Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

Published: April 30, 2024

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) Procedural guidelines Perform all experimental steps at room temperature. For all steps, perform the dilutions with Tris Buffer Saline (TBS; see Table of Materials). Perform the wash in TBS for all the st…

Representative 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 previously developed for cryopreserved sections was applied. CD8+ T cells are located outside the germinal center, and non-CD8+ T cells expressing PD-…

Divulgaciones

The authors have nothing to disclose.

Materials

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 24×60 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

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Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations. J. Vis. Exp. (Pending Publication), e22139, doi: (2024).

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