Determining optimal cytotoxic activity of human Her2neu specific CD8 T cells by comparing the Cr51 release assay to the xCELLigence system.
Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase release , BLT esterase activity and surface expression of CD107. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.
Department of Immunology, College of Medicine, Mayo Clinic, USA.
Courtney L
CL
Erskine
Andrea M
AM
Henle
Keith L
KL
Knutson
D001943:Breast Neoplasms / Q000276:immunology / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002860:Chromium Radioisotopes / Q000737:chemistry; D018920:Coculture Techniques; D003584:Cytological Techniques / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D017097:Electric Impedance; D018984:Epitopes, T-Lymphocyte / Q000097:blood / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000097:blood / Q000276:immunology; D006801:Humans; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000097:blood / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000201:enzymology* / Q000276:immunology*
D002860:Chromium Radioisotopes; D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human