Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry

Main Article Content

Rachel A. Cunningham
Martha Holland
Emily McWilliams
Frank Stephen Hodi
Mariano Severgnini

Keywords

checkpoint markers, flow cytometry, immuno-phenotype, immunotherapy

Abstract

As checkpoint inhibitor immunotherapies gain traction among cancer researchers and clinicians, the need grows for assays that can definitively phenotype patient immune cells. Herein, we present an 8-color flow cytometry panel for lineage and immune checkpoint markers and validate it using healthy human donor peripheral blood mononuclear cells (PBMCs). Flow cytometry data was generated on a BD LSR Fortessa and supported by Luminex multiplex soluble immunoassay. Our data showed significant variation between donors at both baseline and different stages of activation, as well as a trend in increasing expression of checkpoint markers on stimulated CD4+ and CD8+ T-cells with time. Soluble immune checkpoint quantification assays revealed that LAG-3, TIM-3, CTLA-4, and PD-1 soluble isoforms are upregulated after stimulation. This 8-color flow cytometry panel, supported here by soluble immunoassay, can be used to identify and evaluate immune checkpoints on T-lymphocytes in cryopreserved human PBMC samples. This panel is ideal for characterizing checkpoint expression in clinical samples for which cryopreservation is necessary.

Downloads

Download data is not yet available.

Metrics

Metrics Loading ...
Abstract 77 | HTML Downloads 234 PDF Downloads 273 Supplementary information Downloads 0

References

1. Tan, W. L., Jain, A., Takano, A., Newell, et al (2016) Novel therapeutic targets on the horizon for lung cancer. Lancet Oncol, 17(8), e347-e362.
2. Freeman GJ, Long AJ, Iwai Y, et al (2000) Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med, 192(7):1027-34.
3. Jin, H. T., Ahmed, R., & Okazaki, T. (2010) Role of PD-1 in regulating T-cell immunity. Negative Co-Receptors and Ligands (pp. 17-37). Springer, Berlin, Heidelberg.
4. Hodi, F. S., O'Day, S. J., McDermott, D. F., Weber, R. W., Sosman, J. A., et al (2010) Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med, 363(8), 711-723.
5. Lipson, E. J., & Drake, C. G. (2011) Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma. Clin Cancer Res, 17(22) 6958-62.
6. Melero, I., Hervas-Stubbs, S., Glennie, M., Pardoll, D. M., & Chen, L. (2007) Immunostimulatory monoclonal antibodies for cancer therapy. Nat Rev Cancer, 7(2), 95.
7. Wolchok, J. D., Hodi, F. S., Weber, J. S., Allison, J. P., et al. (2013) Development of ipilimumab: a novel immunotherapeutic approach for the treatment of advanced melanoma. Ann NY Acad Sci, 1291(1), 1-13.
8. Dine, J., Gordon, R., Shames, Y., Kasler, M. K., & Barton-Burke, M (2017). Immune Checkpoint Inhibitors: An Innovation in Immunotherapy for the Treatment and Management of Patients with Cancer. Asia Pac J Oncol Nurs, 4(2), 127-135.
9. Massard, C., Gordon, M. S., Sharma, S., Rafii, S., Wainberg, Z. A., et al. (2016) Safety and efficacy of durvalumab (MEDI4736), an anti-programmed cell death ligand-1 immune checkpoint inhibitor, in patients with advanced urothelial bladder cancer. J Clin Oncol, 34(26), 3119-3125.
10. Rosenberg, J. E., Hoffman-Censits, J., Powles, T., Van Der Heijden, M. S., et al. (2016) Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet, 387(10031), 1909-1920.
11. Kaufman, H. L., Russell, J., Hamid, O., Bhatia, S., et al. (2016) Avelumab in patients with chemotherapy-refractory metastatic Merkel cell carcinoma: a multicentre, single-group, open-label, phase 2 trial. Lancet Oncol, 17(10), 1374-1385.
12. Amatore, F., Gorvel, L., & Olive, D. (2018) Inducible Co-Stimulator (ICOS) as a potential therapeutic target for anti-cancer therapy. Expert Opin Ther Targets, 22(4), 343-351.
13. Du, W., Yang, M., Turner, A., Xu, C., Ferris, R. et al. (2017) TIM-3 as a Target for Cancer Immunotherapy and Mechanisms of Action. Int J Mol Sci, 18(3), 645.
14. Goldberg, M. V., & Drake, C. G. (2010) LAG-3 in cancer immunotherapy. Cancer Immunol Immunother (pp. 269-278). Springer, Berlin, Heidelberg.
15. Andrews, L. P., Marciscano, A. E., Drake, C. G., & Vignali, D. A. (2017) LAG 3 (CD 223) as a cancer immunotherapy target. Immunol Rev, 276(1), 80-96.
16. Shimizu, T., Fuchimoto, Y., Okita, H., Fukuda, K., et al. (2018) A curative treatment strategy using tumor debulking surgery combined with immune checkpoint inhibitors for advanced pediatric solid tumors: An in vivo study using a murine model of osteosarcoma. J Pediatr Surg. In Press.
17. Fan, X., Quezada, S. A., Sepulveda, M. A., Sharma, P., & Allison, J. P. (2014) Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy. J Exp Med, 211(4), 715-725.
18. Ascierto, P. A., Melero, I., Bhatia, S., Bono, P., et al. (2017) Initial efficacy of anti-lymphocyte activation gene-3 (anti–LAG-3; BMS-986016) in combination with nivolumab (nivo) in pts with melanoma (MEL) previously treated with anti–PD-1/PD-L1 therapy. J Clin Oncol, 35, no 15 9520-9520.
19. Rühle, P. F., Fietkau, R., Gaipl, U. S., & Frey, B. (2016). Development of a modular assay for detailed immunophenotyping of peripheral human whole blood samples by multicolor flow cytometry. Int J Mol Sci, 17(8), 1316.
20. Donaubauer, A. J., Rühle, P. F., Becker, I., Fietkau, R., Gaipl, U. S., & Frey, B. (2019). One-Tube Multicolor Flow Cytometry Assay (OTMA) for Comprehensive Immunophenotyping of Peripheral Blood. Methods Mol Biol, 1904, 189.
21. Patel, T., Cunningham, A., Holland, M., Daley, J., Lazo, S., et al. (2018) Development of an 8-color antibody panel for functional phenotyping of human CD8+ cytotoxic T cells from peripheral blood mononuclear cells. Cytotechnology, 70(1), 1-11.
22. Sridharan, V., Margalit, D. N., Lynch, S. A., Severgnini, M., et al. (2016). Definitive chemoradiation alters the immunologic landscape and immune checkpoints in head and neck cancer. Br J Cancer, 115(2), 252.
23. Ben‐Ami, E., Barysauskas, C. M., Solomon, S., Tahlil, K., Malley, R., et al. (2017). Immunotherapy with single agent nivolumab for advanced leiomyosarcoma of the uterus: results of a phase 2 study. Cancer, 123(17), 3285-3290.
24. Holland, M., Cunningham, R., Seymour, L., Kleinsteuber, K., et al. (2018) Separation, banking, and quality control of peripheral blood mononuclear cells from whole blood of melanoma patients. Cell Tissue Bank, In Press.
25. Clayton, K. L., Douglas-Vail, M. B., Rahman, A. N. U., Medcalf, K. E., et al (2015) Soluble T cell immunoglobulin mucin domain 3 is shed from CD8+ T cells by the sheddase ADAM10, is increased in plasma during untreated HIV infection, and correlates with HIV disease progression. J Virol, 89(7), 3723-3736.
26. Zhu, X., & Lang, J. (2017) Soluble PD-1 and PD-L1: predictive and prognostic significance in cancer. Oncotarget, 8(57), 97671.
27. Esposito, L., Hunter, K. M., Clark, J., Rainbow, D. B. et al. (2014) Investigation of soluble and transmembrane CTLA-4 isoforms in serum and microvesicles. J Immunol, 1303389.
28. Rodig, S. J., Gusenleitner, D., Jackson, D. G., Gjini, E., Giobbie-Hurder, A., et al. (2018). MHC proteins confer differential sensitivity to CTLA-4 and PD-1 blockade in untreated metastatic melanoma. Sci Transl Med, 10(450), eaar3342.

Most read articles by the same author(s)