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

The overall goal of this multiplexed immunofluorescence analysis is to automatically count cells based on their phenotype. This method allows multifluorescence and automatic cell counting to analyze the phenotype and function of tumor immune cells within a tumor microenvironment. This technology facilitated the generation of two types of composite prognosis for predicted by your markers derived directly from the tumor microenvironment.

Generally, individuals new to this method may struggle because the pheno typing procedure is very long and the resulting raw data requires further processing. We first had the idea for this method when we attempting to characterize the phenotype and immunological interactions of annhodge miranty cells. After thawing, use a paper towel to carefully dry the slides around the tissue sections and then circle the tissues with a hydrophobic barrier pen.

When the barrier has dried, fix the samples in 100%acetone for five minutes, followed by two minutes of air drying, then wash the slides in TBS for 10 minutes. Pretreat the slides with 3 drops of 0.1%Avidin for 10 minutes in the dark, then tap or flick the slides to cover the tissue with the Avidin solution. Add 3 drops of 01%biotin to each sample, then tap or flick the biotin across the samples followed by a TBS wash as just demonstrated.

Then block any nonspecific binding with 100 microliters of 5%normal serum in TBS for 30 minutes at room temperature. At the end of the incubation, tap the slides to remove the excess serum and incubate the slides in 100 microliters of the primary antibody cocktail of interest for one hour in a humidified chamber. Wash the antibodies labeled slides in TBST for five minutes.

After drying, label the cells with 100 microliters of the secondary antibody cocktail of interest for 30 minutes in the humidified chamber, followed by a 10 minute wash in TBS. Incubate the dried secondary antibody labeled slides with 100 microliters of the tertiary antibody cocktail of interest for 30 minutes followed by 10 minutes of a TBS wash. At the end of the wash, dry the slides and mount the tissues with a dapi supplemented mounting medium.

Then clover each slide with a cover slip and let the mounting medium set for at least two hours. To scan the slides, open the microscope software and load the image scanning protocol. Load the first slide onto the microscope stage.

Then in the control bar, click set exposure and adjust the exposure time for each filter until a sufficient but not saturating signal is obtained. Click start in the upper panel and open the LabID folder. Enter the ID of the first slide and click next.

To acquire the overview of the slide under bright field light at a 4x magnification, click monochrome imaging and find specimen. To select the are of tissue to be scanned, press the control key and use the cursor to select or deselect the regions of interest to be scanned at the 4x magnification. To obtain a fluorescent red/blue/green image of each region, click low power imagine.

For high power field selection, press the control key and select at least five regions of interest that correspond to the areas of the tissue that will be scanned at the 20x magnification. Then, to obtain a multi spectral image acquisition of each field, click high power imagine, and click data storage to store the images in the appropriate lab ID folder. To create a new project under the file menu, select new project, under the find feature menu, select cell segmentation, and under the pheno typing menu, select pheno typing, then click configure.

In image format, select multi spectral. In sample format, choose fluorescence. To integrate the representative images into the project, under the file menu click open image and select 10-30 representative images of the whole series.

To remove the auto florescence, open the unstained slide, and select the library source and the appropriate fluorophores. Click auto florescence and select the area of auto florescence on the blank slide. To generate a composite image, select prepare all to integrate the fluorescence library and the auto fluorescent spectra.

Click the eye icon to open the view editor panel and deselect autoflourescence to trim the autoflourescence signal. Select an appropriate color for each marker. To segment the cells, in the compartment menu, select nuclei and membrane.

Under the nuclei menu, set DAPI as the nuclear counter stain. Click segment cells and check the segmentation of the nuclei in the cell membranes. To phenotype the cells, under the phenotype menu, click add.

To create the cell phenotype categories, select more than five representative for Fluorophor and Fluorophor combination, and click train classifier. The classifier is creating an algorithm that attributes a phenotype for each cell with a confidence interval. Zoom and check the phenotypes of the cells.

Then click phenotype all to save the phenotype algorithm and the image project, under the file menu select save project. Then name the project and save it. To perform a batch analysis of the series, select bath analysis and select the saved project.

Select a folder for storing the batched data. Add the images to be analyzed then click run. At the end of the analysis check the quality of the composite image and verify the pheno typing of all of the images within the series.

Analysis of the tumor infiltrating CD8 positive T-cells, on a clear renal cell carcinoma specimen by fluorescence multi spectral imaging reveals approximately half of the CD8 positive T-cells to be single positive for PD-1 and about 1/3 to be double positive for PD-1 and Tim-3. After integrating these various cell signals on a tissue section, measurement of the PD-1 florescence intensity on PD-1 positive Tim-3 positive versus PD-1 positive, Tim-3 negative CD8 positive T-cells, reveals that the PD-1 MFI is higher when Tim-3 is co expressed reinforcing the functional relevance of Tim-3 expression. In this representative experiment, 66%of the renal tumor patients were also positive for PD-1 Ligand expression and 100%were positive for the Tim-3 ligand Galactin-9.

Clinical scoring of the tumor tissues from renal cell carcinoma patients, also demonstrated the positive correlation between the number and percentage of tumor infiltrating CD-8 positive T-cells expressing PD-1 and Tim-3 and the prognostic scoring parameters but not PD-1 positive CD-8 not expressing Tim-3. Interestingly, renal cell carcinoma patients with CD-8 positive T-cells co expressing PD1 with and Tim 3 above the median percentage were more likely to relapse while the expression of PD-1 without Tim-3 co expression was found not to be correlated with patient relapse. While I attempted this procedure it's important to remember to review the phenotype of the cells with two separated investigators including a pathologist if possible.

Following this procedure other matters like for cytometry can be performed to answer additional questions about the functional impact of self phenotype after x-vivo stimulation. After it's development, this technique paved the way for researchers in the field of cancer to explore immune responses in effected organs.