Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Summary

Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8⁺ T cells. This reproducible and reliable technique is suitable for large cohort analyses.

Abstract

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment.

The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8⁺ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

Introduction

In the past few years, immunotherapy has emerged as a very promising treatment for many types of cancers, including RCC. Particularly, immunotherapy based on the inhibition of inhibitory checkpoints like PD-1 and CTLA-4 has been reported to be clinically effective^1,2,3,4,5,6. Monoclonal antibodies against CTLA-4, PD-1, or Program Death Ligand 1 (PD-L1) are already approved in several cancers and lead to long lasting clinical responses in more than 20% of patients

Protocol

This study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee (CPP Ile de France nr. 2012-05-04). Informed consent was obtained from the participant included in the cohorts.

1. Tissue Material

1. Collect RCC tissue samples on the day of surgery. Handle the surgical specimen at room temperature (pathology department).
2. Collect a tumor sample of approximately 0.5 cm x 0.5 cm x 0.5 cm size in a dry tube. Snap freeze in liquid nitrogen and store at -80 °C.
3. Coat the samples in optimal cutting temperature compound. Section the samples with a cryostat at -20 ....

Results

Using the general protocol described above, we aimed to quantify intratumoral CD8⁺ T cells co-expressing the inhibitory receptors PD-1 and Tim-3 in frozen tissues from patients with RCC, and to correlate the results with clinical outcomes²⁵.

Optimization of the CD8/PD-1/Tim-3 Staining:

Different species of antibodies .......

Discussion

Modifications and Troubleshooting:

The tissue quality is an important parameter; it can be easily checked by hematoxylin and eosin coloration.

One advantage of the technique is the possibility of multiplexed stainings, but to avoid fluorophore spillover, it recommended to choose emission wavelengths with delta of 10 nm minimum. Here, because we had 4 stainings including DAPI, we chose to spread out the wavelengths (DAPI: 460 nm, AF488: 519 nm, Cyani.......

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 Salin TBS Tablets	Takara, Bio Inc.	TAKT9141Z	pH7.6 100 tablet
Tris Buffer Salin 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