Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

Abstract

Source: Granier, C., et al. Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1⁺ Tim-3⁺ CD8⁺ T Cells. J. Vis. Exp. (2018).

This video demonstrates an in-situ fluorescence multispectral imaging technique to analyze different subpopulations of infiltrating T cells in tumor tissue. A renal cell carcinoma tissue cryosection is treated with antibodies to label CD8+ T cells that express specific inhibitory receptors and exhibit an immunotherapy-resistant phenotype. The labeled section is visualized using a multispectral microscope to identify the distinct CD8+ T cell subpopulations.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines

1. In Situ Immunofluorescence Staining of tumor-infiltrating lymphocytes (TILs)

1. Procedural guidelines
   1. Perform all experimental steps at room temperature.
   2. For all steps, perform the dilutions with Tris Buffer Saline (TBS; see Table of Materials).
   3. Perform the wash in TBS for all the steps except after the primary antibody incubation. For the latter, use Tris Buffer Saline Tween20 (TBST, see Table of Materials). For buff....

Results

Figure 1: CD8-PD-1-Tim-3 immunostaining on a tonsil paraffin-embedded tissue. Paraffin-embedded tissue sections instead of cryopreserved sections were selected for triple immunostaining. After dewaxing and rehydration, the same protocol of staining .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vectra 3 Automated Quantitative Pathology Imaging	Perkin Elmer	CLS142338
inForm cell analysis 2.1.	Perkin Elmer	CLS135781
R software	https://www.r-project.org
Dakopen delimiting pen	Dako	S2002
Tris Buffer Saline TBS Tablets	Takara, Bio Inc.	TAKT9141Z	pH7.6 100 tablet
Tris Buffer Saline Tween 20 TBS(+Tween20)	Takara, Bio Inc.	TAKT9142Z	pH 7.6 100 tablets
Biotin blocking system	Dako	X0590	Avidin 0.1% and Biotin 0.01%
Normal donkey serum	Jackson Immunoresearch	017-000-001	5% vol./vol. concentration
Fluoroshield with DAPI	Sigma-aldrich	F6057	1.5 µg/mL concentration
Knittel glass coverslip	Knittel Gläser,	100039	24x60 mm 100 cover slips
Rabbit anti-CD8 Clone P17-V	novus	NBP1-79055	use at 4µg/mL
Mouse anti-PD-1 Clone NAT	abcam	ab52587	use at 2 µg/mL
Goat anti-Tim-3	R&D	AF2365	use at 3 µg/mL
Rabbit anti-PD-L1 Clone SP142	Roche	7309457001	use at 1 µg/mL
Mouse AF647 labeled pan- Keratin Clone C11	Cell Signalling	4528	use at 0.5 µg/mL
Goat anti-human gal9	R&D	AF2045	use at 0.3 µg/mL
Cyan 5 conjugated donkey anti-rabbit	Jackson Immunoresearch	711-175-152	use at 5 µg/mL
Biotinylated F(ab'2) donkey anti-mouse IgG	Jackson Immunoresearch	715-066-150	use at 3 µg/mL
Alexa Fluor488 conjugated donkey anti-goat IgG	abcam	ab150133	use at 5 µg/mL
Cy3 labeled streptavidin	Amersham	PA43001	use at 3 µg/mL
Negative control mouse IgG1	Dako	X0931	use at 2 µg/mL
IgG from goat serum	Sigma-aldrich	I5256	use at 3 µg/mL
IgG from rabbit serum	Sigma-aldrich	I5006	use at 4µg/mL