New flow cytometric assays for monitoring cell-mediated cytotoxicity

Abstract

The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the ⁵¹Cr-release assay and IFN-γ ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the ⁵¹Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency.

Keywords::

• cell-mediated cytotoxicity
• cytotoxic T lymphocyte
• multiparameter flow cytometry
• natural killer cell
• vaccine

Disclosure

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

Financial & competing interests disclosure

This project has been funded in whole or in part with federal funds from the National Cancer Institute, NIH, under Contract No. HHSN261200800001E. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Notes

AM: Acetoxymethyl; BCECF: 2´-7´-biscarboxyethyl-5(6)-carboxyfluorescein; BLT: Benzyloxycarbonyl-l-lysine thiobenzyl ester; CFDA: Carboxyfluorescein diacetate; CTL: Cytotoxic T lymphocyte; ELISpot: Enzyme-linked immunospot assay; GrB: Granzyme B; JAM: Just another method; LDA: Limited dilutions assay; LDH: Lactate dehydrogenase; MTT: Methylthiazolyldiphenyl-tetrazolium bromide; MUH: 4-methylumbelliferyl heptanoate.