Abstract
Cancer immunotherapy, which blocks immune checkpoint molecules, is an effective therapeutic strategy for human cancer patients through restoration of tumor-infiltrating (TI) cell function. However, evaluating the efficacy of immune checkpoint inhibitors (ICIs) is difficult because no standard in vitro assay for ICI efficacy evaluation exists. Additionally, blocking a particular immune checkpoint receptor (ICR) is insufficient to restore T cell functionality, because other ICRs still transduce inhibitory signals. Therefore, limiting inhibitory signals transduced via other ICRs is needed to more accurately assess the efficacy of ICIs targeting a particular immune checkpoint. Here, we introduce a newly developed in vitro coculture assay using human peripheral blood mononuclear cells (hPBMCs) and engineered human cancer cell lines. We enriched CD8+ T cells from hPBMCs of healthy donors through low-dose T cell receptor stimulation and cytokine (human IL-2 and IL-7) addition. These enriched CD8+ T cells were functional and expressed multiple ICRs, especially TIM-3 and TIGIT. We also established immune checkpoint ligand (ICL) knockout (KO) cancer cell lines with the CRISPR-Cas9 system. Then, we optimized the in vitro coculture assay conditions to evaluate ICI efficacy. For example, we selected the most effective anti-TIM-3 antibody through coculture of TIM-3+CD8+ T cells with PD-L1-/-PVR-/- cancer cells. In summary, we developed a mechanism-based in vitro coculture assay with hPBMCs and ICL KO cancer cell lines, which could be a useful tool to identify promising ICIs by providing reliable ICI efficacy information.
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Data availability
All data generated and analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- CRISPR:
-
Clustered regularly interspaced short palindromic repeats
- Cas9:
-
CRISPR-associated protein 9
- hPBMC:
-
Human peripheral blood mononuclear cell
- ICI:
-
Immune checkpoint inhibitor
- ICL:
-
Immune checkpoint ligand
- ICR:
-
Immune checkpoint receptor
- TI:
-
Tumor-infiltrating
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Funding
The authors declare no competing financial interests. This study was supported by grants funded by the Ministry of Food and Drug Safety (18182MFDS408) and the Ministry of Science and ICT (MSIT) (2017R1A5A1014560, 2019M3A9B6065221). This study was also supported by Korean Health Technology R&D Project through the Korean Health Industry Development Institute (KHIDI) funded by the Ministry of Health and Welfare (HV20C0144) and Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HN21C1410). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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MJK and S-JH designed and interpreted the study, wrote the manuscript and edited the manuscript. MJK performed the experiments and analyzed the data. KHH and BRL assisted with experiments. S-JH supervised the study. All authors approved the final version of the article, including the authorship list.
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The studies were approved by the Institutional Review Board of Yonsei University Severance Hospital with IRB no. 4-2016-0788 for a single patient with NSCLC and a single patient with HNSCC. All patients who participated in these studies provided written informed consent prior to enrollment and sample collection at Yonsei University Severance Hospital. The research conformed to the principles of the Helsinki Declaration.
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Supplementary Information
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262_2022_3201_MOESM1_ESM.pdf
Supplementary Figure 1 Comparison between the CD8+ T cell enrichments by the existing protocol and the newly developed protocol. hPBMCs were isolated from the peripheral blood of healthy donors. For the existing protocol using αCD3/CD28 dynabeads, isolated hPBMCs were cultured with αCD3/CD28 dynabeads (the ratio of 1:1) and hIL-2 (10 ng/ml). For the newly developed protocol using soluble anti-CD3 antibodies, isolated hPBMCs were cultured with anti-CD3 antibodies (1 μg/ml, soluble), hIL-2 (10 ng/ml), and hIL-7 (10 ng/ml) in non-tissue 24-well culture plates. (a) Representative FACS plot of singlets, lymphocytes, live cells and T cells of the cultured hPBMCs for 15 days according to the indicated protocol. The experiment was performed with hPBMCs from three donors. The data are representative of a single donor. (PDF 436 KB)
262_2022_3201_MOESM2_ESM.pdf
Supplementary Figure 2 Comparison between the CD8+ T cell enrichments by using hPBMCs of healthy donors and cancer patients. hPBMCs were isolated from the peripheral blood of healthy donors, a single patient with NSCLC, and a single patient with HNSCC. Isolated hPBMCs were cultured with anti-CD3 antibodies (1 μg/ml, soluble), hIL-2 (10 ng/ml), and hIL-7 (10 ng/ml) in non-tissue 24-well culture plates. (a) Representative FACS plot of the T cell frequency at the indicated time point after culture initiation. (b) Kinetics of the number of total cells among the cultured hPBMCs. (c) Kinetics of the frequency of T cells, and the number of T cells among the cultured hPBMCs. The experiment was performed with hPBMCs from healthy donors, a single patient with NSCLC, and a single patient with HNSCC. (PDF 332 KB)
262_2022_3201_MOESM3_ESM.pdf
Supplementary Figure 3 Naïve/memory phenotype of enriched CD8+ T cells. hPBMCs were isolated from the peripheral blood of healthy donors. Isolated hPBMCs were cultured with anti-CD3 antibodies (1 μg/ml, soluble), hIL-2 (10 ng/ml), and hIL-7 (10 ng/ml) in non-tissue 24-well culture plates. (a) Representative FACS plot of naïve/memory phenotype of cultured CD8+ T cells at the indicated time point after culture initiation. (b) Quantification of the percentage of effector memory (EM), effector memory expressing CD45RA (EMRA), central memory (CM), and naïve CD8+ T cells at the indicated time point after culture initiation. (c) Representative FACS plot of the ICR expression patterns of CD8+ T cells enriched 15 days according to naïve/memory phenotype. (d) Representative FACS plot of naïve/memory CD8+ T cell frequency in PD-1+ and PD-1-CD8+ T cells at 15 days after culture initiation (left). Quantification of the percentage of naïve/memory CD8+ T cell frequency in PD-1+ and PD-1-CD8+ T cells at 15 days after culture initiation (right). The experiment was performed with hPBMCs from two donors. The data are representative of triplicate samples from a single donor. The data were analyzed by two-tailed unpaired Student’s t-test (b, d). The error bars indicate the means ± SEMs. **p < 0.01; ****p < 0.0001. (PDF 938 KB)
262_2022_3201_MOESM4_ESM.pdf
Supplementary Figure 4 Degranulation in ICR-positive CD8+ T cells was augmented by ICIs. Before coculture, CD8+ T cells enriched for 21 days were preincubated with isotype control or ICR-blocking antibodies for 20 min in a 37°C incubator. Preincubated CD8+ T cells were cocultured with HCC4006 cell lines for 6 hr at a ratio of 1:1 under restimulation with the anti-CD3 antibody (20 μg/ml, plate-coated), the anti-CD28 antibody (5 μg/ml, plate-coated), hIL-2 (10 ng/ml), and hIL-7 (10 ng/ml). (a) Representative FACS plots of cytokine secretion and degranulation in PD-1+CD8+ T cells cultured with and without anti-PD-1 antibodies. (b) Rate of increase in PD-1+CD8+ T cell degranulation induced by anti-PD-1 antibody treatment. (c) Representative FACS plots of cytokine secretion and degranulation in TIGIT+CD8+ T cells cultured with and without anti-TIGIT antibodies. (d) Rate of increase in TIGIT+CD8+ T cell degranulation induced by anti-TIGIT antibody treatment. The data were concatenated in each group (a, c). The data were analyzed by two-tailed unpaired Student’s t-test (b,d). The error bars indicate the means ± SEMs. **p < 0.01; ****p < 0.0001. (PDF 496 KB)
262_2022_3201_MOESM5_ESM.pdf
Supplementary Figure 5 Comparison between the increased cytokine secretion in TIGIT+CD8+ T cells in the ratio of 1:1 and 0.1:1. Before coculture, CD8+ T cells enriched for 21 days were preincubated with isotype control or TIGIT-blocking antibodies for 20 min in a 37°C incubator. Preincubated CD8+ T cells were cocultured with HCC4006 cell lines for 6 hr at a ratio of 1:1 and 0.1:1 under restimulation with the anti-CD3 antibody (20 μg/ml, plate-coated), the anti-CD28 antibody (5 μg/ml, plate-coated), hIL-2 (10 ng/ml), and hIL-7 (10 ng/ml). (a) Representative FACS plot of cytokine secreting-TIGIT+CD8+ T cells cultured with and without anti-TIGIT antibodies in the ratio of 1:1 and 0.1:1. (b) Rate of increase in TIGIT+CD8+ T cell function induced by anti-TIGIT antibodies in the ratio of 1:1 and 0.1:1. (c) Representative FACS plot of degranulation in TIGIT+CD8+ T cells cultured with and without anti-TIGIT antibodies in the ratio of 1:1 and 0.1:1. (d) Rate of increase in TIGIT+CD8+ T cell degranulation induced by anti-TIGIT antibodies in the ratio of 1:1 and 0.1:1. The data were concatenated in each group (a, c). The data were analyzed by two-tailed unpaired Student’s t-test (b,d). The error bars indicate the means ± SEMs. ***p < 0.001; ****p < 0.0001. (PDF 490 KB)
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Kim, M.J., Hong, K.H., Lee, B.R. et al. Establishment of a mechanism-based in vitro coculture assay for evaluating the efficacy of immune checkpoint inhibitors. Cancer Immunol Immunother 71, 2777–2789 (2022). https://doi.org/10.1007/s00262-022-03201-9
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DOI: https://doi.org/10.1007/s00262-022-03201-9