Chronological measurement of cytotoxic CD8 T cell activity using a bioluminescence-based cell viability assay

doi:10.21203/rs.3.rs-2102989/v1

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Chronological measurement of cytotoxic CD8 T cell activity using a bioluminescence-based cell viability assay

https://doi.org/10.21203/rs.3.rs-2102989/v1

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Purpose

Cytotoxic T cells (CTLs) are an effector subset of activated CD8 T cells that play important role in the antitumor immune response. Although CTL cytotoxicity has been studied in vitro using various experimental protocols in which responder CTLs are added to kill the target tumor cells, methodologies for monitoring CTL activity in chronological order have not been fully developed. We attempted to develop a method for measuring CTL activity is measured using a real-time luminometer.

Methods

Splenocytes from B16-F10-bearing mice were harvested and cultured with mouse melanoma B16-F10 or Lewis lung carcinoma (LLC) cells. Flow cytometry was used to evaluate the efficiency of CTL expansion in terms of the frequency of CD44⁺ and CD62L⁻ cells in CD8⁺ cell subsets. CTL activity was assessed using a firefly luciferase-based bioluminescence method with splenic CD8 cells as the responder and luciferase-expressing cells as the target.

Results

The in vitro coculture of B16-F10-bearing spleen cells with B16-F10 cells produced a higher percentage of CTLs than with LLC cells, indicating that B16-F10-specific CTLs proliferated from tumor-bearing spleen cells. According to the time-lapse analysis the bioluminescence signal of luciferase-expressing B16-F10 cells was inhibited after 48 h by in vitro cultured CD8 cells derived from melanoma B16-F10-bearing mouse spleens, suggesting that B16-F10-reactive CTLs suppressed the target cell growth.

Conclusion

This simple bioluminescence-based assay is a useful method for monitoring the time course of CTL activity on the growth inhibition of luciferase-expressing cells.

bioluminescence

CD8 T cell

cytotoxic T cell

luciferase

melanoma

Cytotoxic T cells, known as cytotoxic T lymphocytes (CTLs), are an effector subset of CD8-positive T cells that recognize autologous antigens expressed on the surface of virus-infected cells or cancer cells by their cell surface T cell receptors. In the cancer − immunity cycle, a series of interactions between cancer and the immune system, tumor-specific CTLs are differentiated from naïve CD8 T lymphocytes in the tumor-draining lymph nodes after recognizing a cognate antigen presented by antigen-presenting cells, such as dendritic cells, and are subsequently recruited to the tumor site through blood vessels and attack target cancer cells (Chen and Mellman, 2013; Zhao et al., 2021). In the target tumor tissue, the tumor-reactive CTLs recognize the cells expressing the same antigen and induce target cell death. Notably, several melanoma antigens can induce an antimelanoma immune response in human. TRP-1, TRP-2, and gp100 are effective targets for tumor-reactive CTLs (Bakker et al., 1994; Wang et al., 1995; Wang et al., 1996), which have been utilized for cancer vaccines and for developing new therapeutic strategies (Yamano et al., 2006; Yan et al., 2014; Rezaei et al., 2021). In contrast, given the low immunogenicity of spontaneous animal tumors, an in vitro method for evaluating the CTL response to specific tumor antigens is required.

To date, several methods for assessing T cell-mediated cytotoxicity have been developed. Because of its high sensitivity, the chromium release assay has been widely used; however, its use is declining due to environmental safety concerns regarding the use of radioactive substances and the short half-life of isotopes. Various methods have been developed to overcome these difficulties. Although labeling variations and spontaneous release depend on target cells, methods based on the direct measurement of nonradioactive dyes or intracytoplasmic molecules released from dead cells have been developed (Rossignol et al., 2017). Flow cytometric measurement has also been established using multicolor fluorescent dyes to discriminate between the target cells and effector cells, enabling target cell–specific cell death (Flieger et al., 1995; Sheehy et al., 2001; Fischer and Mackensen, 2003). Firefly luciferase-based measurements can be applied to various biological activities in vitro and in vivo, including gene expression, protein–protein interaction, and signal transduction (Cheng et al., 2010; Luker and Luker, 2011). The advantage of a bioluminescent assay is its ability to track cell viability in real time without damaging cell survival. We previously performed a coculture assay that monitored mouse melanoma viability by bioluminescence and evaluated antimelanoma CTL response in chronological order (Nishizawa et al., 2021). We observed a slight decrease in the bioluminescence of target melanoma cells in the presence of stimulated splenocytes with CTL properties; however, whether the stimulated splenocytes specifically induce target cell death in vitro is debatable. Here we present an optimized assay protocol using purified splenic CD8 cells that improved data accuracy.

Cell cultures

A murine melanoma cell line B16-F10 and Lewis lung carcinoma cell line (LLC) were obtained from the American Type Culture Collection and kindly provided by Dr. K. Moriwaki of Osaka Medical College, respectively. Cells were cultured in Dulbecco's modified eagle's medium (DMEM, FUJIFILM Wako Pure Chemicals, Osaka, Japan) supplemented with 5% (v/v) fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol, 0.1 mM non-essential amino acids, and 10 mM HEPES (Nacalai Tesque, Inc., Kyoto, Japan).

Preparation of splenocytes from tumor-bearing mice

The 6-week-old C57BL/6N mice (Japan SLC) were subcutaneously injected on the back with B16-F10 cells (1 × 10⁶ cells). The spleen of the injected mice was harvested two weeks after injection. Splenocytes of the 8-week-old tumor-bearing or control mice were mechanically released from the tissue, treated with 0.83% NH₄Cl, 0.17 M Tris-Cl pH 7.65 for erythrocyte hemolysis, and resuspended with RPMI1640 (FUJIFILM Wako Pure Chemicals) supplemented with 10% FCS. Mice were housed in a temperature- and humidity-controlled facility on a 12 h light-dark cycle and anesthetized with isoflurane and euthanized before dissection.

Flow cytometry

Cells were treated with HB-197 cell culture supernatant containing an anti-CD16/32 (Fc-γ receptor) antibody. The cell suspension was treated with 2 µg/ml FITC-conjugated anti-mouse CD8a (clone 53 − 6.7, BioLegend), 1 µg/ml PE-conjugated anti-mouse/human CD44 (clone IM7, eBioscience), and 2.5 µg/ml APC-conjugated anti-mouse CD62L (clone MEL-14, BioLegend) monoclonal antibodies (mAbs) on ice for 30 minutes. For detection of major histocompatibility complex (MHC) class I expression, 5 µg/ml FITC-conjugated anti-mouse H-2Db (clone 28-14-8, eBioscience) was used. The data analysis and interpretation were performed on a BD LSR AriaII and the FlowJo software (BD Biosciences).

In vitro activation of CD8 splenocytes by a mixed culture with B16-F10 cells

The B16-F10 cells were seeded onto a 6-well cell culture plate at 1 × 10⁶ cells/well, incubated overnight, and treated with 10 µg/ml mitomycin C (MMC, Wako) for 3 h. The MMC-treated cells were plated at 5 × 10⁴ cells per well on a 24-well plate and cultured in DMEM supplemented with 10% FSC and 20 ng/ml recombinant mouse IFN-γ (BioLegend) for two days. Splenic CD8 T cells were enriched using the EasySep mouse CD8⁺ T cell isolation kit (ST-19853, Veritas). The purified CD8 T cells (4 ×10⁵ cells, over 98% purity) were resuspended in RPMI1640 containing 10% FCS and 100 U/ml recombinant mouse IL-2 (BioLegend), added to 5 × 10⁴ MMC-treated B16-F10 cells, and cultured for three days in a CO2 incubator.

Bioluminescence measurement

The B16-F10 or LLC cells expressing the firefly luciferase (fLuc) gene (fLuc-B16-F10 and fLuc-LLC, respectively) were obtained by transfection with the pGL3-Control Vector (E1741, Promega) at 1 µg/10⁵ cells by the PEI-MAX (Polysciences) according to the manufacturer's instructions. The transfected cells were seeded at 3 × 10⁵ cells in a 35-mm cell culture dish (TrueLine TR4000, Nippon Genetics Co., Ltd.) and cultured overnight in a CO₂ incubator at 37°C and subjected to puromycin (FUJIFILM Wako Pure Chemicals) or A23187 (Sigma) treatment. Cells were cultured in a 4-mL phenol red-free culture medium supplemented with 5% FCS and 150 µg/ml D-luciferin (VivoGlo Luciferin, Promega) in a 35-mm cell culture dish sealed with parafilm (Bermis Flexible Packaging). For the measurement of cell-mediated cytotoxicity, 1 × 10⁷ of splenic-derived CD8 T cells were added to 1 × 10⁵ of target cells in a 35-mm cell culture dish. Bioluminescence was measured by a real-time luminometer (Kronos Dio, Atto) at 37^oC for 48 hours. The data were analyzed by Mann-Whitney's U test using StatPlus software version v7 (AnalystSoft Inc.).

CTL induction in spleen of B16-F10-bearing mouse spleens

Figure 1A shows the study’s framework. To generate CTLs reactive to B16-F10 target cells, we obtained splenocytes from tumor-bearing mice of 14 days after the B16-F10 allograft. The CD8⁺ CD44⁺ CD62L⁻ CTL subset in the spleen was distinguished by forward scatter (FSC), side scatter (SSC), and CD44 and CD62L expression patterns (Fig. 1B, top). The CTL percentage was increased from 18–30%, whereas the CD44⁻ CD62L⁺ naïve T cell percentage was reduced from approximately 66–46% in tumor-bearing splenocytes compared with nontumor-bearing splenocytes, suggesting that B16-F10 allograft-generated B16-F10-reactive effector CD8 T cells were generated in the tumor-bearing mouse spleen (Fig. 1B, bottom).

CTL expansion in mouse splenocytes by in vitro culture

We next analyzed the efficiency of CTL expansion in splenocytes cocultured with the stimulator B16-F10 cells. As reported previously, H-2D^b expression was rarely detected in B16-F10 cells (Seliger et al., 2001; Merritt et al., 2004; Nagato et al., 2014), whereas it significantly increased after exposure to recombinant IFN-γ (Fig. 2A). In LLC, intrinsic H-2D^b expression was detected in the absence of IFN-γ, and it was significantly increased after 2 days of exposure to IFN-γ (Fig. 2A). For the following coculture experiments, we pre-treated B16-F10 cells with IFN-γ to induce the expression levels of MHC class I molecules. In both tumor-bearing and control spleens, the percentage of CD8 T cells was at comparable levels with or without coculture, suggesting that coculture does not affect CD8 composition in splenocytes (Fig. 2B). To examine whether coculture induces expansion of the tumor-specific CTL expansion, we compared the efficiency of CTL expansion by coculture with mouse LLC and B16-F10 cells. In the coculture of B16-F10-tumor-bearing spleen cells, a higher CTL percentage was observed with B16-F10 cells compared with LLC cells, whereas it was less prominent in nontumor-bearing spleen cells, suggesting that melanoma-specific CTLs proliferated from tumor-bearing spleen cells by a coculture with B16-F10 cells (Fig. 2C).

Cytotoxicity analysis of splenocyte-derived CTLs against B16-F10

The CTL killing assay was performed by the fLuc-based bioluminescence method using spleen-derived CTLs as responder cells and fLuc-expressing target cells, respectively. Our previous report showed that the luminescence value is inversely proportional to the survival rate of fLuc-B16-F10 cell (Nishizawa et al., 2021). Consistent with previous data, treatments of fLuc-B16-F10 cells with an antibiotic puromycin or a calcium ionophore A23187 caused a concentration-dependent decrease in the bioluminescence signal (Fig. 3A). We subsequently examined the effect of the responder CTLs on the target cell viability (Fig. 3B). During the treatment, there was a gradual increase in bioluminescence in the responder-free fLuc-B16-F10 cells. The signal was significantly decreased with cocultured CD8 T cells (53% ± 5.5) compared with that of non-cocultured cells (87% ± 8.6) 48 h after the start of measurement, suggesting that CTLs in the cocultured CD8 T cells inhibit the target cell proliferation. To determine whether cocultured CD8 T cells selectively inhibit B16-F10 proliferation, the firefly luciferase-expressing LLC cells, fLuc-LLC, were used as target cells. The fLuc-LLC cells exhibited maximum signal after 20-h without responder cells. The cocultured CD8 T cells with B16-F10 cells did not inhibit fLuc-LLC cell growth 20 h after measurement compared with non-cocultured cells, although the cocultured CD8 T cells slightly increased the growth of LLC cells at early phase compared with that of non-cocultured cells and target cell only (Fig. 3C). These results indicate that the responder CTLs selectively inhibit B16-F10 proliferation.

Several studies have demonstrated that the cytotoxic activity of antigen-specific CTLs can be evaluated by a reduction in cell-associated luciferase activity in various types of murine tumor cell lines (Fu et al., 2010; Olivo Pimentel et al., 2020). Our previous report showed that fLuc-B16-F10 cell viability was slightly impaired by in vitro cultured splenocytes derived from B16-F10-bearing mice (Nishizawa et al., 2021). From this result, we speculated that the target cell death was caused by the in vitro activated splenocytes, but we were uncertain whether the lowered target cell proliferation was due to the specific effect of the splenocyte-derived CTLs. Using purified splenic CD8 T cells as a source of CTLs, we demonstrated that the bioluminescence signal was lowered by cocultured CD8 T cells with melanoma cells compared with coculture-free CD8 T cells, suggesting that the melanoma-reactive effector CD8 cells differentiated in vitro impaired target cell proliferation.

Despite this useful and simple method for measuring target cell viability, some issues must be addressed. First, the sensitivity of measuring cell viability is low compared with previously reported CTL assays. A study regarding B16 cell survival by fluorescence time-lapse imaging reported a reduction in cell viability to approximately 10% during 4 h of coculturing with CTLs (Qi et al., 2019). Our experimental protocol with B16-F10 did not cause a dramatic decrease in the luminescence level even after 40 h of adding activated CD8 cells, suggesting that the efficacy of CTL cytotoxicity is low. The difference in the effect of CTL may be due to the lesser number of injected cells, the number of sites, and the repeat of injection compared with the previous report, which compromised the extent of the acquired immunity against melanoma cells. Alternatively, the B16 cells in the previous report may have higher immunogenicity and cause an accelerated CTL differentiation, which is possibly not identical to the B16-F10 in our study. Regarding CTL activity levels, a previous coculture study reported much less CTL cytotoxicity in colon and lung carcinoma cells (Olivo Pimentel et al., 2020), suggesting that the extent of antigen-specific CTLs may vary depending on cell type. Second, we detected a certain extent of a nonspecific effect on cell proliferation in LLC cells by adding of the responder cells. We speculate that the change in cell density in culture wells due to the addition of responder cells may have affected cell viability. To accurately assess CTL activity, each experimental setting, including conditions of cell injection in vivo and coculture in vitro, must be optimized.

Among the various methods that have been established for evaluating CTL activity, the bioluminescence-based assay is suitable for detecting cell viability, as it causes minimal cell damage even after repeated measurements. The fLuc-B16-F10 cells showed a significant reduction in luminescence signals with A23187 over 3 µM or puromycin over 2 µM, which are confirmed to be drug-dependent cell death by other methods (Schlapbach and Fontana, 1997; Glass-Marmor et al., 1999), suggesting that the luminescence value accurately reflects the survival rate. Notably, because this method does not directly reflect cell death but rather the activity of the remaining live cells, it is unsuitable for quantifying CTL activity. This method, when combined with other quantitative methods, is extremely useful for experiments analyzing temporal changes in CTL activities, such as comparative studies on the effects of immunomodulatory drugs and cells.

In conclusion, we demonstrated that the modified luciferase-based CTL assay protocol established in this study enabled us to monitor cell viability chronologically with high reproducibility. The luciferase-based method can be used to investigate CTL activity against poorly immunogenic tumor cells, such as B16-F10.

Acknowledgments

We appreciate the Division of Joint Research Center, Kindai University for granting access to a flow cytometer; Enago (www.enago.jp) for its English language review.

Funding

This work is supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant-in-Aid for Scientific Research, 19K07278 and 22K06823 to H.H.), and an external funding support research grant from Graduating School of Science and Engineering, Kindai University, Japan.

Competing Interests

The authors have no relevant financial or non-financial interest to disclose.

Author contributions

All authors contributed to the study conception and design. Data collection and analysis were performed by Ryota Hayashi, Hayato Nakatani, Hinami Kawahata, Ryonosuke Fujie, and Kaoru Kurowarabe. The original draft of the manuscript was written by Haruko Hayasaka. All authors read and approved the final manuscript.

Ethics approval

The protocols for animal experiments were approved by the ethics review committee for animal experimentation of Kindai University (KASE-2021-001).

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Chronological measurement of cytotoxic CD8 T cell activity using a bioluminescence-based cell viability assay

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Cell cultures

Preparation of splenocytes from tumor-bearing mice

Flow cytometry

In vitro activation of CD8 splenocytes by a mixed culture with B16-F10 cells

Bioluminescence measurement

Results

CTL induction in spleen of B16-F10-bearing mouse spleens

CTL expansion in mouse splenocytes by in vitro culture

Cytotoxicity analysis of splenocyte-derived CTLs against B16-F10

Discussion

Declarations

References

Status:

Version 1