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 (51)Cr-release assay and IFN-gamma 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 (51)Cr-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.

Figure 1. Cytotoxicity assays based on flow cytometry

(1) Labeling cells with fluorescent dyes, transfection with GFP or fluorescent antigens. Labeling can be done using: fluorescein isothiocyanate labeling [31]; dioctadecyloxacarbocyanine perchlorate₍₃₎ (DiOC18₍₃₎) [32,33,103]; calcein acetoxymethyl ester [104]; 2′7′-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF) [105]; carboxyfluorescein succinimidyl ester or chloromethyl-benzoyl-aminotetramethyl-rhodamine [37,44,67,106]; PKH dyes [34,38,39,43,45,46,70,107]; transfection, transduction with GFP or fluorescent antigens [–112]. (2) Detection of caspase activation and/or GrB substrate cleavage in target cells, via staining with mAb to activated caspases [23,113,114]; detection of cleaved fluorigenic caspase substrate [–26,28] or detection of cleaved fluorigenic GrB substrate [27,28]. (3) Detection of target apoptosis by annexin V binding to ‘flipped’ phosphatidylserine [,,,,,,,,,–118]. (4) Uptake of DNA intercalating fluorescent agents due to increased cell membrane permeability of dying target cells. Fluorescent agents are propidium iodide [–34,37,41,105,107,108]; TO-PRO^®3 [38,39] or 7AAD [–46,67,118]. (5) Analysis of effector cells degranulation with anti-CD107 mAb staining [,–,–,,–121]. (6) Cytokine flow cytometry. Provides valuable information regarding the functionality of antigen-specific T cells, but does not accurately examine the ability of the CD8⁺ T cells to elicit cytotoxic activity [,,,,,,–125]. (7) Effectors staining with MHC tetramers. Provides valuable information regarding the frequency and phenotype of antigen-specific T cells, but does not accurately examine the ability of the CD8⁺ T cells to elicit cytotoxic activity [,,,,,,,,–128]. (8) Phenotypic and activation markers staining [29,40,41,56,67,97,129]. (9) Detection of uptake of target membrane or antigens by effector cells [–133]. (10) Detection of effector–target conjugate formation. Conjugate formation between target cells and cytotoxic cells while evaluates effector frequency has been shown to occur independently of killing [67,134,135]. 7AAD: 7-Aminoactinomycin D; GFP: Green fluorescent protein; GrB: Granzyme B; mAb: Monoclonal antibody; PKH: Paul Karl Horan; PS: Phosphatidylserine; TNF: Tumor necrosis factor.

Figure 2. Application of a flow cytometric cytotoxicity assay for monitoring a cancer vaccine trial

Target cell death is measured using annexin V binding to PKH67-positive cells and CTL degranulation determined by CD107a staining on the CD8-positive cells. The gp100:209-specific CTLs were generated by incubation of melanoma patients’ peripheral blood mononuclear cells for 1 week with g209 peptide in the presence of IL-7 and IL-2. APC: Antigen-presenting cell; CTL: Cytotoxic T lymphocyte; GrB: Granzyme B; PE: Phycoerythrin; PKH: Paul Karl Horan; PS: Phosphatidylserine.