Table Info

Table 2.

Staining conditions for panel 1 (epithelial cancer tissue)

HIER buffer (pH)	HIER incubation time	Primary antibody	Antibody dilution^*	Antibody incubation time	Secondary antibody	Secondary antibody incubation time	Opal™	Opal™ dilution	Opal™ incubation time
AR9	15 min	CD3e	1:100	30 min	HRP	10 min	520	1:100	10 min
AR9	15 min	BTN3A1	1:1,000	Overnight	HRP	10 min	540	1:100	10 min
AR9	15 min	Granzyme B	1:125	30 min	HRP	10 min	570	1:100	10 min
AR9	15 min	BTN2A1	1:500	Overnight	HRP	10 min	620	1:100	10 min
AR9	15 min	Pan-γδ	1:100	30 min	HRP	10 min	650	1:100	10 min
AR9	15 min	PanCK	1:40	30 min	HRP	10 min	690	1:50	10 min

This panel includes markers for CD3, Pan-γδ TCR, granzyme B, BTN2A1, BTN3A1, PanCK, and DAPI. *

using the following stock concentrations: CD3e: not determined (supplied as cell culture supernatant); BTN3A1: 0.1 mg/mL; granzyme B: 6 μg/mL; BTN2A1: 50 μg/mL; Pan-γδ: 0.1 mg/mL; PanCK: 80 μg/mL

Declarations

Acknowledgments

The Olivia Newton-John Cancer Research Institute acknowledges the support of the Victorian Government Operational Infrastructure Support Program, and the Ian Potter Foundation for providing funds to purchase the Vectra System. We would also like to thank Dr Khashaya Asadi, Austin Pathologist, for the provision of lung cancer FFPE tissue slides.

Author contributions

JDGD, LTQ, ET, SO and AB contributed to the concept, design and execution of the method; JDGD wrote the original manuscript; LTQ, SO and AB reviewed and edited the manuscript; JDGD and AB supervised the project; AB acquired funding for the project. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest

AB is the recipient of research funding for work on butyrophilins in cancer; AB and SO are inventors on multiple patents related to the topic. No other authors declare a conflict of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent to publication

Not applicable.

Availability of data and materials

Not applicable.

Funding

JDGD is supported by Cure Cancer Australia through the Cancer Australia Priority—Driven Cancer Research Scheme (#1187815). The contents of the published material are solely the responsibility of La Trobe University and do not reflect the views of Cancer Australia. AB is the recipient of a Fellowship from the Victorian Government Department of Health and Human Services acting through the Victorian Cancer Agency. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright

References

Tyler CJ, Doherty DG, Moser B, Eberl M. Human Vγ9/Vδ2 T cells: innate adaptors of the immune system. Cell Immunol. 2015;296:10–21. [DOI] [PubMed]

Park JH, Lee HK. Function of γδ T cells in tumor immunology and their application to cancer therapy. Exp Mol Med. 2021;53:318–27. [DOI] [PubMed] [PMC]

Kabelitz D, Serrano R, Kouakanou L, Peters C, Kalyan S. Cancer immunotherapy with γδ T cells: many paths ahead of us. Cell Mol Immunol. 2020;17:925–39. [DOI] [PubMed] [PMC]

Meraviglia S, Lo Presti E, Tosolini M, La Mendola C, Orlando V, Todaro M, et al. Distinctive features of tumor-infiltrating γδ T lymphocytes in human colorectal cancer. Oncoimmunology. 2017;6:e1347742. [DOI] [PubMed] [PMC]

Presti E Lo, Pizzolato G, Corsale AM, Caccamo N, Sireci G, Dieli F, et al. γδ T cells and tumor microenvironment: from immunosurveillance to tumor evasion. Front Immunol. 2018;9:1395. [DOI] [PubMed] [PMC]

Harly C, Guillaume Y, Nedellec S, Peigné CM, Mönkkönen H, Mönkkönen J, et al. Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset. Blood. 2012;120:2269–79. [DOI] [PubMed] [PMC]

Rigau M, Ostrouska S, Fulford TS, Johnson DN, Woods K, Ruan Z, et al. Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells. Science. 2020;367:eaay5516. [DOI] [PubMed]

Rigau M, Uldrich AP, Behren A. Targeting butyrophilins for cancer immunotherapy. Trends Immunol. 2021;42:670–80. [DOI] [PubMed]

Hunyady B, Krempels K, Harta G, Mezey E. Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining. J Histochem Cytochem. 1996;44:1353–62. [DOI] [PubMed]

10.

Tóth ZE, Mezey E. Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. J Histochem Cytochem. 2007;55:545–54. [DOI] [PubMed]

11.

Stack EC, Wang C, Roman KA, Hoyt CC. Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods. 2014;70:46–58. [DOI] [PubMed]