A simple and sensitive method for measuring tumor-specific T cell cytotoxicity

Abstract

A simple and sensitive method to quantitatively measure the cytolytic effect of tumor-specific T killer cells is highly desirable for basic and clinical studies. Chromium (51Cr) release assay has been the "gold standard" for quantifying cytolytic activities of cytotoxic T lymphocytes (CTLs) against target cells and this method is still being used in many laboratories. However, a major drawback of this method is the use of radioactive materials, which is inconvenient to handle because of environmental safety concerns and expensive due to the short half-life of the isotope. Consequently, several nonradioactive methods have been reported recently. Here we report a new method that we recently developed for quantifying antigen-specific cytolytic activity of CTLs. This method fully exploits the high sensitivity and the relative simplicity of luciferase quantitative assay. We initially expected the released luciferase in the supernatant to be the adequate source for monitoring cell death. However, to our total surprise, incubation of these killer T cells with the tumor cell targets did not result in significant release of luciferase in the culture medium. Instead, we found that the remaining luciferase inside the cells could accurately reflect the overall cell viability.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Quantification of luciferase expression from serially diluted 4T1-Her2 cells.

A. Cells were seeded at a large range of density (from 10,000 to 50 per well) and were assayed for luciferase expression 2 h later. B. The assay was focused on the low cell number wells. The numbers above each bar indicate the actual luciferase units detected.

Figure 2. Pearson's correlation coefficient analysis.

4T1-Her2 cells were seeded in 96 well plates at increasing numbers and the luciferase level was determined after 2 hours. Both cell number and luciferase quantity were converted into percentage by dividing the individual figure with the maximal cell number seeded (100,000) or the luciferase reading from the well with the maximal cell number. The correlation coefficient (R) is indicated in the figure.

Figure 3. Low natural release of luciferase from 4T1-Her2 cells.

Initially 10,000 4T1-Her2 cells were seeded. The supernatants were collected at the indicated times for measurement of luciferase.

Figure 4. Luciferase was undetectable in the supernatant during T cell-mediated 4T1-Her2 cytolysis.

A. Cell-associated and supernatant luciferase activity during cTCR-splenocyte mediated cytolysis of 4T1-Her2 cells. cTCR-splenocytes were added to 4T1-Her2 cells at a ratio of 10∶1. Supernatants and cells were harvested at the indicated time for quantification of luciferase activity. B. 4T1-Her2 monolayer without cTCR-transduced splenocytes. C. Seventy-two h after cTCR-tranduced splenocytes were added to 4T1-Her2 monolayer.

Figure 5. Reduction in cell-associated luciferase activity closely reflects the killing activity of T killer cells against tumor targets.

Mock-transduced (A) or cTCR-transduced (B) splenocytes were added to 4T1-Her2 cells at the indicated ratio. Cells were harvested 72 later for quantification of luciferase activity. C. Using the formula: % specific luc reduction = (% luc reduction from engrafted T-cell)/(% luc reduction from control T-cell)×100, the data from A and B were converted into percentage of specific luciferase release and plotted.