EF1α, rather than CMV promoter, is suitable for luciferase tag expression in target cells for in vitro cytotoxicity assays of CAR-T cells

Abstract

Precise assessment of the cytotoxic activity of engineered immune cell therapeutics, such as chimeric antigen receptor-engineered T (CAR-T) cells, is essential for their development and quality control. However, luciferase (Luc)-based viability assays, which evaluate target cell viability by overexpressing Luc tags and measuring chemiluminescent signals, may yield biased results depending on the promoter driving Luc expression. This study demonstrates that CAR-T cells can enhance cytomegalovirus (CMV) promoter-driven transcription in target cells via the interferon-gamma (IFN-γ)/nuclear factor κB (NF-κB) signaling pathway, leading to elevated Luc expression and a discrepancy between chemiluminescent signals and actual target cell death. These findings underscore the limitations of CMV promoters in functional protein overexpression systems in the context of engineered T cell killing of target cells due to their susceptibility to transcriptional interference. Statistical analyses indicate that Luc expression driven by the elongation factor-1 alpha (EF1α) promoter exhibits the highest concordance with flow cytometry-based quantification across three CAR-T cytotoxicity assay platforms, making it a more reliable choice for evaluating CAR-T cell cytotoxicity. This study highlights the necessity of selecting appropriate promoters to ensure accurate Luc-based cytotoxicity assessments and provides critical insights for standardizing detection methodologies in CAR-T cell evaluation.

Keywords: CAR-T cell therapy; CMV promoter; EF1α promoter; cytotoxicity assay; luciferase.

Graphical abstract

Figure 1

The Luc reporter driven by the CMV promoter is not suitable for evaluating the cytotoxicity of CAR-T cells against target cells (A) Luc-based chemiluminescence assay was used to assess the cytotoxic efficacy of CAR-T cells under standardized experimental conditions. PDPN CAR-T cells, CD19 CAR-T cells, and mouse Her2 CAR-T cells were co-cultured with HGC-27^CMV−Luc cells, Nalm6^CMV−Luc cells, and B16F10-Her2^CMV−Luc cells at indicated E:T ratios, respectively. Residual luciferase activity in target cells was measured. The relative luciferase activity of each experimental group (n ≥ 3) was normalized against the negative control (NC) group at corresponding time points. (B) The cytotoxic effects of PDPN CAR-T cells against HGC-27^CMV−Luc cells were assessed by flow cytometry-based absolute cell counting, and the statistical correlation analysis between Luc-based measurements and absolute target cell counts obtained via flow cytometry was constructed. (C) The cytotoxic effects of CD19 CAR-T cells against Nalm6^CMV−Luc cells were assessed by flow cytometry-based absolute cell counting, and the statistical correlation analysis between Luc-based measurements and absolute target cell counts obtained via flow cytometry was constructed. (D) The cytotoxic effects of mouse Her2 CAR-T cells against B16F10-Her2^CMV−Luc cells were assessed by flow cytometry-based absolute cell counting, and the statistical correlation analysis between Luc-based measurements and absolute target cell counts obtained via flow cytometry was constructed. For luciferase activity assays in (A) and cell vialibility assays in (B)–(D), the data are shown as mean ± SD.

Figure 2

CAR-T cells induce an abnormal increase in Luc chemiluminescence values through the secretion of IFN-γ (A) Effects of mouse Her2 CAR-T cells, Her2 CAR-T^{IFN-γ KO} cells, Her2 CAR-T^{GzmB KO} cells, and their corresponding negative control mock T cells on the Luc chemiluminescence values in B16F10-Her2^CMV−Luc cells at indicated E:T ratios (n ≥ 3 per group). (B) Mouse IFN-γ levels in blank culture medium and in the supernatant of B16F10-Her2^CMV−Luc cells after Her2 CAR-T cell challenge (n = 4 per group). (C) Effects of mouse Her2 CAR-T cells and Her2 CAR-T^{IFN-γ KO} cells on the Luc chemiluminescence values in B16F10-Her2^CMV−Luc cells with or without the co-culture supernatant of Her2 CAR-T cells and B16F10-Her2 cells at indicated E:T ratios (n ≥ 3 per group). Data shown are mean ± SD from a single representative assay.

Figure 3

CAR-T cells enhance the transcriptional activity of the CMV promoter through the secretion of IFN-γ (A) Relative Luc mRNA levels in various target cells were quantified by qPCR 24 h after challenged by their corresponding CAR-T cells at indicated E:T ratios (n = 4 per group). (B) IFN-γ levels in blank culture medium or in the co-culture supernatant of CAR-T cells and their corresponding target cells (left) were measured by ELISA, and relative Luc mRNA levels in various target cells (right) were quantified by qPCR 24 h after challenged by the co-culture supernatant (n = 4 per group). (C) Relative Luc chemiluminescence value and relative Luc mRNA levels were measured in HGC-27^CMV−Luc cells, Nalm6^CMV−Luc cells, and B16F10-Her2^CMV−Luc cells after challenged by corresponding species of IFN-γ for 24 h (n = 4 per group). (D) Relative Luc mRNA levels in B16F10-Her2^CMV−Luc cells were quantified by qPCR after 24 h of CAR-T cell challenge (n = 4 per group). (E) Effects of 666-15 (1 μM) and JSH-23 (10 μM) on the Luc chemiluminescence values in HGC-27^CMV−Luc cells, Nalm6^CMV−Luc cells, and B16F10-Her2^CMV−Luc cells after challenged by PDPN CAR-T cells, CD19 CAR-T cells, and mouse Her2 CAR-T cells for 24 h, respectively (n ≥ 3 per group). Data shown are mean ± SD from a single representative assay.

Figure 4

Luc chemiluminescence value and residual cell viability in various Luc-labeled target cells driven by multiple promoters after CAR-T cell challenge (A) Effects of PDPN CAR-T cells on target cell Luc chemiluminescence value and residual target cell viability (determined by flow cytometry-based absolute target cell count) in Luc-labeled HGC-27 cells driven by multiple promoters at E:T ratios of 0.1:1 and 0.2:1 (n ≥ 3 per group). (B) Effects of CD19 CAR-T cells on target cell Luc chemiluminescence value and residual target cell viability (determined by flow cytometry-based absolute target cell count) in Luc-labeled Nalm6 cells driven by multiple promoters at E:T ratios of 0.25:1 and 0.5:1 (n ≥ 3 per group). (C) Effects of mouse Her2 CAR-T cells on target cell Luc chemiluminescence value and residual target cell viability (determined by flow cytometry-based absolute target cell count) in Luc-labeled B16F10-Her2 cells driven by multiple promoters at E:T ratios of 0.25:1 and 0.5:1 (n ≥ 3 per group). Data shown are mean ± SD from a single representative assay.