PBMC as effectors that the JAM DNA fragmentation assay can measure cytotoxic activity of woodchuck cells and that its sensitivity is comparable to or greater than that of the 51 Cr-release assay, as was also observed by others. 40 The ability of woodchuck PBMC to kill P815 cells was previously documented using the 51 Cr-release cytotoxicity assay. 34 Accordingly, the JAM assay was applied to examine hepatocyte cytotoxicity by using constant numbers of hepatocytes as effectors and increasing numbers of P815 or LS102.9 cells as targets.…”

Donor double‐negative Treg promote allogeneic mixed chimerism and tolerance

“…Any contaminated T cells and macrophages were further depleted by antibody-bonded MACS beads (Miltenyi Biotec) after flow cytometry analysis. We applied the JAM assay to measure DNA fragment and death of target cells [68][69][70]. Activated NK cells were labeled with 10 lCi/mL of [ 3 H]thymidine at 37 C overnight, washed and then used as targets.…”

A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

“…A simple and sensitive method to measure their activity would greatly benefit basic and clinical studies. For a long time, chromium ( 51 Cr) release assay has remained as the “gold standard” for quantifying cytolytic activities of CTLs against target cells and this method is still being used in many laboratories around the world [2], [3]. However, a major drawback of the 51 Cr release assay is the use of radioactive materials, which are inconvenient to handle because of environmental safety concerns and expensive due to the short half-life of the isotope.…”